生物学（英文版）：1

1 Unraveling the Mystery of How Geckos Defy Gravity Science is most fun when it tickles your imagination. This is particularly true when you see something your common sense tells you just can’t be true. Imagine, for example, you are lying on a bed in a tropical hotel room. A little lizard, a blue gecko about the size of a toothbrush, walks up the wall beside you and upside down across the ceiling, stopping for a few moments over your head to look down at you, and then trots over to the far wall and down. There is nothing at all unusual in what you have just imagined. Geckos are famous for strolling up walls in this fashion. How do geckos perform this gripping feat? Investi- gators have puzzled over the adhesive properties of geckos for decades. What force prevents gravity from dropping the gecko on your nose? The most reasonable hypothesis seemed suction— salamanders’ feet form suction cups that let them climb walls, so maybe geckos’ do too. The way to test this is to see if the feet adhere in a vacuum, with no air to create suction. Salamander feet don’t, but gecko feet do. It’s not suction. How about friction? Cockroaches climb using tiny hooks that grapple onto irregularities in the surface, much as rock- climbers use crampons. Geckos, however, happily run up walls of smooth polished glass that no cockroach can climb. It’s not friction. Electrostatic attraction? Clothes in a dryer stick together because of electrical charges created by their rubbing to- gether. You can stop this by adding a “static remover” like a Cling-free sheet that is heavily ionized. But a gecko’s feet still adhere in ionized air. It’s not electrostatic attraction. Could it be glue? Many insects use adhesive secretions from glands in their feet to aid climbing. But there are no glands cells in the feet of a gecko, no secreted chemicals, no footprints left behind. It’s not glue. There is one tantalizing clue, however, the kind that ex- perimenters love. Gecko feet seem to get stickier on some surfaces than others. They are less sticky on low-energy surfaces like Teflon, and more sticky on surfaces made of polar molecules. This suggests that geckos are tapping directly into the molecular structure of the surfaces they walk on! Tracking down this clue, Kellar Autumn of Lewis & Clark College in Portland, Oregon, and Robert Full of the University of California, Berkeley, took a closer look at gecko feet. Geckos have rows of tiny hairs called setae on the bottoms of their feet, like the bristles of some trendy toothbrush. When you look at these hairs under the micro- scope, the end of each seta is divided into 400 to 1000 fine projections called spatulae. There are about half a million of these setae on each foot, each only one-tenth the diameter of a human hair. Autumn and Full put together an interdisciplinary team of scientists and set out to measure the force produced by a single seta. To do this, they had to overcome two significant experimental challenges: Isolating a single seta. No one had ever isolated a single seta before. They succeeded in doing this by surgically plucking a hair from a gecko foot under a microscope and bonding the hair onto a microprobe. The microprobe was fitted into a specially designed micromanipulator that can move the mounted hair in various ways. Measuring a very small force. Previous research had shown that if you pull on a whole gecko, the adhesive force sticking each of the gecko’s feet to the wall is about 10 Newtons (N), which is like supporting 1 kg. Because each foot has half a million setae, this predicts that a sin- gle seta would produce about 20 microNewtons of force. That’s a very tiny amount to measure. To attempt the measurement, Autumn and Full recruited a mechanical engineer from Stanford, Thomas Kenny. Kenny is an ex- pert at building instruments that can measure forces at the atomic level. Part I The Origin of Living Things Defying gravity. This gecko lizard is able to climb walls and walk upside down across ceilings. Learning how geckos do this is a fascinating bit of experimental science. Real People Doing Real Science The Experiment Once this team had isolated a seta and placed it in Kenny’s device, “We had a real nasty surprise,” says Autumn. For two months, pushing individual seta against a surface, they couldn’t get the isolated hair to stick at all! This forced the research team to stand back and think a bit. Finally it hit them. Geckos don’t walk by pushing their feet down, like we do. Instead, when a gecko takes a step, it pushes the palm of the foot into the surface, then uncurls its toes, sliding them backwards onto the surface. This shoves the forest of tips sideways against the surface. Going back to their instruments, they repeated their ex- periment, but this time they oriented the seta to approach the surface from the side rather than head-on. This had the effect of bringing the many spatulae on the tip of the seta into direct contact with the surface. To measure these forces on the seta from the side, as well as the perpendicular forces they had already been measur- ing, the researchers constructed a micro-electromechanical cantilever. The apparatus consisted of two piezoresistive layers deposited on a silicon cantilever to detect force in both parallel and perpendicular angles. The Results With the seta oriented properly, the experiment yielded re- sults. Fantastic results. The attachment force measured by the machine went up 600-fold from what the team had been measuring before. A single seta produced not the 20 microNewtons of force predicted by the whole-foot mea- surements, but up to an astonishing 200 microNewtons (see graph above)! Measuring many individual seta, adhe- sive forces averaged 194+25 microNewtons. Two hundred microNewtons is a tiny force, but stupen- dous for a single hair only 100 microns long. Enough to hold up an ant. A million hairs could support a small child. A little gecko, ceiling walking with 2 million of them (see photos above), could theoretically carry a 90-pound backpack—talk about being over-engineered. If a gecko’s feet stick that good, how do geckos ever become unstuck? The research team experimented with unattaching individual seta; they used yet another micro- instrument, this one designed by engineer Ronald Fearing also from U.C. Berkeley, to twist the hair in various ways. They found that tipped past a critical angle, 30 degrees, the attractive forces between hair and surface atoms weaken to nothing. The trick is to tip a foot hair until its projections let go. Geckos release their feet by curling up each toe and peeling it off, just the way we remove tape. What is the source of the powerful adhesion of gecko feet? The experiments do not reveal exactly what the attractive force is, but it seems almost certain to involve interactions at the atomic level. For a gecko’s foot to stick, the hundreds of spatulae at the tip of each seta must butt up squarely against the surface, so the individual atoms of each spatula can come into play. When two atoms approach each other very closely—closer than the diameter of an atom—a subtle nu- clear attraction called Van der Waals forces comes into play. These forces are individually very weak, but when lots of them add their little bits, the sum can add up to quite a lot. Might robots be devised with feet tipped with artificial setae, able to walk up walls? Autumn and Full are working with a robotics company to find out. Sometimes science is not only fun, but can lead to surprising advances. To explore this experiment further, go to the Virtual Lab at www.mhhe.com/raven6/vlab1.mhtml 12 Time (s) 345 20 0 -20 40 60 Force ( μ N) 80 0 Begin parallel pulling Seta pulled off sensor The sliding step experiment. The adhesive force of a single seta was measured. An initial push perpendicularly put the seta in contact with the sensor. Then, with parallel pulling, the force continued to increase over time to a value of 60 microNewtons (after this, the seta began to slide and pulled off the sensor). In a large number of similar experiments, adhesion forces typically approach 200 microNewtons. Closeup look at a gecko’s foot. The setae on a gecko’s foot are arranged in rows, and point backwards, away from the toenail. Each seta branches into several hundred spatulae (inset photo). 3 1 The Science of Biology Concept Outline 1.1 Biology is the science of life. Properties of Life. Biology is the science that studies living organisms and how they interact with one another and their environment. 1.2 Scientists form generalizations from observations. The Nature of Science. Science employs both deductive reasoning and inductive reasoning. How Science Is Done. Scientists construct hypotheses from systematically collected objective data. They then perform experiments designed to disprove the hypotheses. 1.3 Darwin’s theory of evolution illustrates how science works. Darwin’s Theory of Evolution. On a round-the-world voyage Darwin made observations that eventually led him to formulate the hypothesis of evolution by natural selection. Darwin’s Evidence. The fossil and geographic patterns of life he observed convinced Darwin that a process of evolution had occurred. Inventing the Theory of Natural Selection. The Malthus idea that populations cannot grow unchecked led Darwin, and another naturalist named Wallace, to propose the hypothesis of natural selection. Evolution After Darwin: More Evidence. In the century since Darwin, a mass of experimental evidence has supported his theory of evolution, now accepted by practically all prac- ticing biologists. 1.4 This book is organized to help you learn biology. Core Principles of Biology. The first half of this text is devoted to general principles that apply to all organisms, the second half to an examination of particular organisms. Y ou are about to embark on a journey—a journey of discovery about the nature of life. Nearly 180 years ago, a young English naturalist named Charles Darwin set sail on a similar journey on board H.M.S. Beagle (figure 1.1 shows a replica of the Beagle). What Darwin learned on his five-year voyage led directly to his development of the theory of evolution by natural selection, a theory that has become the core of the science of biology. Darwin’s voyage seems a fitting place to begin our exploration of biology, the scientific study of living organisms and how they have evolved. Before we begin, however, let’s take a moment to think about what biology is and why it’s important. FIGURE 1.1 A replica of the Beagle, off the southern coast of South America. The famous English naturalist, Charles Darwin, set forth on H.M.S. Beagle in 1831, at the age of 22. 4 Part I The Origin of Living Things Properties of Life In its broadest sense, biology is the study of living things—the science of life. Living things come in an astounding variety of shapes and forms, and biologists study life in many differ- ent ways. They live with gorillas, collect fossils, and listen to whales. They isolate viruses, grow mushrooms, and ex- amine the structure of fruit flies. They read the messages encoded in the long molecules of heredity and count how many times a hummingbird’s wings beat each second. What makes something “alive”? Anyone could deduce that a galloping horse is alive and a car is not, but why? We cannot say, “If it moves, it’s alive,” because a car can move, and gelatin can wiggle in a bowl. They certainly are not alive. What characteristics do define life? All living organ- isms share five basic characteristics: 1. Order. All organisms consist of one or more cells with highly ordered structures: atoms make up mole- cules, which construct cellular organelles, which are contained within cells. This hierarchical organization continues at higher levels in multicellular organisms and among organisms (figure 1.2). 2. Sensitivity. All organisms respond to stimuli. Plants grow toward a source of light, and your pupils dilate when you walk into a dark room. 3. Growth, development, and reproduction. All or- ganisms are capable of growing and reproducing, and they all possess hereditary molecules that are passed to their offspring, ensuring that the offspring are of the same species. Although crystals also “grow,” their growth does not involve hereditary molecules. 4. Regulation. All organisms have regulatory mecha- nisms that coordinate the organism’s internal func- tions. These functions include supplying cells with nu- trients, transporting substances through the organism, and many others. 5. Homeostasis. All organisms maintain relatively constant internal conditions, different from their envi- ronment, a process called homeostasis. All living things share certain key characteristics: order, sensitivity, growth, development and reproduction, regulation, and homeostasis. 1.1 Biology is the science of life. FIGURE 1.2 Hierarchical organization of living things. Life is highly orga- nized—from small and simple to large and complex, within cells, within multicellular organisms, and among organisms. Organelle Macromolecule Molecule Cell WITHIN CELLS Chapter 1 The Science of Biology 5 AMONG ORGANISMS Ecosystem Community Species Population WITHIN MULTICELLULAR ORGANISMS Tissue Organ Organ system Organism 6 Part I The Origin of Living Things The Nature of Science Biology is a fascinating and important subject, because it dramatically affects our daily lives and our futures. Many biologists are working on problems that critically affect our lives, such as the world’s rapidly expanding population and diseases like cancer and AIDS. The knowledge these biolo- gists gain will be fundamental to our ability to manage the world’s resources in a suitable manner, to prevent or cure diseases, and to improve the quality of our lives and those of our children and grandchildren. Biology is one of the most successful of the “natural sci- ences,” explaining what our world is like. To understand biology, you must first understand the nature of science. The basic tool a scientist uses is thought. To understand the nature of science, it is useful to focus for a moment on how scientists think. They reason in two ways: deductively and inductively. Deductive Reasoning Deductive reasoning applies general principles to predict specific results. Over 2200 years ago, the Greek Era- tosthenes used deductive reasoning to accurately estimate the circumference of the earth. At high noon on the longest day of the year, when the sun’s rays hit the bottom of a deep well in the city of Syene, Egypt, Eratosthenes mea- sured the length of the shadow cast by a tall obelisk in Al- exandria, about 800 kilometers to the north. Because he knew the distance between the two cities and the height of the obelisk, he was able to employ the principles of Euclid- ean geometry to correctly deduce the circumference of the earth (figure 1.3). This sort of analysis of specific cases us- ing general principles is an example of deductive reasoning. It is the reasoning of mathematics and philosophy and is used to test the validity of general ideas in all branches of knowledge. General principles are constructed and then used as the basis for examining specific cases. Inductive Reasoning Inductive reasoning uses specific observations to construct general scientific principles. Webster’s Dictionary defines sci- ence as systematized knowledge derived from observation and experiment carried on to determine the principles un- derlying what is being studied. In other words, a scientist determines principles from observations, discovering gen- eral principles by careful examination of specific cases. In- ductive reasoning first became important to science in the 1600s in Europe, when Francis Bacon, Isaac Newton, and others began to use the results of particular experiments to infer general principles about how the world operates. If you release an apple from your hand, what happens? The apple falls to the ground. From a host of simple, specific observations like this, Newton inferred a general principle: all objects fall toward the center of the earth. What New- ton did was construct a mental model of how the world works, a family of general principles consistent with what he could see and learn. Scientists do the same today. They use specific observations to build general models, and then test the models to see how well they work. Science is a way of viewing the world that focuses on objective information, putting that information to work to build understanding. 1.2 Scientists form generalizations from observations. FIGURE 1.3 Deductive reasoning: How Eratosthenes estimated the cir- cumference of the earth using deductive reasoning. 1. On a day when sunlight shone straight down a deep well at Syene in Egypt, Eratosthenes measured the length of the shadow cast by a tall obelisk in the city of Alexandria, about 800 kilometers away. 2. The shadow’s length and the obelisk’s height formed two sides of a triangle. Using the recently developed principles of Euclidean geometry, he calculated the angle, a, to be 7° and 12′, exactly 1 50 of a circle (360°). 3. If angle a = 1 50 of a circle, then the distance between the obelisk (in Alexandria) and the well (in Syene) must equal 1 50 of the circumference of the earth. 4. Eratosthenes had heard that it was a 50-day camel trip from Alexandria to Syene. Assuming that a camel travels about 18.5 kilometers per day, he estimated the distance between obelisk and well as 925 kilometers (using different units of measure, of course). 5. Eratosthenes thus de- duced the circumference of the earth to be 50 H11003 925 H11005 46,250 kilometers. Modern measurements put the distance from the well to the obelisk at just over 800 kilometers. Employ- ing a distance of 800 kilometers, Era- tosthenes’s value would have been 50 × 800 H11005 40,000 kilometers. The actual circumference is 40,075 kilometers. Sunlight at midday Dista nc e b e tw e e n cities = 80 0 k m Well Light rays parallel Height of obelisk Length of shadow a a How Science Is Done How do scientists establish which general principles are true from among the many that might be true? They do this by systematically testing alternative proposals. If these proposals prove inconsistent with experimental observa- tions, they are rejected as untrue. After making careful ob- servations concerning a particular area of science, scien- tists construct a hypothesis, which is a suggested explanation that accounts for those observations. A hy- pothesis is a proposition that might be true. Those hy- potheses that have not yet been disproved are retained. They are useful because they fit the known facts, but they are always subject to future rejection if, in the light of new information, they are found to be incorrect. Testing Hypotheses We call the test of a hypothesis an experiment (figure 1.4). Suppose that a room appears dark to you. To under- stand why it appears dark, you propose several hypotheses. The first might be, “There is no light in the room because the light switch is turned off.” An alternative hypothesis might be, “There is no light in the room because the light- bulb is burned out.” And yet another alternative hypothe- sis might be, “I am going blind.” To evaluate these hy- potheses, you would conduct an experiment designed to eliminate one or more of the hypotheses. For example, you might test your hypotheses by reversing the position of the light switch. If you do so and the light does not come on, you have disproved the first hypothesis. Something other than the setting of the light switch must be the reason for the darkness. Note that a test such as this does not prove that any of the other hypotheses are true; it merely dem- onstrates that one of them is not. A successful experiment is one in which one or more of the alternative hypotheses is demonstrated to be inconsistent with the results and is thus rejected. As you proceed through this text, you will encounter many hypotheses that have withstood the test of experiment. Many will continue to do so; others will be revised as new observations are made by biologists. Biology, like all science, is in a constant state of change, with new ideas appearing and replacing old ones. Chapter 1 The Science of Biology 7 FIGURE 1.4 How science is done. This diagram il- lustrates the way in which scientific in- vestigations proceed. First, scientists make observations that raise a particular question. They develop a number of potential explanations (hypotheses) to answer the question. Next, they carry out experiments in an attempt to eliminate one or more of these hypotheses. Then, predictions are made based on the remaining hypotheses, and further experiments are carried out to test these predictions. As a result of this process, the least unlikely hypothesis is selected. Observation Question Experiment Hypothesis 1 Hypothesis 2 Hypothesis 3 Hypothesis 4 Hypothesis 5 Potential hypotheses Remaining possible hypotheses Last remaining possible hypothesis Reject hypotheses 1 and 4 Reject hypotheses 2 and 3 Experiment Experiment 1 Hypothesis 2 Hypothesis 3 Hypothesis 5 Hypothesis 5 Predictions Predictions confirmed Experiment 2 Experiment 3 Experiment 4 Establishing Controls Often we are interested in learning about processes that are influenced by many factors, or variables. To evaluate alter- native hypotheses about one variable, all other variables must be kept constant. This is done by carrying out two ex- periments in parallel: in the first experiment, one variable is altered in a specific way to test a particular hypothesis; in the second experiment, called the control experiment, that variable is left unaltered. In all other respects the two exper- iments are identical, so any difference in the outcomes of the two experiments must result from the influence of the variable that was changed. Much of the challenge of experi- mental science lies in designing control experiments that isolate a particular variable from other factors that might in- fluence a process. Using Predictions A successful scientific hypothesis needs to be not only valid but useful—it needs to tell you something you want to know. A hypothesis is most useful when it makes predic- tions, because those predictions provide a way to test the va- lidity of the hypothesis. If an experiment produces results inconsistent with the predictions, the hypothesis must be re- jected. On the other hand, if the predictions are supported by experimental testing, the hypothesis is supported. The more experimentally supported predictions a hypothesis makes, the more valid the hypothesis is. For example, Ein- stein’s hypothesis of relativity was at first provisionally ac- cepted because no one could devise an experiment that in- validated it. The hypothesis made a clear prediction: that the sun would bend the path of light passing by it. When this prediction was tested in a total eclipse, the light from background stars was indeed bent. Because this result was unknown when the hypothesis was being formulated, it pro- vided strong support for the hypothesis, which was then ac- cepted with more confidence. Developing Theories Scientists use the word theory in two main ways. A “theo- ry” is a proposed explanation for some natural phenome- non, often based on some general principle. Thus one speaks of the principle first proposed by Newton as the “theory of gravity.” Such theories often bring together concepts that were previously thought to be unrelated, and offer unified explanations of different phenomena. Newton’s theory of gravity provided a single explanation for objects falling to the ground and the orbits of planets around the sun. “Theory” is also used to mean the body of interconnected concepts, supported by scientific rea- soning and experimental evidence, that explains the facts in some area of study. Such a theory provides an indis- pensable framework for organizing a body of knowledge. For example, quantum theory in physics brings together a set of ideas about the nature of the universe, explains ex- perimental facts, and serves as a guide to further questions and experiments. To a scientist, such theories are the solid ground of sci- ence, that of which we are most certain. In contrast, to the general public, theory implies just the opposite—a lack of knowledge, or a guess. Not surprisingly, this difference often results in confusion. In this text, theory will always be used in its scientific sense, in reference to an accepted gen- eral principle or body of knowledge. To suggest, as many critics outside of science do, that evolution is “just a theory” is misleading. The hypothesis that evolution has occurred is an accepted scientific fact; it is supported by overwhelming evidence. Modern evolutionary theory is a complex body of ideas whose importance spreads far beyond explaining evolution; its ramifications permeate all areas of biology, and it provides the conceptual frame- work that unifies biology as a science. Research and the Scientific Method It used to be fashionable to speak of the “scientific meth- od” as consisting of an orderly sequence of logical “ei- ther/or” steps. Each step would reject one of two mutually incompatible alternatives, as if trial-and-error testing would inevitably lead one through the maze of uncertain- ty that always impedes scientific progress. If this were in- deed so, a computer would make a good scientist. But sci- ence is not done this way. As British philosopher Karl Popper has pointed out, successful scientists without ex- ception design their experiments with a pretty fair idea of how the results are going to come out. They have what Popper calls an “imaginative preconception” of what the truth might be. A hypothesis that a successful scientist tests is not just any hypothesis; rather, it is an educated guess or a hunch, in which the scientist integrates all that he or she knows and allows his or her imagination full play, in an attempt to get a sense of what might be true (see Box: How Biologists Do Their Work). It is because insight and imagination play such a large role in scientific progress that some scientists are so much better at science than others, just as Beethoven and Mozart stand out among most other composers. Some scientists perform what is called basic research, which is intended to extend the boundaries of what we know. These individuals typically work at universities, and their research is usually financially supported by their in- stitutions and by external sources, such as the government, industry, and private foundations. Basic research is as di- verse as its name implies. Some basic scientists attempt to find out how certain cells take up specific chemicals, while others count the number of dents in tiger teeth. The infor- mation generated by basic research contributes to the growing body of scientific knowledge, and it provides the scientific foundation utilized by applied research. Scien- tists who conduct applied research are often employed in 8 Part I The Origin of Living Things some kind of industry. Their work may involve the manu- facturing of food additives, creating of new drugs, or test- ing the quality of the environment. After developing a hypothesis and performing a series of experiments, a scientist writes a paper carefully describing the experiment and its results. He or she then submits the paper for publication in a scientific journal, but before it is published, it must be reviewed and accepted by other scien- tists who are familiar with that particular field of research. This process of careful evaluation, called peer review, lies at the heart of modern science, fostering careful work, precise description, and thoughtful analysis. When an important discovery is announced in a paper, other scientists attempt to reproduce the result, providing a check on accuracy and honesty. Nonreproducible results are not taken seriously for long. The explosive growth in scientific research during the second half of the twentieth century is reflected in the enormous number of scientific journals now in existence. Although some, such as Science and Nature, are devoted to a wide range of scientific disciplines, most are extremely specialized: Cell Motility and the Cytoskeleton, Glycoconju- gate Journal, Mutation Research, and Synapse are just a few examples. The scientific process involves the rejection of hypotheses that are inconsistent with experimental results or observations. Hypotheses that are consistent with available data are conditionally accepted. The formulation of the hypothesis often involves creative insight. Chapter 1 The Science of Biology 9 How Biologists Do Their Work learn why the ginkgo trees drop all their leaves simultaneously, a scientist would first formulate several possible answers, called hypotheses: Hypothesis 1: Ginkgo trees possess an inter- nal clock that times the release of leaves to match the season. On the day Nemerov de- scribes, this clock sends a “drop” signal (perhaps a chemical) to all the leaves at the same time. Hypothesis 2: The individual leaves of ginkgo trees are each able to sense day length, and when the days get short enough in the fall, each leaf responds independently by falling. Hypothesis 3: A strong wind arose the night before Nemerov made his observation, blowing all the leaves off the ginkgo trees. Next, the scientist attempts to eliminate one or more of the hypotheses by conduct- ing an experiment. In this case, one might cover some of the leaves so that they can- not use light to sense day length. If hypoth- esis 2 is true, then the covered leaves should not fall when the others do, because they are not receiving the same informa- tion. Suppose, however, that despite the covering of some of the leaves, all the leaves still fall together. This result would eliminate hypothesis 2 as a possibility. Ei- ther of the other hypotheses, and many others, remain possibilities. This simple experiment with ginkgoes points out the essence of scientific progress: science does not prove that cer- tain explanations are true; rather, it proves that others are not. Hypotheses that are inconsistent with experimental results are rejected, while hypotheses that are not proven false by an experiment are provi- sionally accepted. However, hypotheses may be rejected in the future when more information becomes available, if they are inconsistent with the new information. Just as finding the correct path through a maze by trying and eliminating false paths, sci- entists work to find the correct explana- tions of natural phenomena by eliminating false possibilities. The Consent Late in November, on a single night Not even near to freezing, the ginkgo trees That stand along the walk drop all their leaves In one consent, and neither to rain nor to wind But as though to time alone: the golden and green Leaves litter the lawn today, that yesterday Had spread aloft their fluttering fans of light. What signal from the stars? What senses took it in? What in those wooden motives so decided To strike their leaves, to down their leaves, Rebellion or surrender? And if this Can happen thus, what race shall be exempt? What use to learn the lessons taught by time, If a star at any time may tell us: Now. Howard Nemerov What is bothering the poet Howard Nem- erov is that life is influenced by forces he cannot control or even identify. It is the job of biologists to solve puzzles such as the one he poses, to identify and try to understand those things that influence life. Nemerov asks why ginkgo trees (figure 1.A) drop all their leaves at once. To find an answer to questions such as this, biolo- gists and other scientists pose possible an- swers and then try to determine which an- swers are false. Tests of alternative possibilities are called experiments. To FIGURE 1.A A ginkgo tree. 10 Part I The Origin of Living Things Darwin’s Theory of Evolution Darwin’s theory of evolution explains and describes how organisms on earth have changed over time and acquired a diversity of new forms. This famous theory provides a good example of how a scientist develops a hypothesis and how a scientific theory grows and wins acceptance. Charles Robert Darwin (1809–1882; figure 1.5) was an English naturalist who, after 30 years of study and obser- vation, wrote one of the most famous and influential books of all time. This book, On the Origin of Species by Means of Natural Selection, or The Preservation of Favoured Races in the Struggle for Life, created a sensation when it was pub- lished, and the ideas Darwin expressed in it have played a central role in the development of human thought ever since. In Darwin’s time, most people be- lieved that the various kinds of organ- isms and their individual structures re- sulted from direct actions of the Creator (and to this day many people still believe this to be true). Species were thought to be specially created and unchangeable, or immutable, over the course of time. In contrast to these views, a number of earlier philosophers had presented the view that living things must have changed during the history of life on earth. Darwin proposed a concept he called natural selection as a coherent, logical explanation for this process, and he brought his ideas to wide public at- tention. His book, as its title indicates, presented a conclusion that differed sharply from conventional wisdom. Al- though his theory did not challenge the existence of a Divine Creator, Darwin argued that this Creator did not simply create things and then leave them forev- er unchanged. Instead, Darwin’s God expressed Himself through the operation of natural laws that produced change over time, or evolution. These views put Darwin at odds with most people of his time, who believed in a literal interpretation of the Bible and ac- cepted the idea of a fixed and constant world. His revolu- tionary theory deeply troubled not only many of his con- temporaries but Darwin himself. The story of Darwin and his theory begins in 1831, when he was 22 years old. On the recommendation of one of his professors at Cambridge University, he was selected to serve 1.3 Darwin’s theory of evolution illustrates how science works. FIGURE 1.5 Charles Darwin. This newly rediscovered photograph taken in 1881, the year before Darwin died, appears to be the last ever taken of the great biologist. as naturalist on a five-year navigational mapping expedition around the coasts of South America (figure 1.6), aboard H.M.S. Beagle (figure 1.7). During this long voyage, Darwin had the chance to study a wide variety of plants and animals on continents and islands and in distant seas. He was able to explore the biological richness of the tropical forests, exam- ine the extraordinary fossils of huge extinct mammals in Patagonia at the southern tip of South America, and observe the remarkable series of related but distinct forms of life on the Galápagos Islands, off the west coast of South America. Such an opportunity clearly played an important role in the development of his thoughts about the nature of life on earth. When Darwin returned from the voyage at the age of 27, he began a long period of study and contemplation. During the next 10 years, he published important books on several different subjects, including the formation of oceanic islands from coral reefs and the geology of South America. He also devoted eight years of study to barnacles, a group of small marine animals with shells that inhabit rocks and pilings, eventually writing a four-volume work on their classification and natural history. In 1842, Darwin and his family moved out of London to a country home at Down, in the county of Kent. In these pleasant surroundings, Darwin lived, studied, and wrote for the next 40 years. Darwin was the first to propose natural selection as an explanation for the mechanism of evolution that produced the diversity of life on earth. His hypothesis grew from his observations on a five-year voyage around the world. Chapter 1 The Science of Biology 11 British Isles Western Isles Europe Africa Indian Ocean Madagascar Mauritius Bourbon Island Cape of Good Hope King George’s Sound Hobart Sydney Australia New Zealand Friendly Islands Phillippine Islands Equator North Pacific Ocean Asia North Atlantic Ocean Cape Verde Marquesas Galápagos Islands Valparaiso Society Islands Straits of Magellan Tierra del FuegoCape Horn Falkland Islands Port Desire South Atlantic Ocean Montevideo Buenos Aires Rio de Janeiro St. Helena Ascension North America Canary Islands Keeling Islands South America Bahia FIGURE 1.6 The five-year voyage of H.M.S. Beagle. Most of the time was spent exploring the coasts and coastal islands of South America, such as the Galápagos Islands. Darwin’s studies of the animals of the Galápagos Islands played a key role in his eventual development of the theory of evolution by means of natural selection. FIGURE 1.7 Cross section of the Beagle. A 10-gun brig of 242 tons, only 90 feet in length, the Beagle had a crew of 74 people! After he first saw the ship, Darwin wrote to his college professor Henslow: “The absolute want of room is an evil that nothing can surmount.” 12 Part I The Origin of Living Things Darwin’s Evidence One of the obstacles that had blocked the acceptance of any theory of evolution in Darwin’s day was the incorrect notion, widely believed at that time, that the earth was only a few thousand years old. Evidence discovered during Darwin’s time made this assertion seem less and less likely. The great geologist Charles Lyell (1797–1875), whose Principles of Geology (1830) Darwin read eagerly as he sailed on the Beagle, outlined for the first time the story of an ancient world of plants and animals in flux. In this world, species were constantly becoming extinct while oth- ers were emerging. It was this world that Darwin sought to explain. What Darwin Saw When the Beagle set sail, Darwin was fully convinced that species were immutable. Indeed, it was not until two or three years after his return that he began to consider seri- ously the possibility that they could change. Nevertheless, during his five years on the ship, Darwin observed a number of phenomena that were of central importance to him in reaching his ultimate conclusion (table 1.1). For example, in the rich fossil beds of southern South America, he observed fossils of extinct armadillos similar to the armadillos that still lived in the same area (figure 1.8). Why would similar living and fossil organisms be in the same area unless the earlier form had given rise to the other? Repeatedly, Darwin saw that the characteristics of simi- lar species varied somewhat from place to place. These geographical patterns suggested to him that organismal lin- eages change gradually as species migrate from one area to another. On the Galápagos Islands, off the coast of Ecua- dor, Darwin encountered giant land tortoises. Surprisingly, these tortoises were not all identical. In fact, local residents and the sailors who captured the tortoises for food could tell which island a particular tortoise had come from just by looking at its shell. This distribution of physical variation suggested that all of the tortoises were related, but that they had changed slightly in appearance after becoming isolated on different islands. In a more general sense, Darwin was struck by the fact that the plants and animals on these relatively young vol- canic islands resembled those on the nearby coast of South America. If each one of these plants and animals had been created independently and simply placed on the Galápagos Islands, why didn’t they resemble the plants and animals of islands with similar climates, such as those off the coast of Africa, for example? Why did they resem- ble those of the adjacent South American coast instead? The fossils and patterns of life that Darwin observed on the voyage of the Beagle eventually convinced him that evolution had taken place. Table 1.1 Darwin’s Evidence that Evolution Occurs FOSSILS 1. Extinct species, such as the fossil armadillo in figure 1.8, most closely resemble living ones in the same area, suggesting that one had given rise to the other. 2. In rock strata (layers), progressive changes in characteristics can be seen in fossils from earlier and earlier layers. GEOGRAPHICAL DISTRIBUTION 3. Lands with similar climates, such as Australia, South Africa, California, and Chile, have unrelated plants and animals, indicating that diversity is not entirely influenced by climate and environment. 4. The plants and animals of each continent are distinctive; all South American rodents belong to a single group, structurally similar to the guinea pigs, for example, while most of the rodents found elsewhere belong to other groups. OCEANIC ISLANDS 5. Although oceanic islands have few species, those they do have are often unique (endemic) and show relatedness to one another, such as the Galápagos tortoises. This suggests that the tortoises and other groups of endemic species developed after their mainland ancestors reached the islands and are, therefore, more closely related to one another. 6. Species on oceanic islands show strong affinities to those on the nearest mainland. Thus, the finches of the Galápagos Islands closely resemble a finch seen on the western coast of South America. The Galápagos finches do not resemble the birds on the Cape Verde Islands, islands in the Atlantic Ocean off the coast of Africa that are similar to the Galápagos. Darwin visited the Cape Verde Islands and many other island groups personally and was able to make such comparisons on the basis of his own observations. FIGURE 1.8 Fossil evidence of evolution. The now-extinct glyptodont (a) was a 2000-kilogram South American armadillo, much larger than the modern armadillo (b), which weighs an average of about 4.5 kilograms. (Drawings are not to scale.) (a) Glyptodont (b) Armadillo Inventing the Theory of Natural Selection It is one thing to observe the results of evolution, but quite another to understand how it happens. Darwin’s great achievement lies in his formulation of the hypothe- sis that evolution occurs because of natural selection. Darwin and Malthus Of key importance to the development of Darwin’s in- sight was his study of Thomas Malthus’s Essay on the Principle of Population (1798). In his book, Malthus pointed out that populations of plants and animals (in- cluding human beings) tend to increase geometrically, while the ability of humans to increase their food supply increases only arithmetically. A geometric progression is one in which the elements increase by a constant factor; for example, in the progression 2, 6, 18, 54, ..., each number is three times the preceding one. An arithmetic progression, in contrast, is one in which the elements in- crease by a constant difference; in the progression 2, 6, 10, 14,..., each number is four greater than the preced- ing one (figure 1.9). Because populations increase geometrically, virtually any kind of animal or plant, if it could reproduce un- checked, would cover the entire surface of the world within a surprisingly short time. Instead, populations of species remain fairly constant year after year, because death limits population numbers. Malthus’s conclusion provided the key ingredient that was necessary for Dar- win to develop the hypothesis that evolution occurs by natural selection. Sparked by Malthus’s ideas, Darwin saw that although every organism has the potential to produce more off- spring than can survive, only a limited number actually do survive and produce further offspring. Combining this observation with what he had seen on the voyage of the Beagle, as well as with his own experiences in breed- ing domestic animals, Darwin made an important associ- ation (figure 1.10): Those individuals that possess supe- rior physical, behavioral, or other attributes are more likely to survive than those that are not so well endowed. By surviving, they gain the opportunity to pass on their favorable characteristics to their offspring. As the fre- quency of these characteristics increases in the popula- tion, the nature of the population as a whole will gradu- ally change. Darwin called this process selection. The driving force he identified has often been referred to as survival of the fittest. Chapter 1 The Science of Biology 13 Geometric progression Arithmetic progression 2 6 18 54 4 6 8 FIGURE 1.9 Geometric and arithmetic progressions. A geometric progression increases by a constant factor (e.g., H11003 2 or H11003 3 or H11003 4), while an arithmetic progression increases by a constant difference (e.g., units of 1 or 2 or 3) . Malthus contended that the human growth curve was geometric, but the human food production curve was only arithmetic. Can you see the problems this difference would cause? FIGURE 1.10 An excerpt from Charles Darwin’s On the Origin of Species. Natural Selection Darwin was thoroughly familiar with variation in domesticated animals and began On the Origin of Species with a detailed discussion of pigeon breeding. He knew that breeders selected certain varieties of pigeons and other animals, such as dogs, to produce certain char- acteristics, a process Darwin called ar- tificial selection. Once this had been done, the animals would breed true for the characteristics that had been select- ed. Darwin had also observed that the differences purposely developed be- tween domesticated races or breeds were often greater than those that sep- arated wild species. Domestic pigeon breeds, for example, show much greater variety than all of the hundreds of wild species of pigeons found throughout the world. Such relation- ships suggested to Darwin that evolu- tionary change could occur in nature too. Surely if pigeon breeders could foster such variation by “artificial selec- tion,” nature could do the same, play- ing the breeder’s role in selecting the next generation—a process Darwin called natural selection. Darwin’s theory thus incorporates the hypothesis of evolution, the pro- cess of natural selection, and the mass of new evidence for both evolution and natural selection that Darwin compiled. Thus, Darwin’s theory provides a simple and direct explanation of biological diversity, or why animals are different in different places: because habitats differ in their requirements and opportunities, the organisms with characteristics favored locally by natural selection will tend to vary in different places. Darwin Drafts His Argument Darwin drafted the overall argument for evolution by natu- ral selection in a preliminary manuscript in 1842. After showing the manuscript to a few of his closest scientific friends, however, Darwin put it in a drawer, and for 16 years turned to other research. No one knows for sure why Darwin did not publish his initial manuscript—it is very thorough and outlines his ideas in detail. Some histo- rians have suggested that Darwin was shy of igniting public criticism of his evolutionary ideas—there could have been little doubt in his mind that his theory of evolution by nat- ural selection would spark controversy. Others have pro- posed that Darwin was simply refining his theory all those years, although there is little evidence he altered his initial manuscript in all that time. Wallace Has the Same Idea The stimulus that finally brought Dar- win’s theory into print was an essay he received in 1858. A young English nat- uralist named Alfred Russel Wallace (1823–1913) sent the essay to Darwin from Malaysia; it concisely set forth the theory of evolution by means of natural selection, a theory Wallace had developed independently of Darwin. Like Darwin, Wallace had been greatly influenced by Malthus’s 1798 essay. Colleagues of Wallace, knowing of Darwin’s work, encouraged him to communicate with Darwin. After re- ceiving Wallace’s essay, Darwin ar- ranged for a joint presentation of their ideas at a seminar in London. Darwin then completed his own book, expand- ing the 1842 manuscript which he had written so long ago, and submitted it for publication. Publication of Darwin’s Theory Darwin’s book appeared in November 1859 and caused an immediate sensation. Many people were deeply disturbed by the suggestion that human beings were descended from the same ancestor as apes (figure 1.11). Darwin did not actually discuss this idea in his book, but it followed directly from the principles he outlined. In a subsequent book, The Descent of Man, Darwin presented the argument directly, building a powerful case that humans and living apes have common an- cestors. Although people had long accepted that humans closely resembled apes in many characteristics, the possibility that there might be a direct evolutionary relationship was un- acceptable to many. Darwin’s arguments for the theory of evolution by natural selection were so compelling, however, that his views were almost completely accepted within the in- tellectual community of Great Britain after the 1860s. The fact that populations do not really expand geometrically implies that nature acts to limit population numbers. The traits of organisms that survive to produce more offspring will be more common in future generations—a process Darwin called natural selection. 14 Part I The Origin of Living Things FIGURE 1.11 Darwin greets his monkey ancestor. In his time, Darwin was often portrayed unsympathetically, as in this drawing from an 1874 publication. Evolution After Darwin: More Evidence More than a century has elapsed since Darwin’s death in 1882. During this period, the evidence supporting his the- ory has grown progressively stronger. There have also been many significant advances in our understanding of how evolution works. Although these advances have not altered the basic structure of Darwin’s theory, they have taught us a great deal more about the mechanisms by which evolution occurs. We will briefly explore some of this evidence here; in chapter 21 we will return to the the- ory of evolution and examine the evidence in more detail. The Fossil Record Darwin predicted that the fossil record would yield inter- mediate links between the great groups of organisms, for example, between fishes and the amphibians thought to have arisen from them, and between reptiles and birds. We now know the fossil record to a degree that was unthink- able in the nineteenth century. Recent discoveries of mi- croscopic fossils have extended the known history of life on earth back to about 3.5 billion years ago. The discovery of other fossils has supported Darwin’s predictions and has shed light on how organisms have, over this enormous time span, evolved from the simple to the complex. For verte- brate animals especially, the fossil record is rich and exhib- its a graded series of changes in form, with the evolutionary parade visible for all to see (see Box: Why Study Fossils?). The Age of the Earth In Darwin’s day, some physicists argued that the earth was only a few thousand years old. This bothered Darwin, be- cause the evolution of all living things from some single original ancestor would have required a great deal more time. Using evidence obtained by studying the rates of ra- dioactive decay, we now know that the physicists of Dar- win’s time were wrong, very wrong: the earth was formed about 4.5 billion years ago. The Mechanism of Heredity Darwin received some of his sharpest criticism in the area of heredity. At that time, no one had any concept of genes or of how heredity works, so it was not possible for Darwin to explain completely how evolution occurs. Theories of he- redity in Darwin’s day seemed to rule out the possibility of genetic variation in nature, a critical requirement of Dar- win’s theory. Genetics was established as a science only at the start of the twentieth century, 40 years after the publica- tion of Darwin’s On the Origin of Species. When scientists began to understand the laws of inheritance (discussed in chapter 13), the heredity problem with Darwin’s theory vanished. Genetics accounts in a neat and orderly way for the production of new variations in organisms. Comparative Anatomy Comparative studies of animals have provided strong evi- dence for Darwin’s theory. In many different types of verte- brates, for example, the same bones are present, indicating their evolutionary past. Thus, the forelimbs shown in figure 1.12 are all constructed from the same basic array of bones, modified in one way in the wing of a bat, in another way in the fin of a porpoise, and in yet another way in the leg of a horse. The bones are said to be homologous in the differ- ent vertebrates; that is, they have the same evolutionary ori- gin, but they now differ in structure and function. This con- trasts with analogous structures, such as the wings of birds and butterflies, which have similar structure and function but different evolutionary origins. Chapter 1 The Science of Biology 15 Human Cat Bat Porpoise Horse FIGURE 1.12 Homology among vertebrate limbs. The forelimbs of these five vertebrates show the ways in which the relative proportions of the forelimb bones have changed in relation to the particular way of life of each organism. Molecular Biology Biochemical tools are now of major importance in efforts to reach a better understanding of how evolution occurs. Within the last few years, for example, evolutionary biolo- gists have begun to “read” genes, much as you are reading this page. They have learned to recognize the order of the “letters” of the long DNA molecules, which are present in every living cell and which provide the genetic information for that organism. By comparing the sequences of “letters” in the DNA of different groups of animals or plants, we can specify the degree of relationship among the groups more precisely than by any other means. In many cases, detailed family trees can then be constructed. The consistent pattern emerging from a growing mountain of data is one of pro- gressive change over time, with more distantly related species showing more differences in their DNA than closely related ones, just as Darwin’s theory predicts. By measuring the degree of difference in the genetic coding, and by inter- preting the information available from the fossil record, we can even estimate the rates at which evolution is occurring in different groups of organisms. Development Twentieth-century knowledge about growth and develop- ment further supports Darwin’s theory of evolution. Strik- ing similarities are seen in the developmental stages of many organisms of different species. Human embryos, for example, go through a stage in which they possess the same structures that give rise to the gills in fish, a tail, and even a stage when the embryo has fur! Thus, the develop- ment of an organism (its ontogeny) often yields informa- tion about the evolutionary history of the species as a whole (its phylogeny). Since Darwin’s time, new discoveries of the fossil record, genetics, anatomy, and development all support Darwin’s theory. 16 Part I The Origin of Living Things by studying modern organisms. But history is complex and unpredictable—and princi- ples of evolution (like natural selection) cannot specify the pathway that life’s histo- ry has actually followed. Paleontology holds the archives of the pathway—the fossil record of past life, with its fascinating histo- ry of mass extinctions, periods of rapid change, long episodes of stability, and con- stantly changing patterns of dominance and diversity. Humans represent just one tiny, largely fortuitous, and late-arising twig on the enormously arborescent bush of life. Paleontology is the study of this grandest of all bushes. geological time, occur by a natural process of evolutionary transformation—“descent with modification,” in Darwin’s words. I was thrilled to learn that humans had arisen from apelike ancestors, who had themselves evolved from the tiny mouselike mammals that had lived in the time of dinosaurs and seemed then so inconspicuous, so unsuc- cessful, and so unpromising. Now, at mid-career (I was born in 1941) I remain convinced that I made the right choice, and committed to learn and convey, as much as I can as long as I can, about evo- lution and the history of life. We can learn a great deal about the process of evolution I grew up on the streets of New York City, in a family of modest means and little for- mal education, but with a deep love of learning. Like many urban kids who be- come naturalists, my inspiration came from a great museum—in particular, from the magnificent dinosaurs on display at the American Museum of Natural History. As we all know from Jurassic Park and other sources, dinomania in young children (I was five when I saw my first dinosaur) is not rare—but nearly all children lose the passion, and the desire to become a pale- ontologist becomes a transient moment between policeman and fireman in a chro- nology of intended professions. But I per- sisted and became a professional paleontol- ogist, a student of life’s history as revealed by the evidence of fossils (though I ended up working on snails rather than dino- saurs!). Why? I remained committed to paleontology because I discovered, still as a child, the wonder of one of the greatest transforming ideas ever discovered by science: evolution. I learned that those dinosaurs, and all crea- tures that have ever lived, are bound to- gether in a grand family tree of physical re- lationships, and that the rich and fascinating changes of life, through billions of years in Why Study Fossils? Flight has evolved three separate times among ver- tebrates. Birds and bats are still with us, but pterosaurs, such as the one pictured, became extinct with the di- nosaurs about 65 million years ago. Stephen Jay Gould Harvard University Chapter 1 The Science of Biology 17 Core Principles of Biology From centuries of biological observation and inquiry, one organizing principle has emerged: biological diversity re- flects history, a record of success, failure, and change ex- tending back to a period soon after the formation of the earth. The explanation for this diversity, the theory of evo- lution by natural selection, will form the backbone of your study of biological science, just as the theory of the covalent bond is the backbone of chemistry, or the theory of quan- tum mechanics is that of physics. Evolution by natural selec- tion is a thread that runs through everything you will learn in this book. Basic Principles The first half of this book is devoted to a description of the basic principles of biology, introduced through a levels-of- organization framework (see figure 1.2). At the molecular, organellar, and cellular levels of organization, you will be in- troduced to cell biology. You will learn how cells are con- structed and how they grow, divide, and communicate. At the organismal level, you will learn the principles of genetics, which deal with the way that individual traits are transmit- ted from one generation to the next. At the population level, you will examine evolution, the gradual change in popula- tions from one generation to the next, which has led through natural selection to the biological diversity we see around us. Finally, at the community and ecosystem levels, you will study ecology, which deals with how organisms in- teract with their environments and with one another to pro- duce the complex communities characteristic of life on earth. Organisms The second half of the book is devoted to an examination of organisms, the products of evolution. It is estimated that at least 5 million different kinds of plants, animals, and micro- organisms exist, and their diversity is incredible (figure 1.13). Later in the book, we will take a particularly detailed look at the vertebrates, the group of animals of which we are mem- bers. We will consider the vertebrate body and how it func- tions, as this information is of greatest interest and impor- tance to most students. As you proceed through this book, what you learn at one stage will give you the tools to understand the next. The core principle of biology is that biological diversity is the result of a long evolutionary journey. 1.4 This book is organized to help you learn biology. Plantae Animalia Fungi Eubacteria Archaebacteria Protista FIGURE 1.13 The diversity of life. Biologists categorize all living things into six major groups called kingdoms: archaebacteria, eubacteria, protists, fungi, plants, and animals. Chapter 1 Summary Questions Media Resources 1.1 Biology is the science of life. 18 Part I The Origin of Living Things ? Living things are highly organized, whether as single cells or as multicellular organisms, with several hier- archical levels. 1. What are the characteristics of living things? 1.2 Scientists form generalizations from observations. ? Science is the determination of general principles from observation and experimentation. ? Scientists select the best hypotheses by using controlled experiments to eliminate alternative hypotheses that are inconsistent with observations. ? A group of related hypotheses supported by a large body of evidence is called a theory. In science, a theory represents what we are most sure about. However, there are no absolute truths in science, and even theories are accepted only conditionally. ? Scientists conduct basic research, designed to gain information about natural phenomena in order to contribute to our overall body of knowledge, and applied research, devoted to solving specific problems with practical applications. 2. What is the difference be- tween deductive and inductive reasoning? What is a hypothesis? 3. What are variables? How are control experiments used in test- ing hypotheses? 4. How does a hypothesis become a theory? At what point does a theory become accepted as an absolute truth, no longer subject to any uncertainty? 5. What is the difference between basic and applied research? 6. Describe the evidence that led Darwin to propose that evolu- tion occurs by means of natural selection. What evidence gathered since the publication of Darwin’s theory has lent further support to the theory? 7. What is the difference be- tween homologous and analo- gous structures? Give an example of each. 8. Can you think of any alterna- tives to levels-of-organization as ways of organizing the mass of information in biology? ? One of the central theories of biology is Darwin’s theory that evolution occurs by natural selection. It states that certain individuals have heritable traits that allow them to produce more offspring in a given kind of environment than other individuals lacking those traits. Consequently, those traits will increase in frequency through time. ? Because environments differ in their requirements and opportunities, the traits favored by natural selection will vary in different environments. ? This theory is supported by a wealth of evidence ac- quired over more than a century of testing and questioning. ? Biological diversity is the result of a long history of evolutionary change. For this reason evolution is the core of the science of biology. ? Considered in terms of levels-of-organization, the science of biology can be said to consist of subdisci- plines focusing on particular levels. Thus one speaks of molecular biology, cell biology, organismal biolo- gy, population biology, and community biology. 1.3 Darwin’s theory of evolution illustrates how science works. 1.4 This book is organized to help you learn biology. ? Art Activity: Biological organization ? Scientists on Science: Why Paleonthology? ? Experiments: Probability and Hypothesis Testing in Biology ? Introduction to Evolution ? Before Darwin ? Voyage of the Beagle ? Natural Selection ? The Process of Natural Selection ? Evidence for Evolution ? Student Research: The Search for Medicinal Plants on Science Articles ? 140 Years Without Darwin Are Enough ? Bird-Killing Cats: Nature’s Way of Making Better Bids http://www.mhhe.com/raven6e http://www.biocourse.com 19 2 The Nature of Molecules Concept Outline 2.1 Atoms are nature’s building material. Atoms. All substances are composed of tiny particles called atoms, each a positively charged nucleus around which orbit negative electrons. Electrons Determine the Chemical Behavior of Atoms. Electrons orbit the nucleus of an atom; the closer an electron’s orbit to the nucleus, the lower its energy level. 2.2 The atoms of living things are among the smallest. Kinds of Atoms. Of the 92 naturally occurring elements, only 11 occur in organisms in significant amounts. 2.3 Chemical bonds hold molecules together. Ionic Bonds Form Crystals. Atoms are linked together into molecules, joined by chemical bonds that result from forces like the attraction of opposite charges or the sharing of electrons. Covalent Bonds Build Stable Molecules. Chemical bonds formed by the sharing of electrons can be very strong, and require much energy to break. 2.4 Water is the cradle of life. Chemistry of Water. Water forms weak chemical associations that are responsible for much of the organization of living chemistry. Water Atoms Act Like Tiny Magnets. Because electrons are shared unequally by the hydrogen and oxygen atoms of water, a partial charge separation occurs. Each water atom acquires a positive and negative pole and is said to be “polar.” Water Clings to Polar Molecules. Because the opposite partial charges of polar molecules attract one another, water tends to cling to itself and other polar molecules and to exclude nonpolar molecules. Water Ionizes. Because its covalent bonds occasionally break, water contains a low concentration of hydrogen (H + ) and hydroxide (OH – ) ions, the fragments of broken water molecules. A bout 10 to 20 billion years ago, an enormous explo- sion likely marked the beginning of the universe. With this explosion began the process of evolution, which eventually led to the origin and diversification of life on earth. When viewed from the perspective of 20 billion years, life within our solar system is a recent development, but to understand the origin of life, we need to consider events that took place much earlier. The same processes that led to the evolution of life were responsible for the evolution of molecules (figure 2.1). Thus, our study of life on earth begins with physics and chemistry. As chemical machines ourselves, we must understand chemistry to begin to understand our origins. FIGURE 2.1 Cells are made of molecules. Specific, often simple, combina- tions of atoms yield an astonishing diversity of molecules within the cell, each with unique functional characteristics. weight will be greater on the earth because the earth’s grav- itational force is greater than the moon’s. The atomic mass of an atom is equal to the sum of the masses of its protons and neutrons. Atoms that occur naturally on earth contain from 1 to 92 protons and up to 146 neutrons. The mass of atoms and subatomic particles is measured in units called daltons. To give you an idea of just how small these units are, note that it takes 602 million million billion (6.02 × 10 23 ) daltons to make 1 gram! A proton weighs ap- proximately 1 dalton (actually 1.009 daltons), as does a neu- tron (1.007 daltons). In contrast, electrons weigh only 1 1840 of a dalton, so their contribution to the overall mass of an atom is negligible. 20 Part I The Origin of Living Things Atoms Any substance in the universe that has mass (see below) and occupies space is defined as matter. All matter is com- posed of extremely small particles called atoms. Because of their size, atoms are difficult to study. Not until early in this century did scientists carry out the first experiments sug- gesting what an atom is like. The Structure of Atoms Objects as small as atoms can be “seen” only indirectly, by using very complex technology such as tunneling microcopy. We now know a great deal about the complexities of atomic structure, but the simple view put forth in 1913 by the Danish physicist Niels Bohr provides a good starting point. Bohr proposed that every atom possesses an orbiting cloud of tiny subatomic particles called electrons whizzing around a core like the plan- ets of a miniature solar system. At the center of each atom is a small, very dense nucleus formed of two other kinds of subatomic particles, protons and neutrons (figure 2.2). Within the nucleus, the cluster of protons and neutrons is held together by a force that works only over short subatomic distances. Each proton car- ries a positive (+) charge, and each electron carries a negative (–) charge. Typically an atom has one electron for each proton. The number of protons (the atom’s atomic number) determines the chemical character of the atom, because it dictates the number of electrons orbiting the nucleus which are available for chemical activity. Neu- trons, as their name implies, possess no charge. Atomic Mass The terms mass and weight are often used interchangeably, but they have slightly different meanings. Mass refers to the amount of a substance, while weight refers to the force gravity exerts on a substance. Hence, an object has the same mass whether it is on the earth or the moon, but its 2.1 Atoms are nature’s building material. FIGURE 2.2 Basic structure of atoms. All atoms have a nucleus consisting of protons and neutrons, except hydrogen, the smallest atom, which has only one proton and no neutrons in its nucleus. Oxygen, for example, has eight protons and eight neutrons in its nucleus. Electrons spin around the nucleus a far distance away from the nucleus. Proton (Positive charge) (No charge) (Negative charge) Neutron Electron Hydrogen 1 Proton 1 Electron Oxygen 8 Protons 8 Neutrons 8 Electrons Isotopes Atoms with the same atomic number (that is, the same num- ber of protons) have the same chemical properties and are said to belong to the same element. Formally speaking, an element is any substance that cannot be broken down to any other substance by ordinary chemical means. However, while all atoms of an element have the same number of protons, they may not all have the same number of neutrons. Atoms of an element that possess different numbers of neutrons are called isotopes of that element. Most elements in nature exist as mixtures of different isotopes. Carbon (C), for example, has three isotopes, all containing six protons (figure 2.3). Over 99% of the carbon found in nature exists as an isotope with six neutrons. Because its total mass is 12 daltons (6 from protons plus 6 from neutrons), this isotope is referred to as carbon-12, and symbolized 12 C. Most of the rest of the natu- rally occurring carbon is carbon-13, an isotope with seven neutrons. The rarest carbon isotope is carbon-14, with eight neutrons. Unlike the other two isotopes, carbon-14 is unsta- ble: its nucleus tends to break up into elements with lower atomic numbers. This nuclear breakup, which emits a signifi- cant amount of energy, is called radioactive decay, and iso- topes that decay in this fashion are radioactive isotopes. Some radioactive isotopes are more unstable than others and therefore decay more readily. For any given isotope, however, the rate of decay is constant. This rate is usually expressed as the half-life, the time it takes for one half of the atoms in a sample to decay. Carbon-14, for example, has a half-life of about 5600 years. A sample of carbon containing 1 gram of carbon-14 today would contain 0.5 gram of car- bon-14 after 5600 years, 0.25 gram 11,200 years from now, 0.125 gram 16,800 years from now, and so on. By determin- ing the ratios of the different isotopes of carbon and other elements in biological samples and in rocks, scientists are able to accurately determine when these materials formed. While there are many useful applications of radioactivity, there are also harmful side effects that must be considered in any planned use of radioactive substances. Radioactive sub- stances emit energetic subatomic particles that have the po- tential to severely damage living cells, producing mutations in their genes, and, at high doses, cell death. Consequently, ex- posure to radiation is now very carefully controlled and regu- lated. Scientists who work with radioactivity (basic re- searchers as well as applied scientists such as X-ray technologists) wear radiation-sensitive badges to monitor the total amount of radioactivity to which they are exposed. Each month the badges are collected and scrutinized. Thus, em- ployees whose work places them in danger of excessive radio- active exposure are equipped with an “early warning system.” Electrons The positive charges in the nucleus of an atom are counter- balanced by negatively charged electrons orbiting at vary- ing distances around the nucleus. Thus, atoms with the same number of protons and electrons are electrically neu- tral, having no net charge. Electrons are maintained in their orbits by their attrac- tion to the positively charged nucleus. Sometimes other forces overcome this attraction and an atom loses one or more electrons. In other cases, atoms may gain additional electrons. Atoms in which the number of electrons does not equal the number of protons are known as ions, and they carry a net electrical charge. An atom that has more protons than electrons has a net positive charge and is called a cation. For example, an atom of sodium (Na) that has lost one electron becomes a sodium ion (Na + ), with a charge of +1. An atom that has fewer protons than elec- trons carries a net negative charge and is called an anion. A chlorine atom (Cl) that has gained one electron becomes a chloride ion (Cl – ), with a charge of –1. An atom consists of a nucleus of protons and neutrons surrounded by a cloud of electrons. The number of its electrons largely determines the chemical properties of an atom. Atoms that have the same number of protons but different numbers of neutrons are called isotopes. Isotopes of an atom differ in atomic mass but have similar chemical properties. Chapter 2 The Nature of Molecules 21 Carbon-12 6 Protons 6 Neutrons 6 Electrons Carbon-13 6 Protons 7 Neutrons 6 Electrons Carbon-14 6 Protons 8 Neutrons 6 Electrons FIGURE 2.3 The three most abundant isotopes of carbon. Isotopes of a particular atom have different numbers of neutrons. Electrons Determine the Chemical Behavior of Atoms The key to the chemical behavior of an atom lies in the ar- rangement of its electrons in their orbits. It is convenient to visualize individual electrons as following discrete circular orbits around a central nucleus, as in the Bohr model of the atom. However, such a simple picture is not realistic. It is not possible to precisely locate the position of any individual electron precisely at any given time. In fact, a particular electron can be anywhere at a given instant, from close to the nucleus to infinitely far away from it. However, a particular electron is more likely to be locat- ed in some positions than in others. The area around a nu- cleus where an electron is most likely to be found is called the orbital of that electron (figure 2.4). Some electron or- bitals near the nucleus are spherical (s orbitals), while oth- ers are dumbbell-shaped (p orbitals). Still other orbitals, more distant from the nucleus, may have different shapes. Regardless of its shape, no orbital may contain more than two electrons. Almost all of the volume of an atom is empty space, be- cause the electrons are quite far from the nucleus relative to its size. If the nucleus of an atom were the size of an ap- ple, the orbit of the nearest electron would be more than 1600 meters away. Consequently, the nuclei of two atoms never come close enough in nature to interact with each other. It is for this reason that an atom’s electrons, not its protons or neutrons, determine its chemical behavior. This also explains why the isotopes of an element, all of which have the same arrangement of electrons, behave the same way chemically. Energy within the Atom All atoms possess energy, defined as the ability to do work. Because electrons are attracted to the positively charged nucleus, it takes work to keep them in orbit, just as it takes work to hold a grapefruit in your hand against the pull of gravity. The grapefruit is said to possess potential energy, the ability to do work, because of its position; if you were to release it, the grapefruit would fall and its energy would be reduced. Conversely, if you were to move the grapefruit to the top of a building, you would increase its potential energy. Similarly, electrons have potential energy of posi- tion. To oppose the attraction of the nucleus and move the electron to a more distant orbital requires an input of en- ergy and results in an electron with greater potential ener- gy. This is how chlorophyll captures energy from light during photosynthesis (chapter 10)—the light excites elec- trons in the chlorophyll. Moving an electron closer to the nucleus has the opposite effect: energy is released, usually as heat, and the electron ends up with less potential energy (figure 2.5). A given atom can possess only certain discrete amounts of energy. Like the potential energy of a grapefruit on a step of a staircase, the potential energy contributed by the posi- tion of an electron in an atom can have only certain values. 22 Part I The Origin of Living Things 1s Orbital x x y z Orbital for energy level K: one spherical orbital (1s) 2s Orbital 2p Orbitals Composite of all p orbitals Orbitals for energy level L: one spherical orbital (2s) and three dumbbell-shaped orbitals (2p) z y FIGURE 2.4 Electron orbitals. The lowest energy level or electron shell, which is nearest the nucleus, is level K. It is occupied by a single s orbital, referred to as 1s. The next highest energy level, L, is occupied by four orbitals: one s orbital (referred to as the 2s orbital) and three p orbitals (each referred to as a 2p orbital). The four L-level orbitals compactly fill the space around the nucleus, like two pyramids set base- to-base. Every atom exhibits a ladder of potential energy values, rather than a continuous spectrum of possibilities, a discrete set of orbits at particular distances from the nucleus. During some chemical reactions, electrons are trans- ferred from one atom to another. In such reactions, the loss of an electron is called oxidation, and the gain of an elec- tron is called reduction (figure 2.6). It is important to real- ize that when an electron is transferred in this way, it keeps its energy of position. In organisms, chemical energy is stored in high-energy electrons that are transferred from one atom to another in reactions involving oxidation and reduction. Because the amount of energy an electron possesses is related to its distance from the nucleus, electrons that are the same distance from the nucleus have the same energy, even if they occupy different orbitals. Such electrons are said to occupy the same energy level. In a schematic dia- gram of an atom (figure 2.7), the nucleus is represented as a small circle and the electron energy levels are drawn as con- centric rings, with the energy level increasing with distance from the nucleus. Be careful not to confuse energy levels, which are drawn as rings to indicate an electron’s energy, with orbitals, which have a variety of three-dimensional shapes and indicate an electron’s most likely location. Electrons orbit a nucleus in paths called orbitals. No orbital can contain more than two electrons, but many orbitals may be the same distance from the nucleus and, thus, contain electrons of the same energy. Chapter 2 The Nature of Molecules 23 Energy released Energy level 3 Energy level 2 Energy level 1 – ML K Energy level 1 Energy absorbed Energy level 2 Energy level 3 + – + + + + + + MLK FIGURE 2.5 Atomic energy levels. When an electron absorbs energy, it moves to higher energy levels farther from the nucleus. When an electron releases energy, it falls to lower energy levels closer to the nucleus. FIGURE 2.6 Oxidation and reduction. Oxidation is the loss of an electron; reduction is the gain of an electron. Oxidation Reduction H11001 – H11001 Helium Nitrogen 7H11001 7n 2H11001 2n KKL Nucleus L M N K Energy le ve l FIGURE 2.7 Electron energy levels for helium and nitrogen. Gold balls represent the electrons. Each concentric circle represents a different distance from the nucleus and, thus, a different electron energy level. 24 Part I The Origin of Living Things 1 H 1 H 3 Li 4 Be 19 K 12 Mg 93 Np 94 Pu 95 Am 96 Cm 97 Bk 98 Cf 99 Es 100 Fm 101 Md 102 No 103 Lr 37 Rb 38 Sr 39 Y 42 Mo 45 Rh 46 Pd 47 Ag 48 Cd 49 In 50 Sn 51 Sb 52 Te 53 I 54 Xe 21 Sc 40 Zr 22 Ti 23 V 24 Cr 25 Mn 27 Co 28 Ni 29 Cu 30 Zn 36 Kr 5 B 6 C 6 C 8 O 2 He 55 Cs 56 Ba 72 Hf 73 Ta 74 W 75 Re 76 Os 77 Ir 78 Pt 79 Au 80 Hg 81 Tl 82 Pb 83 Bi 84 Po 85 At 86 Rn 87 Fr 88 Ra 57 La 89 Ac 104 105 106 107 108 109 58 Ce 59 Pr 60 Nd 61 Pm 62 Sm 63 Eu 64 Gd 65 Tb 66 Dy 67 Ho 68 Er 69 Tm 70 Yb 71 Lu 90 Th 91 Pa 92 U (Lanthanide series) (Actinide series) 11 Na 20 Ca 41 Nb 43 Tc 44 Ru 26 Fe 13 Al 31 Ga 32 Ge 14 Si 7 N 15 P 33 As 16 S 35 Br 34 Se 9 F 18 Ar 10 Ne 17 Cl 110 FIGURE 2.8 Periodic table of the elements. In this representation, the frequency of elements that occur in the earth’s crust is indicated by the height of the block. Elements found in significant amounts in living organisms are shaded in blue. Kinds of Atoms There are 92 naturally occurring elements, each with a dif- ferent number of protons and a different arrangement of electrons. When the nineteenth-century Russian chemist Dmitri Mendeleev arranged the known elements in a table according to their atomic mass (figure 2.8), he discovered one of the great generalizations in all of science. Mendeleev found that the elements in the table exhibited a pattern of chemical properties that repeated itself in groups of eight el- ements. This periodically repeating pattern lent the table its name: the periodic table of elements. The Periodic Table The eight-element periodicity that Mendeleev found is based on the interactions of the electrons in the outer en- ergy levels of the different elements. These electrons are called valence electrons and their interactions are the basis for the differing chemical properties of the elements. For most of the atoms important to life, an outer energy level can contain no more than eight electrons; the chemi- cal behavior of an element reflects how many of the eight positions are filled. Elements possessing all eight elec- trons in their outer energy level (two for helium) are inert, or nonreactive; they include helium (He), neon (Ne), argon (Ar), krypton (Kr), xenon (Xe), and radon (Rn). In sharp contrast, elements with seven electrons (one fewer than the maximum number of eight) in their outer energy level, such as fluorine (F), chlorine (Cl), and bromine (Br), are highly reactive. They tend to gain the extra electron needed to fill the energy level. Elements with only one electron in their outer energy level, such as lithium (Li), sodium (Na), and potassium (K), are also very reactive; they tend to lose the single electron in their outer level. Mendeleev’s periodic table thus leads to a useful generali- zation, the octet rule (Latin octo, “eight”) or rule of eight: atoms tend to establish completely full outer energy levels. Most chemical behavior can be predicted quite accurately from this simple rule, combined with the tendency of at- oms to balance positive and negative charges. 2.2 The atoms of living things are among the smallest. Distribution of the Elements Of the 92 naturally occurring elements on earth, only 11 are found in organisms in more than trace amounts (0.01% or higher). These 11 elements have atomic numbers less than 21 and, thus, have low atomic masses. Table 2.1 lists the levels of various elements in the human body; their levels in other organisms are similar. Inspection of this table suggests that the distribution of elements in living systems is by no means accidental. The most common elements inside or- ganisms are not the elements that are most abundant in the earth’s crust. For example, silicon, aluminum, and iron con- stitute 39.2% of the earth’s crust, but they exist in trace amounts in the human body. On the other hand, carbon at- oms make up 18.5% of the human body but only 0.03% of the earth’s crust. Ninety-two elements occur naturally on earth; only eleven of them are found in significant amounts in living organisms. Four of them—oxygen, hydrogen, carbon, nitrogen—constitute 96.3% of the weight of your body. Chapter 2 The Nature of Molecules 25 Table 2.1 The Most Common Elements on Earth and Their Distribution in the Human Body Approximate Percent of Percent of Earth’s Crust Human Body Element Symbol Atomic Number by Weight by Weight Importance or Function Oxygen Silicon Aluminum Iron Calcium Sodium Potassium Magnesium Hydrogen Manganese Fluorine Phosphorus Carbon Sulfur Chlorine Vanadium Chromium Copper Nitrogen Boron Cobalt Zinc Selenium Molybdenum Tin Iodine O Si Al Fe Ca Na K Mg H Mn F P C S Cl V Cr Cu N B Co Zn Se Mo Sn I 8 14 13 26 20 11 19 12 1 25 9 15 6 16 17 23 24 29 7 5 27 30 34 42 50 53 46.6 27.7 6.5 5.0 3.6 2.8 2.6 2.1 0.14 0.1 0.07 0.07 0.03 0.03 0.01 0.01 0.01 0.01 Trace Trace Trace Trace Trace Trace Trace Trace 65.0 Trace Trace Trace 1.5 0.2 0.4 0.1 9.5 Trace Trace 1.0 18.5 0.3 0.2 Trace Trace Trace 3.3 Trace Trace Trace Trace Trace Trace Trace Required for cellular respiration; component of water Critical component of hemoglobin in the blood Component of bones and teeth; trig- gers muscle contraction Principal positive ion outside cells; important in nerve function Principal positive ion inside cells; im- portant in nerve function Critical component of many energy- transferring enzymes Electron carrier; component of water and most organic molecules Backbone of nucleic acids; important in energy transfer Backbone of organic molecules Component of most proteins Principal negative ion outside cells Key component of many enzymes Component of all proteins and nucleic acids Key component of some enzymes Key component of many enzymes Component of thyroid hormone 26 Part I The Origin of Living Things Na Sodium atom Sodium ion Chlorine atom Chloride ion+ – Na + Cl Cl H11002 (a) FIGURE 2.9 The formation of ionic bonds by sodium chloride. (a) When a sodium atom donates an electron to a chlorine atom, the sodium atom becomes a positively charged sodium ion, and the chlorine atom becomes a negatively charged chloride ion. (b) Sodium chloride forms a highly regular lattice of alternating sodium ions and chloride ions. NaCl crystal Cl H11002 Cl H11002 Cl H11002 Cl H11002 Cl H11002 Na H11001 Na H11001 Na H11001 Na H11001 (b) Ionic Bonds Form Crystals A group of atoms held together by energy in a stable associ- ation is called a molecule. When a molecule contains atoms of more than one element, it is called a compound. The atoms in a molecule are joined by chemical bonds; these bonds can result when atoms with opposite charges attract (ionic bonds), when two atoms share one or more pairs of electrons (covalent bonds), or when atoms interact in other ways. We will start by examining ionic bonds, which form when atoms with opposite electrical charges (ions) attract. A Closer Look at Table Salt Common table salt, sodium chloride (NaCl), is a lattice of ions in which the atoms are held together by ionic bonds (figure 2.9). Sodium has 11 electrons: 2 in the inner energy level, 8 in the next level, and 1 in the outer (valence) level. The valence electron is unpaired (free) and has a strong ten- dency to join with another electron. A stable configuration can be achieved if the valence electron is lost to another atom that also has an unpaired electron. The loss of this electron results in the formation of a positively charged sodium ion, Na + . The chlorine atom has 17 electrons: 2 in the inner energy level, 8 in the next level, and 7 in the outer level. Hence, one of the orbitals in the outer energy level has an unpaired electron. The addition of another electron to the outer level fills that level and causes a negatively charged chloride ion, Cl – , to form. When placed together, metallic sodium and gaseous chlorine react swiftly and explosively, as the sodium atoms donate electrons to chlorine, forming Na + and Cl – ions. Be- cause opposite charges attract, the Na + and Cl – remain asso- ciated in an ionic compound, NaCl, which is electrically neutral. However, the electrical attractive force holding NaCl together is not directed specifically between particular Na + and Cl – ions, and no discrete sodium chloride mole- cules form. Instead, the force exists between any one ion and all neighboring ions of the opposite charge, and the ions ag- gregate in a crystal matrix with a precise geometry. Such ag- gregations are what we know as salt crystals. If a salt such as NaCl is placed in water, the electrical attraction of the water molecules, for reasons we will point out later in this chapter, disrupts the forces holding the ions in their crystal matrix, causing the salt to dissolve into a roughly equal mixture of free Na + and Cl – ions. An ionic bond is an attraction between ions of opposite charge in an ionic compound. Such bonds are not formed between particular ions in the compound; rather, they exist between an ion and all of the oppositely charged ions in its immediate vicinity. 2.3 Chemical bonds hold molecules together. Covalent Bonds Build Stable Molecules Covalent bonds form when two atoms share one or more pairs of valence electrons. Consider hydrogen (H) as an example. Each hydrogen atom has an unpaired electron and an unfilled outer energy level; for these reasons the hy- drogen atom is unstable. When two hydrogen atoms are close to each other, however, each atom’s electron can orbit both nuclei. In effect, the nu- clei are able to share their electrons. The result is a diatomic (two-atom) molecule of hydrogen gas (figure 2.10). The molecule formed by the two hy- drogen atoms is stable for three reasons: 1. It has no net charge. The di- atomic molecule formed as a result of this sharing of electrons is not charged, because it still contains two protons and two electrons. 2. The octet rule is satisfied. Each of the two hydrogen atoms can be considered to have two or- biting electrons in its outer energy level. This satisfies the octet rule, because each shared electron orbits both nuclei and is included in the outer energy level of both atoms. 3. It has no free electrons. The bonds between the two atoms also pair the two free electrons. Unlike ionic bonds, covalent bonds are formed between two specific atoms, giving rise to true, discrete molecules. While ionic bonds can form regular crys- tals, the more specific associations made possible by covalent bonds allow the formation of complex molecular structures. Covalent Bonds Can Be Very Strong The strength of a covalent bond depends on the number of shared electrons. Thus double bonds, which satisfy the oc- tet rule by allowing two atoms to share two pairs of elec- trons, are stronger than single bonds, in which only one electron pair is shared. This means more chemical energy is required to break a double bond than a single bond. The strongest covalent bonds are triple bonds, such as those that link the two nitrogen atoms of nitrogen gas molecules. Covalent bonds are represented in chemical formulations as lines connecting atomic symbols, where each line between two bonded atoms represents the sharing of one pair of electrons. The structural formulas of hydrogen gas and oxygen gas are H—H and OH33527O, respectively, while their molecular formulas are H 2 and O 2 . Molecules with Several Covalent Bonds Molecules often consist of more than two atoms. One reason that larger mole- cules may be formed is that a given atom is able to share electrons with more than one other atom. An atom that requires two, three, or four additional electrons to fill its outer energy level completely may acquire them by sharing its elec- trons with two or more other atoms. For example, the carbon atom (C) contains six electrons, four of which are in its outer energy level. To satisfy the octet rule, a carbon atom must gain ac- cess to four additional electrons; that is, it must form four covalent bonds. Be- cause four covalent bonds may form in many ways, carbon atoms are found in many different kinds of molecules. Chemical Reactions The formation and breaking of chemi- cal bonds, the essence of chemistry, is called a chemical reaction. All chemi- cal reactions involve the shifting of at- oms from one molecule or ionic com- pound to another, without any change in the number or identity of the atoms. For convenience, we refer to the origi- nal molecules before the reaction starts as reactants, and the molecules result- ing from the chemical reaction as prod- ucts. For example: A — B + C — D → A — C + B + D reactants products The extent to which chemical reactions occur is influ- enced by several important factors: 1. Temperature. Heating up the reactants increases the rate of a reaction (as long as the temperature isn’t so high as to destroy the molecules). 2. Concentration of reactants and products. Reac- tions proceed more quickly when more reactants are available. An accumulation of products typically speeds reactions in the reverse direction. 3. Catalysts. A catalyst is a substance that increases the rate of a reaction. It doesn’t alter the reaction’s equi- librium between reactants and products, but it does shorten the time needed to reach equilibrium, often dramatically. In organisms, proteins called enzymes catalyze almost every chemical reaction. A covalent bond is a stable chemical bond formed when two atoms share one or more pairs of electrons. Chapter 2 The Nature of Molecules 27 FIGURE 2.10 Hydrogen gas. (a) Hydrogen gas is a diatomic molecule composed of two hydrogen atoms, each sharing its electron with the other. (b) The flash of fire that consumed the Hindenburg occurred when the hydrogen gas that was used to inflate the dirigible combined explosively with oxygen gas in the air to form water. H 2 (hydrogen gas) Covalent bond + + – – (a) (b) Chemistry of Water Of all the molecules that are common on earth, only wa- ter exists as a liquid at the relatively low temperatures that prevail on the earth’s surface, three-fourths of which is covered by liquid water (figure 2.11). When life was origi- nating, water provided a medium in which other molecules could move around and interact without being held in place by strong covalent or ionic bonds. Life evolved as a result of these interactions, and it is still inextricably tied to water. Life began in water and evolved there for 3 bil- lion years before spreading to land. About two-thirds of any organism’s body is composed of water, and no organ- ism can grow or reproduce in any but a water-rich envi- ronment. It is no accident that tropical rain forests are bursting with life, while dry deserts appear almost lifeless except when water becomes temporarily plentiful, such as after a rainstorm. The Atomic Structure of Water Water has a simple atomic structure. It consists of an oxy- gen atom bound to two hydrogen atoms by two single cova- lent bonds (figure 2.12a). The resulting molecule is stable: it satisfies the octet rule, has no unpaired electrons, and carries no net electrical charge. The single most outstanding chemical property of wa- ter is its ability to form weak chemical associations with only 5 to 10% of the strength of covalent bonds. This property, which derives directly from the structure of wa- ter, is responsible for much of the organization of living chemistry. The chemistry of life is water chemistry. The way in which life first evolved was determined in large part by the chemical properties of the liquid water in which that evolution occurred. 28 Part I The Origin of Living Things FIGURE 2.11 Water takes many forms. As a liquid, water fills our rivers and runs down over the land to the sea. (a) The iceberg on which the penguins are holding their meeting was formed in Antarctica from a huge block of ice that broke away into the ocean water. (b) When water cools below 0°C, it forms beautiful crystals, familiar to us as snow and ice. However, water is not always plentiful. (c) At Badwater, in Death Valley, California, there is no hint of water except for the broken patterns of dried mud. (a) (b) (c) FIGURE 2.12 Water has a simple molecular structure. (a) Each molecule is composed of one oxygen atom and two hydrogen atoms. The oxygen atom shares one electron with each hydrogen atom. (b) The greater electronegativity of the oxygen atom makes the water molecule polar: water carries two partial negative charges (δ – ) near the oxygen atom and two partial positive charges (δ + ), one on each hydrogen atom. δ – δ – δ – δ – δ + δ + δ + δ + 104.5° Oxygen Hydrogen Hydrogen Bohr model Ball-and-stick model H H 8+ 8n + + O (a) (b) 2.4 Water is the cradle of life. Water Atoms Act Like Tiny Magnets Both the oxygen and the hydrogen atoms attract the electrons they share in the covalent bonds of a water molecule; this attraction is called elec- tronegativity. However, the oxygen atom is more electronegative than the hydrogen atoms, so it attracts the electrons more strongly than do the hydrogen atoms. As a result, the shared electrons in a water molecule are far more likely to be found near the oxygen nucleus than near the hydrogen nuclei. This stronger attrac- tion for electrons gives the oxygen atom two partial negative charges (δ – ), as though the elec- tron cloud were denser near the oxygen atom than around the hydrogen atoms. Because the water molecule as a whole is electrically neu- tral, each hydrogen atom carries a partial positive charge (δ + ). The Greek letter delta (δ) signifies a partial charge, much weaker than the full unit charge of an ion. What would you expect the shape of a water molecule to be? Each of water’s two covalent bonds has a partial charge at each end, δ – at the oxygen end and δ + at the hydrogen end. The most stable arrangement of these charges is a tetrahe- dron, in which the two negative and two positive charges are approximately equidistant from one another (figure 2.12b). The oxygen atom lies at the center of the tetrahedron, the hydrogen atoms occupy two of the apexes, and the partial negative charges occupy the other two apexes. This results in a bond angle of 104.5° between the two covalent oxygen- hydrogen bonds. (In a regular tetrahedron, the bond angles would be 109.5°; in water, the partial negative charges occu- py more space than the hydrogen atoms, and, therefore, they compress the oxygen-hydrogen bond angle slightly.) The water molecule, thus, has distinct “ends,” each with a partial charge, like the two poles of a magnet. (These partial charges are much less than the unit charges of ions, how- ever.) Molecules that exhibit charge separation are called polar molecules because of their magnet-like poles, and water is one of the most polar molecules known. The polarity of water underlies its chemistry and the chemistry of life. Polar molecules interact with one another, as the δ – of one molecule is attracted to the δ + of another. Because many of these interactions involve hydrogen atoms, they are called hydrogen bonds (figure 2.13). Each hydrogen bond is individually very weak and transient, lasting on average only 100,000 1 ,000,000 second (10 –11 sec). However, the cumula- tive effects of large numbers of these bonds can be enor- mous. Water forms an abundance of hydrogen bonds, which are responsible for many of its important physical properties (table 2.2). The water molecule is very polar, with ends that exhibit partial positive and negative charges. Opposite charges attract, forming weak linkages called hydrogen bonds. Chapter 2 The Nature of Molecules 29 Hydrogen atom Hydrogen bond An organic molecule Oxygen atom δ – δ + FIGURE 2.13 Structure of a hydrogen bond. Table 2.2 The Properties of Water Property Explanation Example of Benefit to Life Cohesion High specific heat High heat of vaporization Lower density of ice High polarity Hydrogen bonds hold water molecules together Hydrogen bonds absorb heat when they break, and release heat when they form, minimizing temperature changes Many hydrogen bonds must be broken for water to evapo- rate Water molecules in an ice crystal are spaced relatively far apart because of hydrogen bonding Polar water molecules are attracted to ions and polar com- pounds, making them soluble Leaves pull water upward from the roots; seeds swell and germinate Water stabilizes the temperature of organisms and the environment Evaporation of water cools body surfaces Because ice is less dense than water, lakes do not freeze solid Many kinds of molecules can move freely in cells, permitting a diverse array of chemical reactions Water Clings to Polar Molecules The polarity of water causes it to be attracted to other polar molecules. When the other molecules are also water, the at- traction is referred to as cohesion. When the other mole- cules are of a different substance, the attraction is called ad- hesion. It is because water is cohesive that it is a liquid, and not a gas, at moderate temperatures. The cohesion of liquid water is also responsible for its surface tension. Small insects can walk on water (figure 2.14) because at the air-water interface all of the hydrogen bonds in water face downward, causing the molecules of the water surface to cling together. Water is adhesive to any substance with which it can form hydrogen bonds. That is why substances containing polar molecules get “wet” when they are immersed in water, while those that are composed of nonpolar molecules (such as oils) do not. The attraction of water to substances like glass with sur- face electrical charges is responsible for capillary action: if a glass tube with a narrow diameter is lowered into a beaker of water, water will rise in the tube above the level of the water in the beaker, because the adhesion of water to the glass surface, drawing it upward, is stronger than the force of gravity, drawing it down. The narrower the tube, the greater the electrostatic forces between the water and the glass, and the higher the water rises (figure 2.15). Water Stores Heat Water moderates temperature through two properties: its high specific heat and its high heat of vaporization. The temperature of any substance is a measure of how rapidly its individual molecules are moving. Because of the many hydrogen bonds that water molecules form with one anoth- er, a large input of thermal energy is required to break these bonds before the individual water molecules can be- gin moving about more freely and so have a higher temper- ature. Therefore, water is said to have a high specific heat, which is defined as the amount of heat that must be ab- sorbed or lost by 1 gram of a substance to change its tem- perature by 1 degree Celsius (°C). Specific heat measures the extent to which a substance resists changing its temper- ature when it absorbs or loses heat. Because polar substanc- es tend to form hydrogen bonds, and energy is needed to break these bonds, the more polar a substance is, the higher is its specific heat. The specific heat of water (1 calo- rie/gram/°C) is twice that of most carbon compounds and nine times that of iron. Only ammonia, which is more polar than water and forms very strong hydrogen bonds, has a higher specific heat than water (1.23 calories/gram/°C). Still, only 20% of the hydrogen bonds are broken as water heats from 0° to 100°C. Because of its high specific heat, water heats up more slowly than almost any other compound and holds its tem- perature longer when heat is no longer applied. This char- acteristic enables organisms, which have a high water con- tent, to maintain a relatively constant internal temperature. The heat generated by the chemical reactions inside cells would destroy the cells, if it were not for the high specific heat of the water within them. A considerable amount of heat energy (586 calories) is re- quired to change 1 gram of liquid water into a gas. Hence, water also has a high heat of vaporization. Because the transition of water from a liquid to a gas requires the input of energy to break its many hydrogen bonds, the evapora- tion of water from a surface causes cooling of that surface. Many organisms dispose of excess body heat by evaporative cooling; for example, humans and many other vertebrates sweat. At low temperatures, water molecules are locked into a crystal-like lattice of hydrogen bonds, forming the solid we call ice (figure 2.16). Interestingly, ice is less dense than liquid water because the hydrogen bonds in ice space the water molecules relatively far apart. This unusual feature enables icebergs to float. Were it otherwise, ice would cover nearly all bodies of water, with only shallow surface melting annually. 30 Part I The Origin of Living Things FIGURE 2.14 Cohesion. Some insects, such as this water strider, literally walk on water. In this photograph you can see the dimpling the insect’s feet make on the water as its weight bears down on the surface. Because the surface tension of the water is greater than the force that one foot brings to bear, the strider glides atop the surface of the water rather than sinking. FIGURE 2.15 Capillary action. Capillary action causes the water within a narrow tube to rise above the surrounding water; the adhesion of the water to the glass surface, which draws water upward, is stronger than the force of gravity, which tends to draw it down. The narrower the tube, the greater the surface area available for adhesion for a given volume of water, and the higher the water rises in the tube. Water Is a Powerful Solvent Water is an effective solvent because of its ability to form hydrogen bonds. Water molecules gather closely around any substance that bears an electrical charge, whether that substance carries a full charge (ion) or a charge separation (polar molecule). For example, sucrose (table sugar) is composed of molecules that contain slightly polar hydroxyl (OH) groups. A sugar crystal dissolves rapidly in water be- cause water molecules can form hydrogen bonds with indi- vidual hydroxyl groups of the sucrose molecules. There- fore, sucrose is said to be soluble in water. Every time a sucrose molecule dissociates or breaks away from the crys- tal, water molecules surround it in a cloud, forming a hy- dration shell and preventing it from associating with oth- er sucrose molecules. Hydration shells also form around ions such as Na + and Cl – (figure 2.17). Water Organizes Nonpolar Molecules Water molecules always tend to form the maximum possi- ble number of hydrogen bonds. When nonpolar molecules such as oils, which do not form hydrogen bonds, are placed in water, the water molecules act to exclude them. The nonpolar molecules are forced into association with one an- other, thus minimizing their disruption of the hydrogen bonding of water. In effect, they shrink from contact with water and for this reason they are referred to as hydropho- bic (Greek hydros, “water” and phobos, “fearing”). In con- trast, polar molecules, which readily form hydrogen bonds with water, are said to be hydrophilic (“water-loving”). The tendency of nonpolar molecules to aggregate in wa- ter is known as hydrophobic exclusion. By forcing the hy- drophobic portions of molecules together, water causes these molecules to assume particular shapes. Different mo- lecular shapes have evolved by alteration of the location and strength of nonpolar regions. As you will see, much of the evolution of life reflects changes in molecular shape that can be induced in just this way. Water molecules, which are very polar, cling to one another, so that it takes considerable energy to separate them. Water also clings to other polar molecules, causing them to be soluble in water solution, but water tends to exclude nonpolar molecules. Chapter 2 The Nature of Molecules 31 Water molecules Stable hydrogen bonds Unstable hydrogen bonds (a) Liquid water (b) Ice FIGURE 2.16 The role of hydrogen bonds in an ice crystal. (a) In liquid water, hydrogen bonds are not stable and constantly break and re- form. (b) When water cools below 0°C, the hydrogen bonds are more stable, and a regular crystalline structure forms in which the four partial charges of one water molecule interact with the opposite charges of other water molecules. Because water forms a crystal latticework, ice is less dense than liquid water and floats. If it did not, inland bodies of water far from the earth’s equator might never fully thaw. Hydration shells Water molecules Salt crystal Na H11001 Cl H11002 Cl H11002 Cl H11002 Cl H11002 Na H11001 Na H11001 Na H11001 FIGURE 2.17 Why salt dissolves in water. When a crystal of table salt dissolves in water, individual Na + and Cl – ions break away from the salt lattice and become surrounded by water molecules. Water molecules orient around Cl – ions so that their partial positive poles face toward the negative Cl – ion; water molecules surrounding Na + ions orient in the opposite way, with their partial negative poles facing the positive Na + ion. Surrounded by hydration shells, Na + and Cl – ions never reenter the salt lattice. Water Ionizes The covalent bonds within a water molecule sometimes break spontaneously. In pure water at 25°C, only 1 out of every 550 million water molecules undergoes this process. When it happens, one of the protons (hydrogen atom nu- clei) dissociates from the molecule. Because the dissociated proton lacks the negatively charged electron it was sharing in the covalent bond with oxygen, its own positive charge is no longer counterbalanced, and it becomes a positively charged ion, H + . The rest of the dissociated water molecule, which has retained the shared electron from the covalent bond, is negatively charged and forms a hydroxide ion (OH – ). This process of spontaneous ion formation is called ionization: H 2 O → OH – +H + water hydroxide ion hydrogenion (proton) At 25°C, a liter of water contains 10,000 1 ,000 (or 10 –7 ) mole of H + ions. (A mole is defined as the weight in grams that corresponds to the summed atomic masses of all of the atoms in a molecule. In the case ofH + , the atomic mass is 1, and a mole of H + ions would weigh 1 gram. One mole of any substance always contains 6.02 × 10 23 mole- cules of the substance.) Therefore, the molar concentra- tion of hydrogen ions (represented as [H + ]) in pure water is 10 –7 mole/liter. Actually, the hydrogen ion usually associ- ates with another water molecule to form a hydronium (H 3 O + ) ion. pH A more convenient way to express the hydrogen ion concen- tration of a solution is to use the pH scale (figure 2.18). This scale defines pH as the negative logarithm of the hy- drogen ion concentration in the solution: pH = –log [H + ] Because the logarithm of the hydrogen ion concentration is simply the exponent of the molar concentration of H + , the pH equals the exponent times –1. Thus, pure water, with an [H + ] of 10 –7 mole/liter, has a pH of 7. Recall that for every H + ion formed when water dissociates, an OH – ion is also formed, meaning that the dissociation of water produces H + and OH – in equal amounts. Therefore, a pH value of 7 indi- cates neutrality—a balance between H + and OH – —on the pH scale. Note that the pH scale is logarithmic, which means that a difference of 1 on the scale represents a tenfold change in hydrogen ion concentration. This means that a solution with a pH of 4 has 10 times the concentration of H + than is present in one with a pH of 5. Acids. Any substance that dissociates in water to increase the concentration of H + ions is called an acid. Acidic solu- tions have pH values below 7. The stronger an acid is, the more H + ions it produces and the lower its pH. For exam- ple, hydrochloric acid (HCl), which is abundant in your stomach, ionizes completely in water. This means that 10 –1 mole per liter of HCl will dissociate to form 10 –1 mole per liter of H + ions, giving the solution a pH of 1. The pH of champagne, which bubbles because of the carbonic acid dissolved in it, is about 2. Bases. A substance that combines with H + ions when dis- solved in water is called a base. By combining with H + ions, a base lowers the H + ion concentration in the solution. Basic (or alkaline) solutions, therefore, have pH values above 7. Very strong bases, such as sodium hydroxide (NaOH), have pH values of 12 or more. 32 Part I The Origin of Living Things 10 H110021 H + Ion Concentration Examples of Solutions Hydrochloric acid Stomach acid 1 pH Value 10 H110022 Lemon juice2 10 H110023 Vinegar, cola, beer3 10 H110024 Tomatoes4 10 H110025 Black coffee Normal rainwater 5 10 H110026 Urine Saliva 6 10 H110027 Pure water Blood 7 10 H110028 Seawater8 10 H110029 Baking soda9 10 H1100210 Great Salt Lake10 10 H1100211 Household ammonia11 10 H1100212 Household bleach 12 10 H1100213 Oven cleaner 13 10 H1100214 Sodium hydroxide14 FIGURE 2.18 The pH scale. The pH value of a solution indicates its concentration of hydrogen ions. Solutions with a pH less than 7 are acidic, while those with a pH greater than 7 are basic. The scale is logarithmic, so that a pH change of 1 means a tenfold change in the concentration of hydrogen ions. Thus, lemon juice is 100 times more acidic than tomato juice, and seawater is 10 times more basic than pure water, which has a pH of 7. Buffers The pH inside almost all living cells, and in the fluid sur- rounding cells in multicellular organisms, is fairly close to 7. Most of the biological catalysts (enzymes) in living systems are extremely sensitive to pH; often even a small change in pH will alter their shape, thereby disrupting their activities and rendering them useless. For this reason it is important that a cell maintain a constant pH level. Yet the chemical reactions of life constantly produce acids and bases within cells. Furthermore, many animals eat sub- stances that are acidic or basic; cola, for example, is a strong (although dilute) acidic solution. Despite such variations in the concentrations of H + and OH – , the pH of an organism is kept at a relatively constant level by buffers (figure 2.19). A buffer is a substance that acts as a reservoir for hy- drogen ions, donating them to the solution when their con- centration falls and taking them from the solution when their concentration rises. What sort of substance will act in this way? Within organisms, most buffers consist of pairs of substances, one an acid and the other a base. The key buffer in human blood is an acid-base pair consisting of carbonic acid (acid) and bicarbonate (base). These two substances in- teract in a pair of reversible reactions. First, carbon dioxide (CO 2 ) and H 2 O join to form carbonic acid (H 2 CO 3 ), which in a second reaction dissociates to yield bicarbonate ion (HCO 3 – ) and H + (figure 2.20). If some acid or other sub- stance adds H + ions to the blood, the HCO 3 – ions act as a base and remove the excess H + ions by forming H 2 CO 3 . Similarly, if a basic substance removes H + ions from the blood, H 2 CO 3 dissociates, releasing more H + ions into the blood. The forward and reverse reactions that interconvert H 2 CO 3 and HCO 3 – thus stabilize the blood’s pH. The reaction of carbon dioxide and water to form car- bonic acid is important because it permits carbon, essential to life, to enter water from the air. As we will discuss in chapter 4, biologists believe that life first evolved in the early oceans. These oceans were rich in carbon because of the reaction of carbon dioxide with water. In a condition called blood acidosis, human blood, which normally has a pH of about 7.4, drops 0.2 to 0.4 points on the pH scale. This condition is fatal if not treated immediately. The reverse condition, blood alkalosis, involves an increase in blood pH of a similar magnitude and is just as serious. The pH of a solution is the negative logarithm of the H + ion concentration in the solution. Thus, low pH values indicate high H + concentrations (acidic solutions), and high pH values indicate low H + concentrations (basic solutions). Even small changes in pH can be harmful to life. Chapter 2 The Nature of Molecules 33 10 0 1 2 3 4 5 6 7 8 9 3 Amount of base added Buffering range pH 452 FIGURE 2.19 Buffers minimize changes in pH. Adding a base to a solution neutralizes some of the acid present, and so raises the pH. Thus, as the curve moves to the right, reflecting more and more base, it also rises to higher pH values. What a buffer does is to make the curve rise or fall very slowly over a portion of the pH scale, called the “buffering range” of that buffer. FIGURE 2.20 Buffer formation. Carbon dioxide and water combine chemically to form carbonic acid (H 2 CO 3 ). The acid then dissociates in water, freeing H + ions. This reaction makes carbonated beverages acidic, and produced the carbon-rich early oceans that cradled life. H 2 O Water CO 2 Carbon dioxide H 2 CO 3 Carbonic acid HCO H11002 3 Bicarbonate ion H H11001 Hydrogen ion – + + + ? The smallest stable particles of matter are protons, neutrons, and electrons, which associate to form atoms. ? The core, or nucleus, of an atom consists of protons and neutrons; the electrons orbit around the nucleus in a cloud. The farther an electron is from the nucleus, the faster it moves and the more energy it possesses. ? The chemical behavior of an atom is largely determined by the distribution of its electrons and in particular by the num- ber of electrons in its outermost (highest) energy level. There is a strong tendency for atoms to have a completely filled outer level; electrons are lost, gained, or shared until this condition is reached. 2.2 The atoms of living things are among the smallest. 34 Part I The Origin of Living Things Chapter 2 Summary Questions Media Resources 2.1 Atoms are nature’s building material. 1. An atom of nitrogen has 7 protons and 7 neutrons. What is its atomic number? What is its atomic mass? How many elec- trons does it have? 2. How do the isotopes of a sin- gle element differ from each other? 3. The half-life of radium-226 is 1620 years. If a sample of mate- rial contains 16 milligrams of ra- dium-226, how much will it con- tain in 1620 years? How much will it contain in 3240 years? How long will it take for the sample to contain 1 milligram of radium-226? ? More than 95% of the weight of an organism consists of oxygen, hydrogen, carbon, and nitrogen, all of which form strong covalent bonds with one another. ? Ionic bonds form when electrons transfer from one atom to another, and the resulting oppositely charged ions attract one another. ? Covalent bonds form when two atoms share elec- trons. They are responsible for the formation of most biologically important molecules. 4. What is the octet rule, and how does it affect the chemical behavior of atoms? 5. What is the difference be- tween an ionic bond and a cova- lent bond? Give an example of each. 2.3 Chemical bonds hold molecules together. 2.4 Water is the cradle of life. ? The chemistry of life is the chemistry of water (H 2 O). The central oxygen atom in water attracts the elec- trons it shares with the two hydrogen atoms. This charge separation makes water a polar molecule. ? A hydrogen bond is formed between the partial posi- tive charge of a hydrogen atom in one molecule and the partial negative charge of another atom, either in another molecule or in a different portion of the same molecule. ? Water is cohesive and adhesive, has a great capacity for storing heat, is a good solvent for other polar molecules, and tends to exclude nonpolar molecules. ? The H + concentration in a solution is expressed by the pH scale, in which pH equals the negative loga- rithm of the H + concentration. 6. What types of atoms partici- pate in the formation of hydro- gen bonds? How do hydrogen bonds contribute to water’s high specific heat? 7. What types of molecules are hydrophobic? What types are hydrophilic? Why do these two types of molecules behave differ- ently in water? 8. What is the pH of a solution that has a hydrogen ion concen- tration of 10 –3 mole/liter? Would such a solution be acidic or basic? ? Atomic Structure ? Basic Chemistry ? Atoms ? Bonds ? Ionic Bonds ? Bonds ? Water ? ph Scale http://www.mhhe.com/raven6e http://www.biocourse.com 35 3 The Chemical Building Blocks of Life Concept Outline 3.1 Molecules are the building blocks of life. The Chemistry of Carbon. Because individual carbon atoms can form multiple covalent bonds, organic molecules can be quite complex. 3.2 Proteins perform the chemistry of the cell. The Many Functions of Proteins. Proteins can be cata- lysts, transporters, supporters, and regulators. Amino Acids Are the Building Blocks of Proteins. Proteins are long chains of various combinations of amino acids. A Protein’s Function Depends on the Shape of the Molecule. A protein’s shape is determined by its amino acid sequence. How Proteins Fold Into Their Functional Shape. The distribution of nonpolar amino acids along a protein chain largely determines how the protein folds. How Proteins Unfold. When conditions such as pH or temperature fluctuate, proteins may denature or unfold. 3.3 Nucleic acids store and transfer genetic information. Information Molecules. Nucleic acids store information in cells. RNA is a single-chain polymer of nucleotides, while DNA possesses two chains twisted around each other. 3.4 Lipids make membranes and store energy. Phospholipids Form Membranes. The spontaneous ag- gregation of phospholipids in water is responsible for the formation of biological membranes. Fats and Other Kinds of Lipids. Organisms utilize a wide variety of water-insoluble molecules. Fats as Food. Fats are very efficient energy storage molecules because of their high proportion of C—H bonds. 3.5 Carbohydrates store energy and provide building materials. Simple Carbohydrates. Sugars are simple carbohydrates, often consisting of six-carbon rings. Linking Sugars Together. Sugars can be linked together to form long polymers, or polysaccharides. Structural Carbohydrates. Structural carbohydrates like cellulose are chains of sugars linked in a way that enzymes cannot easily attack. M olecules are extremely small compared with the fa- miliar world we see about us. Imagine: there are more water molecules in a cup than there are stars in the sky. Many other molecules are gigantic, compared with wa- ter, consisting of thousands of atoms. These atoms are or- ganized into hundreds of smaller molecules that are linked together into long chains (figure 3.1). These enormous molecules, almost always synthesized by living things, are called macromolecules. As we shall see, there are four gen- eral types of macromolecules, the basic chemical building blocks from which all organisms are assembled. FIGURE 3.1 Computer-generated model of a macromolecule. Pictured is an enzyme responsible for releasing energy from sugar. This complex molecule consists of hundreds of different amino acids linked into chains that form the characteristic coils and folds seen here. Biological Macromolecules Some organic molecules in organisms are small and sim- ple, containing only one or a few functional groups. Oth- ers are large complex assemblies called macromolecules. In many cases, these macromolecules are polymers, mole- cules built by linking together a large number of small, similar chemical subunits, like railroad cars coupled to form a train. For example, complex carbohydrates like starch are polymers of simple ring-shaped sugars, pro- teins are polymers of amino acids, and nucleic acids (DNA and RNA) are polymers of nucleotides. Biological macromolecules are traditionally grouped into four major categories: proteins, nucleic acids, lipids, and carbohy- drates (table 3.1). 36 Part I The Origin of Living Things The Chemistry of Carbon In chapter 2 we discussed how atoms combine to form molecules. In this chapter, we will focus on organic mole- cules, those chemical compounds that contain carbon. The frameworks of biological molecules consist predominantly of carbon atoms bonded to other carbon atoms or to atoms of oxygen, nitrogen, sulfur or hydrogen. Because carbon atoms possess four valence electrons and so can form four covalent bonds, molecules containing carbon can form straight chains, branches, or even rings. As you can imag- ine, all of these possibilities generate an immense range of molecular structures and shapes. Organic molecules consisting only of carbon and hydro- gen are called hydrocarbons. Covalent bonds between car- bon and hydrogen are energy-rich. We use hydrocarbons from fossil fuels as a primary source of energy today. Propane gas, for example, is a hydrocarbon consisting of a chain of three carbon atoms, with eight hydrogen atoms bound to it: H H H ||| H—C—C—C—H | || H H H Because carbon-hydrogen covalent bonds store consider- able energy, hydrocarbons make good fuels. Gasoline, for example, is rich in hydrocarbons. Functional Groups Carbon and hydrogen atoms both have very similar elec- tronegativities, so electrons in C—C and C—H bonds are evenly distributed, and there are no significant differences in charge over the molecular surface. For this reason, hy- drocarbons are nonpolar. Most organic molecules that are produced by cells, however, also contain other atoms. Be- cause these other atoms often have different electronegativ- ities, molecules containing them exhibit regions of positive or negative charge, and so are polar. These molecules can be thought of as a C—H core to which specific groups of atoms called functional groups are attached. For example, a hydrogen atom bonded to an oxygen atom (—OH) is a functional group called a hydroxyl group. Functional groups have definite chemical properties that they retain no matter where they occur. The hydroxyl group, for example, is polar, because its oxygen atom, being very electronegative, draws electrons toward itself (as we saw in chapter 2). Figure 3.2 illustrates the hydroxyl group and other biologically important functional groups. Most chemical reactions that occur within organisms involve the transfer of a functional group as an intact unit from one molecule to another. 3.1 Molecules are the building blocks of life. Hydroxyl Carbonyl Carboxyl Amino Sulfhydryl Phosphate Methyl Carbohydrates, alcohols Amino acids, vinegar Group Structural Formula Ball-and- Stick Model Found In: Formaldehyde Ammonia Proteins, rubber Phospholipids, nucleic acids, ATP Methane gas HS O – P O – O O HC H H OH O OH C H H N C O H H O O – PO H N S H O O – H H C H H O C O O C FIGURE 3.2 The primary functional chemical groups. These groups tend to act as units during chemical reactions and confer specific chemical properties on the molecules that possess them. Amino groups, for example, make a molecule more basic, while carboxyl groups make a molecule more acidic. Building Macromolecules Although the four categories of macromolecules contain dif- ferent kinds of subunits, they are all assembled in the same fundamental way: to form a covalent bond between two sub- unit molecules, an —OH group is removed from one sub- unit and a hydrogen atom (H) is removed from the other (figure 3.3a). This condensation reaction is called a dehy- dration synthesis, because the removal of the —OH group and H during the synthesis of a new molecule in effect con- stitutes the removal of a molecule of water (H 2 O). For every subunit that is added to a macromolecule, one water mole- cule is removed. Energy is required to break the chemical bonds when water is extracted from the subunits, so cells must supply energy to assemble macromolecules. These and other biochemical reactions require that the reacting sub- stances be held close together and that the correct chemical bonds be stressed and broken. This process of positioning and stressing, termed catalysis, is carried out in cells by a special class of proteins known as enzymes. Cells disassemble macromolecules into their constituent subunits by performing reactions that are essentially the re- verse of dehydration—a molecule of water is added instead of removed (figure 3.3b). In this process, which is called hydrolysis (Greek hydro, “water” + lyse, “break”), a hydro- gen atom is attached to one subunit and a hydroxyl group to the other, breaking a specific covalent bond in the macromolecule. Hydrolytic reactions release the energy that was stored in the bonds that were broken. Polymers are large molecules consisting of long chains of similar subunits joined by dehydration reactions. In a dehydration reaction, a hydroxyl (—OH) group is removed from one subunit and a hydrogen atom (H) is removed from the other. Chapter 3 The Chemical Building Blocks of Life 37 Table 3.1 Macromolecules Macromolecule Subunit Function Example PROTEINS Globular Structural NUCLEIC ACIDS DNA RNA LIPIDS Fats Phospholipids Prostaglandins Steroids Terpenes CARBOHYDRATES Starch, glycogen Cellulose Chitin Hemoglobin Hair; silk Chromosomes Messenger RNA Butter; corn oil; soap Lecithin Prostaglandin E (PGE) Cholesterol; estrogen Carotene; rubber Potatoes Paper; strings of celery Crab shells Amino acids Amino acids Nucleotides Nucleotides Glycerol and three fatty acids Glycerol, two fatty acids, phosphate, and polar R groups Five-carbon rings with two nonpolar tails Four fused carbon rings Long carbon chains Glucose Glucose Modified glucose Catalysis; transport Support Encodes genes Needed for gene expression Energy storage Cell membranes Chemical messengers Membranes; hormones Pigments; structural Energy storage Cell walls Structural support H 2 O H 2 O HO HO H HO H HHHO Energy Dehydration synthesis HO H HHO Energy Hydrolysis (a) (b) FIGURE 3.3 Making and breaking macromolecules. (a) Biological macromolecules are polymers formed by linking subunits together. The covalent bond between the subunits is formed by dehydration synthesis, an energy-requiring process that creates a water molecule for every bond formed. (b) Breaking the bond between subunits requires the returning of a water molecule with a subsequent release of energy, a process called hydrolysis. The Many Functions of Proteins We will begin our discussion of macromolecules that make up the bodies of organisms with proteins (see table 3.1). The proteins within living organisms are immensely diverse in structure and function (table 3.2 and figure 3.4). 1. Enzyme catalysis. We have already encountered one class of proteins, enzymes, which are biological catalysts that facilitate specific chemical reactions. Be- cause of this property, the appearance of enzymes was one of the most important events in the evolution of life. Enzymes are globular proteins, with a three- dimensional shape that fits snugly around the chemi- cals they work on, facilitating chemical reactions by stressing particular chemical bonds. 2. Defense. Other globular proteins use their shapes to “recognize” foreign microbes and cancer cells. These cell surface receptors form the core of the body’s hormone and immune systems. 3. Transport. A variety of globular proteins transport specific small molecules and ions. The transport pro- tein hemoglobin, for example, transports oxygen in the blood, and myoglobin, a similar protein, transports oxygen in muscle. Iron is transported in blood by the protein transferrin. 38 Part I The Origin of Living Things 3.2 Proteins perform the chemistry of the cell. Table 3.2 The Many Functions of Proteins Function Class of Protein Examples Use Metabolism (Catalysis) Defense Cell recognition Transport throughout body Membrane transport Structure/Support Motion Osmotic regulation Regulation of gene action Regulation of body functions Storage Enzymes Immunoglobulins Toxins Cell surface antigens Globins Transporters Fibers Muscle Albumin Repressors Hormones Ion binding Hydrolytic enzymes Proteases Polymerases Kinases Antibodies Snake venom MHC proteins Hemoglobin Myoglobin Cytochromes Sodium-potassium pump Proton pump Anion channels Collagen Keratin Fibrin Actin Myosin Serum albumin lac repressor Insulin Vasopressin Oxytocin Ferritin Casein Calmodulin Cleave polysaccharides Break down proteins Produce nucleic acids Phosphorylate sugars and proteins Mark foreign proteins for elimination Block nerve function “Self” recognition Carries O 2 and CO 2 in blood Carries O 2 and CO 2 in muscle Electron transport Excitable membranes Chemiosmosis Transport Cl– ions Cartilage Hair, nails Blood clot Contraction of muscle fibers Contraction of muscle fibers Maintains osmotic concentration of blood Regulates transcription Controls blood glucose levels Increases water retention by kidneys Regulates uterine contractions and milk production Stores iron, especially in spleen Stores ions in milk Binds calcium ions 4. Support. Fibrous, or threadlike, proteins play struc- tural roles; these structural proteins (see figure 3.4) in- clude keratin in hair, fibrin in blood clots, and col- lagen, which forms the matrix of skin, ligaments, tendons, and bones and is the most abundant protein in a vertebrate body. 5. Motion. Muscles contract through the sliding mo- tion of two kinds of protein filament: actin and myo- sin. Contractile proteins also play key roles in the cell’s cytoskeleton and in moving materials within cells. 6. Regulation. Small proteins called hormones serve as intercellular messengers in animals. Proteins also play many regulatory roles within the cell, turning on and shutting off genes during development, for exam- ple. In addition, proteins also receive information, act- ing as cell surface receptors. Proteins carry out a diverse array of functions, including catalysis, defense, transport of substances, motion, and regulation of cell and body functions. Chapter 3 The Chemical Building Blocks of Life 39 (a) (b) (c) (d) (e) FIGURE 3.4 Some of the more common structural proteins. (a) Collagen: strings of a tennis racket from gut tissue; (b) fibrin: scanning electron micrograph of a blood clot (3000×); (c) keratin: a peacock feather; (d) silk: a spider’s web; (e) keratin: human hair. Amino Acids Are the Building Blocks of Proteins Although proteins are complex and versatile molecules, they are all polymers of only 20 amino acids, in a specific order. Many scientists believe amino acids were among the first molecules formed in the early earth. It seems highly likely that the oceans that existed early in the history of the earth contained a wide variety of amino acids. Amino Acid Structure An amino acid is a molecule containing an amino group (—NH 2 ), a carboxyl group (—COOH), and a hydrogen atom, all bonded to a central carbon atom: R | H 2 N—C—COOH | H Each amino acid has unique chemical properties deter- mined by the nature of the side group (indicated by R) cova- lently bonded to the central carbon atom. For example, when the side group is —CH 2 OH, the amino acid (serine) is polar, but when the side group is —CH 3 , the amino acid (alanine) is nonpolar. The 20 common amino acids are grouped into five chemical classes, based on their side groups: 1. Nonpolar amino acids, such as leucine, often have R groups that contain —CH 2 or —CH 3 . 2. Polar uncharged amino acids, such as threonine, have R groups that contain oxygen (or only —H). 3. Ionizable amino acids, such as glutamic acid, have R groups that contain acids or bases. 4. Aromatic amino acids, such as phenylalanine, have R groups that contain an organic (carbon) ring with al- ternating single and double bonds. 5. Special-function amino acids have unique individual properties; methionine often is the first amino acid in a chain of amino acids, proline causes kinks in chains, and cysteine links chains together. Each amino acid affects the shape of a protein differently depending on the chemical nature of its side group. Portions of a protein chain with numerous nonpolar amino acids, for example, tend to fold into the interior of the protein by hy- drophobic exclusion. Proteins Are Polymers of Amino Acids In addition to its R group, each amino acid, when ionized, has a positive amino (NH 3 + ) group at one end and a nega- tive carboxyl (COO – ) group at the other end. The amino and carboxyl groups on a pair of amino acids can undergo a condensation reaction, losing a molecule of water and forming a covalent bond. A covalent bond that links two amino acids is called a peptide bond (figure 3.5). The two amino acids linked by such a bond are not free to rotate around the N—C linkage because the peptide bond has a partial double-bond character, unlike the N—C and C—C bonds to the central carbon of the amino acid. The stiffness of the peptide bond is one factor that makes it possible for chains of amino acids to form coils and other regular shapes. A protein is composed of one or more long chains, or polypeptides, composed of amino acids linked by peptide bonds. It was not until the pioneering work of Frederick Sanger in the early 1950s that it became clear that each kind of protein had a specific amino acid sequence. Sanger succeeded in determining the amino acid sequence of insu- lin and in so doing demonstrated clearly that this protein had a defined sequence, the same for all insulin molecules in the solution. Although many different amino acids occur in nature, only 20 commonly occur in proteins. Figure 3.6 illustrates these 20 “common” amino acids and their side groups. A protein is a polymer containing a combination of up to 20 different kinds of amino acids. The amino acids fall into five chemical classes, each with different properties. These properties determine the nature of the resulting protein. 40 Part I The Origin of Living Things H — H — N — C — OH O — C — H — H H 2 O — H — N — C — OH O — C — H — Amino acidAmino acid H — H — N — C — O — — — — — C — H — H — N — C — OH O — — — — — C — H — Polypeptide chain RR RR FIGURE 3.5 The peptide bond. A peptide bond forms when the —NH 2 end of one amino acid joins to the —COOH end of another. Because of the partial double-bond nature of peptide bonds, the resulting peptide chain cannot rotate freely around these bonds. Chapter 3 The Chemical Building Blocks of Life 41 Proline (Pro) CH 2 CH — C — OH — — H N — — — — CH 2 CH 2 — O Methionine (Met) CH 2 H 2 N — C — C — OH — — HO — — — CH 2 — S — CH 3 Cysteine (Cys) CH 2 H 2 N — C — C — OH — — HO — — — SH SPECIAL STRUCTURAL PROPERTY CH 3 H 2 N — C — C — OH — — HO — — CH H 2 N — C — C — OH — — HO — — — — CH 3 CH 3 CH 2 H 2 N — C — C — OH — — HO — — — — CH 3 CH 3 — CH CH 2 H 2 N — C — C — OH — — HO — — — CH 3 H — C — CH 3 — CH 2 H 2 N — C — C — OH — — HO — — — CH 2 H 2 N — C — C — OH — — HO NH — C — —— — — — — Alanine (Ala) Leucine (Leu) Isoleucine (Ile) Phenylalanine (Phe) Tryptophan (Trp) CH 2 H 2 N — C — C — OH — — HO — — OH — H — C — OH H 2 N — C — C — OH — — HO — — CH 3 — CH 2 H 2 N — C — C — OH — — HO — — — CH 2 — C — NH 2 — — O CH 2 H 2 N — C — C — OH — — HO OH — — —— — C H 2 N — C — C — OH — — HO — NH 2 — — — — — CH 2 O H H 2 N — C — C — OH — — HO — — Tyrosine (Tyr) Glutamine (Gln) Asparagine (Asn) Threonine (Thr) Serine (Ser) Glycine (Gly) CH 2 H 2 N — C — C — OH — — HO — — — CH 2 — C — — — O O – CH 2 H 2 N — C — C — OH — — HO CH 2 — NH — CH 2 — — — — — — CNH 2 + — NH 2 CH 2 H 2 N — C — C — OH — — HO — — — CH 2 — NH 3 + — CH 2 CH 2 — CH 2 H 2 N — C — C — OH — — HO — C — N — — — — HC — N — CH H H 2 N — C — C — OH — — HO — — CH 2 — C — — — O O – Glutamic acid (Glu) Aspartic acid (Asp) Histidine (His) Lysine (Lys) Arginine (Arg) Ionizable (charged) Polar uncharged Nonpolar NONAROMATIC AROMATIC H + — — Valine (Val) FIGURE 3.6 The 20 common amino acids. Each amino acid has the same chemical backbone, but differs in the side, or R, group it possesses. Six of the amino acids are nonpolar because they have —CH 2 or —CH 3 in their R groups. Two of the six are bulkier because they contain ring structures, which classifies them also as aromatic. Another six are polar because they have oxygen or just hydrogen in their R groups; these amino acids, which are uncharged, differ from one another in how polar they are. Five other amino acids are polar and, because they have a terminal acid or base in their R group, are capable of ionizing to a charged form. The remaining three have special chemical properties that allow them to help form links between protein chains or kinks in proteins. A Protein’s Function Depends on the Shape of the Molecule The shape of a protein is very important because it determines the protein’s function. If we picture a polypeptide as a long strand similar to a reed, a pro- tein might be the basket woven from it. Overview of Protein Structure Proteins consist of long amino acid chains folded into complex shapes. What do we know about the shape of these proteins? One way to study the shape of something as small as a protein is to look at it with very short wavelength energy—with X rays. X-ray diffraction is a painstaking procedure that allows the investigator to build up a three- dimensional picture of the position of each atom. The first protein to be analyzed in this way was myoglobin, soon followed by the related protein hemoglobin. As more and more proteins were add- ed to the list, a general principle became evident: in every protein studied, essentially all the internal amino acids are nonpolar ones, amino acids such as leucine, valine, and phenylalanine. Water’s tenden- cy to hydrophobically exclude nonpolar molecules literally shoves the nonpolar portions of the amino acid chain into the protein’s interior. This posi- tions the nonpolar amino acids in close contact with one another, leaving little empty space inside. Polar and charged amino acids are restricted to the surface of the protein except for the few that play key functional roles. Levels of Protein Structure The structure of proteins is traditionally discussed in terms of four levels of structure, as primary, sec- ondary, tertiary, and quaternary (figure 3.7). Because of progress in our knowledge of protein structure, two additional levels of structure are increasingly distinguished by molecular biologists: motifs and do- mains. Because these latter two elements play im- portant roles in coming chapters, we introduce them here. Primary Structure. The specific amino acid se- quence of a protein is its primary structure. This sequence is determined by the nucleotide se- quence of the gene that encodes the protein. Be- cause the R groups that distinguish the various amino acids play no role in the peptide backbone of proteins, a protein can consist of any sequence of amino acids. Thus, a protein containing 100 amino acids could form any of 20 100 different ami- no acid sequences (that’s the same as 10 130 , or 1 42 Part I The Origin of Living Things N N N N N H H H H H C C C C O O O C C C C C C O C O N N H H C O C C O C H N N H O O C C C β-pleated sheet α helix C OHO N HH Tertiary structure Secondary structure Primary structure Quaternary structure (c) (b) (a) (d) H H H H H H H H H HO O O O O O O OO O C C C C C C C C C C C C C C C C C C C C C CN N N N N N N N N N FIGURE 3.7 Levels of protein structure. The amino acid sequence of a protein is called its (a) primary structure. Hydrogen bonds form between nearby amino acids, producing (b) fold-backs called beta-pleated sheets and coils called alpha helices. These fold-backs and coils constitute the protein’s secondary structure. A globular protein folds up on itself further to assume a three- dimensional (c) tertiary structure. Many proteins aggregate with other polypeptide chains in clusters; this clustering is called the (d) quaternary structure of the protein. followed by 130 zeros—more than the number of atoms known in the universe). This is an important property of proteins because it permits such great diversity. Secondary Structure. The amino acid side groups are not the only portions of proteins that form hydrogen bonds. The —COOH and —NH 2 groups of the main chain also form quite good hydrogen bonds—so good that their interactions with water might be expected to offset the tendency of nonpolar sidegroups to be forced into the protein interior. Inspection of the protein structures deter- mined by X-ray diffraction reveals why they don’t—the polar groups of the main chain form hydrogen bonds with each other! Two patterns of H bonding occur. In one, hy- drogen bonds form along a single chain, linking one amino acid to another farther down the chain. This tends to pull the chain into a coil called an alpha (α) helix. In the other pattern, hydrogen bonds occur across two chains, linking the amino acids in one chain to those in the other. Often, many parallel chains are linked, forming a pleated, sheet- like structure called a β-pleated sheet. The folding of the amino acid chain by hydrogen bonding into these charac- teristic coils and pleats is called a protein’s secondary structure. Motifs. The elements of secondary structure can combine in proteins in characteristic ways called motifs, or some- times “supersecondary structure.” One very common motif is the β α β motif, which creates a fold or crease; the so- called “Rossmann fold” at the core of nucleotide binding sites in a wide variety of proteins is a β α β α β motif. A sec- ond motif that occurs in many proteins is the β barrel, a β sheet folded around to form a tube. A third type of motif, the α turn α motif, is important because many proteins use it to bind the DNA double helix. Tertiary Structure. The final folded shape of a globular protein, which positions the various motifs and folds non- polar side groups into the interior, is called a protein’s ter- tiary structure. A protein is driven into its tertiary structure by hydrophobic interactions with water. The final folding of a protein is determined by its primary structure—by the chemical nature of its side groups. Many proteins can be fully unfolded (“denatured”) and will spontaneously refold back into their characteristic shape. The stability of a protein, once it has folded into its 3-D shape, is strongly influenced by how well its interior fits together. When two nonpolar chains in the interior are in very close proximity, they experience a form of molecular attraction called van der Waal’s forces. Individually quite weak, these forces can add up to a strong attraction when many of them come into play, like the combined strength of hundreds of hooks and loops on a strip of Velcro. They are effective forces only over short distances, however; there are no “holes” or cavities in the interior of proteins. That is why there are so many different nonpolar amino acids (alanine, valine, leucine, isoleucine). Each has a dif- ferent-sized R group, allowing very precise fitting of non- polar chains within the protein interior. Now you can un- derstand why a mutation that converts one nonpolar ami- no acid within the protein interior (alanine) into another (leucine) very often disrupts the protein’s stability; leucine is a lot bigger than alanine and disrupts the precise way the chains fit together within the protein interior. A change in even a single amino acid can have profound effects on pro- tein shape and can result in loss or altered function of the protein. Domains. Many proteins in your body are encoded within your genes in functional sections called exons (exons will be discussed in detail in chapter 15). Each exon-encoded sec- tion of a protein, typically 100 to 200 amino acids long, folds into a structurally independent functional unit called a domain. As the polypeptide chain folds, the domains fold into their proper shape, each more-or-less independent of the others. This can be demonstrated experimentally by ar- tificially producing the fragment of polypeptide that forms the domain in the intact protein, and showing that the frag- ment folds to form the same structure as it does in the intact protein. A single polypeptide chain connects the domains of a protein, like a rope tied into several adjacent knots. Often the domains of a protein have quite separate functions—one domain of an enzyme might bind a cofactor, for example, and another the enzyme’s substrate. Quaternary Structure. When two or more polypeptide chains associate to form a functional protein, the individ- ual chains are referred to as subunits of the protein. The subunits need not be the same. Hemoglobin, for example, is a protein composed of two α-chain subunits and two β- chain subunits. A protein’s subunit arrangement is called its quaternary structure. In proteins composed of sub- units, the interfaces where the subunits contact one an- other are often nonpolar, and play a key role in transmit- ting information between the subunits about individual subunit activities. A change in the identity of one of these amino acids can have profound effects. Sickle cell hemoglobin is a mutation that alters the identity of a single amino acid at the corner of the β subunit from polar glutamate to nonpolar valine. Putting a nonpolar amino acid on the surface creates a “sticky patch” that causes one hemoglobin molecule to stick to another, forming long nonfunctional chains and leading to the cell sickling characteristic of this hereditary disorder. Protein structure can be viewed at six levels: 1. the amino acid sequence, or primary structure; 2. coils and sheets, called secondary structure; 3. folds or creases, called motifs; 4. the three-dimensional shape, called tertiary structure; 5. functional units, called domains; and 6. individual polypeptide subunits associated in a quaternary structure. Chapter 3 The Chemical Building Blocks of Life 43 How Proteins Fold Into Their Functional Shape How does a protein fold into a specific shape? Nonpolar amino acids play a key role. Until recently, investigators thought that newly made proteins fold spontaneously as hydrophobic interac- tions with water shove nonpolar amino acids into the protein interior. We now know this is too simple a view. Protein chains can fold in so many different ways that trial and error would simply take too long. In addition, as the open chain folds its way toward its final form, nonpolar “sticky” interior portions are exposed during intermediate stages. If these intermediate forms are placed in a test tube in the same protein environ- ment that occurs in a cell, they stick to other unwanted protein partners, form- ing a gluey mess. Chaperonins How do cells avoid this? A vital clue came in studies of unusual mutations that prevented viruses from replicating in E. coli bacterial cells—it turned out the virus proteins could not fold prop- erly! Further study revealed that nor- mal cells contain special proteins called chaperonins that help new proteins fold correctly (figure 3.8). When the E. coli gene encoding its chaperone pro- tein is disabled by mutation, the bacte- ria die, clogged with lumps of incor- rectly folded proteins. Fully 30% of the bacteria’s proteins fail to fold to the right shape. Molecular biologists have now identified more than 17 kinds of proteins that act as molecular chaperones. Many are heat shock proteins, produced in greatly elevated amounts if a cell is exposed to elevated temperature; high temperatures cause proteins to unfold, and heat shock chaperonins help the cell’s proteins refold. There is considerable controversy about how chaper- onins work. It was first thought that they provided a pro- tected environment within which folding could take place unhindered by other proteins, but it now seems more like- ly that chaperonins rescue proteins that are caught in a wrongly folded state, giving them another chance to fold correctly. When investigators “fed” a deliberately mis- folded protein called malate dehydrogenase to chaper- onins, the protein was rescued, refolding to its active shape. Protein Folding and Disease There are tantalizing suggestions that chaperone protein deficiencies may play a role in certain diseases by failing to facilitate the intricate folding of key proteins. Cystic fibrosis is a hereditary disorder in which a mutation disables a pro- tein that plays a vital part in moving ions across cell mem- branes. In at least some cases, the vital membrane protein appears to have the correct amino acid sequence, but fails to fold to its final form. It has also been speculated that chaper- one deficiency may be a cause of the protein clumping in brain cells that produces the amyloid plaques characteristic of Alzheimer’s disease. Proteins called chaperones aid newly produced proteins to fold properly. 44 Part I The Origin of Living Things Correctly folded protein Unfolded protein Chaperone proteins present Chaperone proteins absent Unfolded Incorrectly folded Enzyme degradation of protein FIGURE 3.8 A current model of protein folding. A newly synthesized protein rapidly folds into characteristic motifs composed of H9251 helices and H9252 sheets, but these elements of structure are only roughly aligned in an open conformation. Subsequent folding occurs more slowly, by trial and error. This process is aided by chaperone proteins, which appear to recognize improperly folded proteins and unfold them, giving them another chance to fold properly. Eventually, if proper folding is not achieved, the misfolded protein is degraded by proteolytic enzymes. How Proteins Unfold If a protein’s environment is altered, the protein may change its shape or even unfold. This pro- cess is called denaturation. Proteins can be de- natured when the pH, temperature, or ionic concentration of the surrounding solution is changed. When proteins are denatured, they are usually rendered biologically inactive. This is particularly significant in the case of enzymes. Because practically every chemical reaction in a living organism is catalyzed by a specific en- zyme, it is vital that a cell’s enzymes remain functional. That is the rationale behind tradi- tional methods of salt-curing and pickling: prior to the ready availability of refrigerators and freezers, the only practical way to keep microor- ganisms from growing in food was to keep the food in a solution containing high salt or vine- gar concentrations, which denatured the en- zymes of microorganisms and kept them from growing on the food. Most enzymes function within a very narrow range of physical parameters. Blood-borne en- zymes that course through a human body at a pH of about 7.4 would rapidly become dena- tured in the highly acidic environment of the stomach. On the other hand, the protein- degrading enzymes that function at a pH of 2 or less in the stomach would be denatured in the basic pH of the blood. Similarly, organisms that live near oceanic hydrothermal vents have enzymes that work well at the temperature of this extreme environment (over 100°C). They cannot survive in cooler waters, because their en- zymes would denature at lower temperatures. Any given organism usually has a “tolerance range” of pH, tempera- ture, and salt concentration. Within that range, its enzymes maintain the proper shape to carry out their biological functions. When a protein’s normal environment is reestablished after denaturation, a small protein may spontaneously re- fold into its natural shape, driven by the interactions be- tween its nonpolar amino acids and water (figure 3.9). Larger proteins can rarely refold spontaneously because of the complex nature of their final shape. It is important to distinguish denaturation from dissociation. The four subunits of hemoglobin (figure 3.10) may dissociate into four individual molecules (two α-globin and two β- globin) without denaturation of the folded globin pro- teins, and will readily reassume their four-subunit quater- nary structure. Every globular protein has a narrow range of conditions in which it folds properly; outside that range, proteins tend to unfold. Chapter 3 The Chemical Building Blocks of Life 45 Reducin agent —N—C— H H O CH 2 — — — — — Disulfide —C—C— — — —— O H H CH 2 — — S — S — Cooling or removal of urea Heating or addition of urea Unfolded ribonuclease Reduced ribonuclease Native ribonuclease FIGURE 3.9 Primary structure determines tertiary structure. When the protein ribonuclease is treated with reducing agents to break the covalent disulfide bonds that cross-link its chains, and then placed in urea or heated, the protein denatures (unfolds) and loses its enzymatic activity. Upon cooling or removal of urea, it refolds and regains its enzymatic activity. This demonstrates that no information but the amino acid sequence of the protein is required for proper folding: the primary structure of the protein determines its tertiary structure. Beta chains Heme group Alpha chains FIGURE 3.10 The four subunits of hemoglobin. The hemoglobin molecule is made of four globin protein subunits, informally referred to as polypeptide chains. The two lower α chains, identical α-globin proteins, are shaded pink; the two upper β chains, identical β- globin proteins, are shaded blue. Information Molecules The biochemical activity of a cell depends on production of a large number of proteins, each with a specific sequence. The ability to produce the correct proteins is passed be- tween generations of organisms, even though the protein molecules themselves are not. Nucleic acids are the information storage devices of cells, just as disks or tapes store the information that com- puters use, blueprints store the information that builders use, and road maps store the information that tourists use. There are two varieties of nucleic acids: deoxyribonucleic acid (DNA; figure 3.11) and ribonucleic acid (RNA). The way in which DNA encodes the information used to as- semble proteins is similar to the way in which the letters on a page encode information (see chapter 14). Unique among macromolecules, nucleic acids are able to serve as templates to produce precise copies of themselves, so that the information that specifies what an organism is can be copied and passed down to its descendants. For this rea- son, DNA is often referred to as the hereditary material. Cells use the alternative form of nucleic acid, RNA, to read the cell’s DNA-encoded information and direct the synthesis of proteins. RNA is similar to DNA in structure and is made as a transcripted copy of portions of the DNA. This transcript passes out into the rest of the cell, where it serves as a blueprint specifying a protein’s amino acid sequence. This process will be described in detail in chapter 15. “Seeing” DNA DNA molecules cannot be seen with an optical micro- scope, which is incapable of resolving anything smaller than 1000 atoms across. An electron microscope can image structures as small as a few dozen atoms across, but still cannot resolve the individual atoms of a DNA strand. This limitation was finally overcome in the last decade with the introduction of the scanning-tunneling micro- scope (figure 3.12). How do these microscopes work? Imagine you are in a dark room with a chair. To determine the shape of the chair, you could shine a flashlight on it, so that the light bounces off the chair and forms an image on your eye. That’s what optical and electron microscopes do; in the lat- ter, the “flashlight” emits a beam of electrons instead of light. You could, however, also reach out and feel the chair’s surface with your hand. In effect, you would be put- ting a probe (your hand) near the chair and measuring how far away the surface is. In a scanning-tunneling microscope, computers advance a probe over the surface of a molecule in steps smaller than the diameter of an atom. 46 Part I The Origin of Living Things 3.3 Nucleic acids store and transfer genetic information. FIGURE 3.11 The first photograph of a DNA molecule. This micrograph, with sketch below, shows a section of DNA magnified a million times! The molecule is so slender that it would take 50,000 of them to equal the diameter of a human hair. FIGURE 3.12 A scanning tunneling micrograph of DNA (false color; 2,000,000×). The micrograph shows approximately three turns of the DNA double helix (see figure 3.15). The Structure of Nucleic Acids Nucleic acids are long polymers of repeating subunits called nucleotides. Each nucleotide consists of three components: a five-carbon sugar (ribose in RNA and deoxyribose in DNA); a phosphate (—PO 4 ) group; and an organic nitrogen- containing base (figure 3.13). When a nucleic acid polymer forms, the phosphate group of one nucleotide binds to the hydroxyl group of another, releasing water and forming a phosphodiester bond. A nucleic acid, then, is simply a chain of five-carbon sugars linked together by phosphodi- ester bonds with an organic base protruding from each sugar (figure 3.14). Two types of organic bases occur in nucleotides. The first type, purines, are large, double-ring molecules found in both DNA and RNA; they are adenine (A) and guanine (G). The second type, pyrimidines, are smaller, single-ring molecules; they include cytosine (C, in both DNA and RNA), thymine (T, in DNA only), and uracil (U, in RNA only). Chapter 3 The Chemical Building Blocks of Life 47 Phosphate group Sugar Nitrogenous base N O 4 5 1 32 P CH 2 O – O O – OH R OH in RNA H in DNA O FIGURE 3.13 Structure of a nucleotide. The nucleotide subunits of DNA and RNA are made up of three elements: a five-carbon sugar, an organic nitrogenous base, and a phosphate group. 5H11032 3H11032 P P P P OH 5-carbon sugar Nitrogenous base Phosphate group Phosphodiester bond HC C NC H N C NH 2 Adenine H 2 NC C N N N C H N C CH O O O O O Guanine H N N CH OC C NC H N C NH 2 Cytosine (both DNA and RNA) Thymine (DNA only) Uracil (RNA only) O CC NC H N C O H H H CH 3 H O CC NC H N C O H H H P U R I N E S P Y R I M I D I N E S (a) (b) FIGURE 3.14 The structure of a nucleic acid and the organic nitrogen-containing bases. (a) In a nucleic acid, nucleotides are linked to one another via phosphodiester bonds, with organic bases protruding from the chain. (b) The organic nitrogenous bases can be either purines or pyrimidines. In DNA, thymine replaces the uracil found in RNA. DNA Organisms encode the information specifying the amino acid sequences of their proteins as sequences of nu- cleotides in the DNA. This method of encoding information is very sim- ilar to that by which the sequences of letters encode information in a sentence. While a sentence written in English consists of a combination of the 26 different letters of the al- phabet in a specific order, the code of a DNA molecule consists of dif- ferent combinations of the four types of nucleotides in specific se- quences such as CGCTTACG. The information encoded in DNA is used in the everyday metabolism of the organism and is passed on to the or- ganism’s descendants. DNA molecules in organisms ex- ist not as single chains folded into complex shapes, like proteins, but rather as double chains. Two DNA polymers wind around each other like the outside and inside rails of a circular staircase. Such a winding shape is called a helix, and a helix composed of two chains winding about one another, as in DNA, is called a double helix. Each step of DNA’s helical staircase is a base- pair, consisting of a base in one chain attracted by hydrogen bonds to a base opposite it on the other chain. These hydrogen bonds hold the two chains together as a duplex (figure 3.15). The base-pairing rules are rigid: adenine can pair only with thymine (in DNA) or with uracil (in RNA), and cytosine can pair only with guanine. The bases that partici- pate in base-pairing are said to be complementary to each other. Addi- tional details of the structure of DNA and how it interacts with RNA in the production of proteins are presented in chapters 14 and 15. RNA RNA is similar to DNA, but with two major chemical differences. First, RNA molecules contain ribose sugars in which the number 2 carbon is bonded to a hydroxyl group. In DNA, this hydroxyl group is replaced by a hy- drogen atom. Second, RNA molecules utilize uracil in 48 Part I The Origin of Living Things O OH 3H11032 end 5H11032 end O O O G C P O O O O O O O P P P P P P P P P C C G G A A T T Sugar-phosphate "backbone" Hydrogen bonds between nitrogenous bases Phosphodiester bond FIGURE 3.15 The structure of DNA. Hydrogen bond formation (dashed lines) between the organic bases, called base-pairing, causes the two chains of a DNA duplex to bind to each other and form a double helix. place of thymine. Uracil has the same structure as thy- mine, except that one of its carbons lacks a methyl (— CH 3 ) group. Transcribing the DNA message into a chemically differ- ent molecule such as RNA allows the cell to tell which is the original information storage molecule and which is the transcript. DNA molecules are always double-stranded (ex- cept for a few single-stranded DNA viruses that will be dis- cussed in chapter 33), while the RNA molecules tran- scribed from DNA are typically single-stranded (figure 3.16). Although there is no chemical reason why RNA can- not form double helices as DNA does, cells do not possess the enzymes necessary to assemble double strands of RNA, as they do for DNA. Using two different molecules, one single-stranded and the other double-stranded, separates the role of DNA in storing hereditary information from the role of RNA in using this information to specify protein structure. Which Came First, DNA or RNA? The information necessary for the synthesis of proteins is stored in the cell’s double-stranded DNA base sequences. The cell uses this information by first making an RNA transcript of it: RNA nucleotides pair with complementary DNA nucleotides. By storing the information in DNA while using a complementary RNA sequence to actually direct protein synthesis, the cell does not expose the information- encoding DNA chain to the dangers of single-strand cleav- age every time the information is used. Therefore, DNA is thought to have evolved from RNA as a means of preserv- ing the genetic information, protecting it from the ongo- ing wear and tear associated with cellular activity. This ge- netic system has come down to us from the very beginnings of life. The cell uses the single-stranded, short-lived RNA tran- script to direct the synthesis of a protein with a specific se- quence of amino acids. Thus, the information flows from DNA to RNA to protein, a process that has been termed the “central dogma” of molecular biology. ATP In addition to serving as subunits of DNA and RNA, nu- cleotide bases play other critical roles in the life of a cell. For example, adenine is a key component of the molecule adenosine triphosphate (ATP; figure 3.17), the energy cur- rency of the cell. It also occurs in the molecules nicotina- mide adenine dinucleotide (NAD + ) and flavin adenine dinu- cleotide (FAD + ), which carry electrons whose energy is used to make ATP. A nucleic acid is a long chain of five-carbon sugars with an organic base protruding from each sugar. DNA is a double-stranded helix that stores hereditary information as a specific sequence of nucleotide bases. RNA is a single-stranded molecule that transcribes this information to direct protein synthesis. Chapter 3 The Chemical Building Blocks of Life 49 P P P P P P P P P P P P P P P P P P P P P P DNA Deoxyribose-phosphate backbone Bases Hydrogen bonding occurs between base-pairs RNA Ribose-phosphate backbone Bases G C G G G C C T A A A A A A T TT G C U U FIGURE 3.16 DNA versus RNA. DNA forms a double helix, uses deoxyribose as the sugar in its sugar-phosphate backbone, and utilizes thymine among its nitrogenous bases. RNA, on the other hand, is usually single-stranded, uses ribose as the sugar in its sugar-phosphate backbone, and utilizes uracil in place of thymine. Triphosphate group 5-carbon sugar Nitrogenous base (adenine) O 4 5 1 32 P CH 2 O O O – P O O O – P O – O O – OH OH O NH 2 N N N N FIGURE 3.17 ATP. Adenosine triphosphate (ATP) contains adenine, a five- carbon sugar, and three phosphate groups. This molecule serves to transfer energy rather than store genetic information. Lipids are a loosely defined group of molecules with one main characteristic: they are insoluble in water. The most familiar lipids are fats and oils. Lipids have a very high pro- portion of nonpolar carbon-hydrogen (C—H) bonds, and so long-chain lipids cannot fold up like a protein to sequester their nonpolar portions away from the surrounding aqueous environment. Instead, when placed in water many lipid mol- ecules will spontaneously cluster together and expose what polar groups they have to the surrounding water while se- questering the nonpolar parts of the molecules together within the cluster. This spontaneous assembly of lipids is of paramount importance to cells, as it underlies the structure of cellular membranes. Phospholipids Form Membranes Phospholipids are among the most important molecules of the cell, as they form the core of all biological membranes. An individual phospholipid is a composite molecule, made up of three kinds of subunits: 1. Glycerol, a three-carbon alcohol, with each carbon bearing a hydroxyl group. Glycerol forms the back- bone of the phospholipid molecule. 2. Fatty acids, long chains of C—H bonds (hydrocarbon chains) ending in a carboxyl (—COOH) group. Two fatty acids are attached to the glycerol backbone in a phospholipid molecule. 3. Phosphate group, attached to one end of the glycerol. The charged phosphate group usually has a charged organic molecule linked to it, such as choline, etha- nolamine, or the amino acid serine. The phospholipid molecule can be thought of as having a polar “head” at one end (the phosphate group) and two long, very nonpolar “tails” at the other. In water, the non- polar tails of nearby phospholipids aggregate away from the water, forming two layers of tails pointed toward each oth- er—a lipid bilayer (figure 3.18). Lipid bilayers are the basic framework of biological membranes, discussed in detail in chapter 6. H | H—C—Fatty acid | H—C—Fatty acid | H—C—Phosphate group | H Because the C—H bonds in lipids are very nonpolar, they are not water-soluble, and aggregate together in water. This kind of aggregation by phospholipids forms biological membranes. 50 Part I The Origin of Living Things 3.4 Lipids make membranes and store energy. Hydrophobic "tails" Hydrophilic "heads" Hydrophobic region Hydrophilic region Hydrophilic region Oil Water Water Water (a) (b) FIGURE 3.18 Phospholipids. (a) At an oil-water interface, phospholipid molecules will orient so that their polar (hydrophilic) heads are in the polar medium, water, and their nonpolar (hydrophobic) tails are in the nonpolar medium, oil. (b) When surrounded by water, phospholipid molecules arrange themselves into two layers with their heads extending outward and their tails inward. Fats and Other Kinds of Lipids Fats are another kind of lipid, but unlike phospholipids, fat molecules do not have a polar end. Fats consist of a glycerol molecule to which is attached three fatty acids, one to each carbon of the glycerol backbone. Because it contains three fatty acids, a fat molecule is called a triglyceride, or, more properly, a triacylglycerol (figure 3.19). The three fatty acids of a triglyceride need not be identical, and often they differ markedly from one another. Organisms store the en- ergy of certain molecules for long periods in the many C—H bonds of fats. Because triglyceride molecules lack a polar end, they are not soluble in water. Placed in water, they spontaneously clump together, forming fat globules that are very large rel- ative to the size of the individual molecules. Because fats are insoluble, they can be deposited at specific locations within an organism. Storage fats are one kind of lipid. Oils such as olive oil, corn oil, and coconut oil are also lipids, as are waxes such as beeswax and earwax (see table 3.1). The hydrocarbon chains of fatty acids vary in length; the most common are even- numbered chains of 14 to 20 carbons. If all of the internal carbon atoms in the fatty acid chains are bonded to at least two hydrogen atoms, the fatty acid is said to be saturated, because it contains the maximum possible number of hydro- gen atoms (figure 3.20). If a fatty acid has double bonds be- tween one or more pairs of successive carbon atoms, the fatty acid is said to be unsaturated. If a given fatty acid has more than one double bond, it is said to be polyunsaturat- ed. Fats made from polyunsaturated fatty acids have low melting points because their fatty acid chains bend at the double bonds, preventing the fat molecules from aligning closely with one another. Consequently, a polyunsaturated fat such as corn oil is usually liquid at room temperature and is called an oil. In contrast, most saturated fats such as those in butter are solid at room temperature. Organisms contain many other kinds of lipids besides fats (see figure 3.19). Terpenes are long-chain lipids that are components of many biologically important pigments, such as chlorophyll and the visual pigment retinal. Rubber is also a terpene. Steroids, another type of lipid found in mem- branes, are composed of four carbon rings. Most animal cell membranes contain the steroid cholesterol. Other ste- roids, such as testosterone and estrogen, function in multi- cellular organisms as hormones. Prostaglandins are a group of about 20 lipids that are modified fatty acids, with two nonpolar “tails” attached to a five-carbon ring. Prostaglan- dins act as local chemical messengers in many vertebrate tissues. Cells contain many kinds of molecules in addition to membrane phospholipids that are not soluble in water. Chapter 3 The Chemical Building Blocks of Life 51 (a) Phospholipid (phosphatidyl choline) HO (c) Terpene (citronellol) (d) Steroid (cholesterol) OP O – O O C CH 3 CH 3 OH CH 3 CH 3 CH CH 2 CH CH 2 CH 2 (CH 2 ) 14 CH 3 CH 3 CO (CH 2 ) 7 (CH 2 ) 7 CH CHCO CH 2 CH 3 CH 3 CH 3 N + H 3 CCH 2 CH 2 CH 2 CH CH 3 CH 3 CH 2 CH 2 CH 2 CH 2 CH CH 2 CH 2 O O H H CHO CHO CHO C O C H H C H H C H H C H H C H H H C O C H H C H H C H H C H H C H H H C O C H H C H H C H H C H H C H H H (b) Triacylglycerol molecule FIGURE 3.19 Lipids. These structures represent four major classes of biologically important lipids: (a) phospholipids, (b) triacylglycerols (triglycerides), (c) terpenes, and (d) steroids. Fats as Food Most fats contain over 40 carbon atoms. The ratio of energy- storing C—H bonds to carbon atoms in fats is more than twice that of carbohydrates (see next section), making fats much more efficient molecules for storing chemical energy. On the average, fats yield about 9 kilocalories (kcal) of chemical energy per gram, as compared with somewhat less than 4 kcal per gram for carbohydrates. All fats produced by animals are saturated (except some fish oils), while most plant fats are unsaturated. The excep- tions are the tropical oils (palm oil and coconut oil), which are saturated despite their fluidity at room temperature. It is possible to convert an oil into a solid fat by adding hy- drogen. Peanut butter sold in stores is usually artificially hydrogenated to make the peanut fats solidify, preventing them from separating out as oils while the jar sits on the store shelf. However, artificially hydrogenating unsaturat- ed fats seems to eliminate the health advantage they have over saturated fats, as it makes both equally rich in C—H bonds. Therefore, it now appears that margarine made from hydrogenated corn oil is no better for your health than butter. When an organism consumes excess carbohydrate, it is converted into starch, glycogen, or fats and reserved for fu- ture use. The reason that many humans gain weight as they grow older is that the amount of energy they need decreas- es with age, while their intake of food does not. Thus, an increasing proportion of the carbohydrate they ingest is available to be converted into fat. A diet rich in fats is one of several factors that are thought to contribute to heart disease, particularly to ath- erosclerosis, a condition in which deposits of fatty tissue called plaque adhere to the lining of blood vessels, blocking the flow of blood. Fragments of plaque, breaking off from a deposit, are a major cause of strokes. Fats are efficient energy-storage molecules because of their high concentration of C—H bonds. 52 Part I The Origin of Living Things O C H C H H C H H C H H C H H C H H C H H C H H C H H C H H C H H C H H C H H C H H C H H C H HHO H C H C H H C H H O C H C H H C H H C H H C H H C H H C H H C H C H H C H C H C H H C H C H C H HO No double bonds between carbon atoms; fatty acid chains fit close together Double bonds present between carbon atoms; fatty acid chains do not fit close together (a) Saturated fat (b) Unsaturated fat FIGURE 3.20 Saturated and unsaturated fats. (a) Palmitic acid, with no double bonds and, thus, a maximum number of hydrogen atoms bonded to the carbon chain, is a saturated fatty acid. Many animal triacylglycerols (fats) are saturated. Because their fatty acid chains can fit closely together, these triacylglycerols form immobile arrays called hard fat. (b) Linoleic acid, with three double bonds and, thus, fewer than the maximum number of hydrogen atoms bonded to the carbon chain, is an unsaturated fatty acid. Plant fats are typically unsaturated. The many kinks the double bonds introduce into the fatty acid chains prevent the triacylglycerols from closely aligning and produce oils, which are liquid at room temperature. Simple Carbohydrates Carbohydrates function as energy-storage molecules as well as structural elements. Some are small, simple molecules, while others form long polymers. Sugars Are Simple Carbohydrates The carbohydrates are a loosely defined group of mole- cules that contain carbon, hydrogen, and oxygen in the molar ratio 1:2:1. Their empirical formula (which lists the atoms in the molecule with subscripts to indicate how many there are of each) is (CH 2 O) n , where n is the number of car- bon atoms. Because they contain many carbon-hydrogen (C—H) bonds, which release energy when they are broken, carbohydrates are well suited for energy storage. Monosaccharides. The simplest of the carbohydrates are the simple sugars, or monosaccharides (Greek mono, “single” + Latin saccharum, “sugar”). Simple sugars may contain as few as three carbon atoms, but those that play the central role in energy storage have six (figure 3.21). The empirical formula of six-carbon sugars is: C 6 H 12 O 6 , or (CH 2 O) 6 Six-carbon sugars can exist in a straight-chain form, but in an aqueous environment they almost always form rings. The most important of these for energy storage is glucose (figure 3.22), a six-carbon sugar which has seven energy- storing C—H bonds. Chapter 3 The Chemical Building Blocks of Life 53 3.5 Carbohydrates store energy and provide building materials. 4 5 1 3 2 H H H HH H OH OH OH O 4 4 4 4 5 5 5 5 6 6 6 1 1 1 1 3 3 2 23 3 2 2 OH H OH O CH 2 OH CH 2 OH CH 2 OH CH 2 OH OH HO O OH OH HO CH 2 OH H H H HO H H H H OH H OH OH OH O H H HO CH 2 OH H H H OH O GalactoseFructoseGlucose RiboseGlyceraldehyde 3-carbon sugar 5-carbon sugars 6-carbon sugars Deoxyribose HH 1 3 2 H H H H OH OH O C C C FIGURE 3.21 Monosaccharides. Monosaccharides, or simple sugars, can contain as few as three carbon atoms and are often used as building blocks to form larger molecules. The five-carbon sugars ribose and deoxyribose are components of nucleic acids (see figure 3.15). The six-carbon sugar glucose is a component of large energy-storage molecules. CH 2 OH CH 2 OH HO H H CCC 324 O H OHH H H H OH H H H H H OH O H O H O H O H O C H 1 O HC 56 H C H H H HO O H HO OH C H CCC 23 4 6 5 3 2 1 4 5 6 1 C H HO H CCO C H HH OH H O H OH C O H H 23 4 5 6 1 H O H O O H O C H O H O H HC H 341256 6 5 4 1 2 3 H CH H O FIGURE 3.22 Structure of the glucose molecule. Glucose is a linear six- carbon molecule that forms a ring shape in solution. The structure of the ring can be represented in many ways; the ones shown here are the most common, with the carbons conventionally numbered (in green) so that the forms can be compared easily. The bold, darker lines represent portions of the molecule that are projecting out of the page toward you—remember, these are three- dimensional molecules! Disaccharides. Many familiar sugars like sucrose are “double sugars,” two monosaccharides joined by a covalent bond (figure 3.23). Called disaccharides, they often play a role in the transport of sugars, as we will discuss shortly. Polysaccharides. Polysaccharides are macromolecules made up of monosaccharide subunits. Starch is a polysac- charide used by plants to store energy. It consists entirely of glucose molecules, linked one after another in long chains. Cellulose is a polysaccharide that serves as a structural building material in plants. It too consists entirely of glucose molecules linked together into chains, and special enzymes are required to break the links. Sugar Isomers Glucose is not the only sugar with the formula C 6 H 12 O 6 . Other common six-carbon sugars such as fructose and ga- lactose also have this same empirical formula (figure 3.24). These sugars are isomers, or alternative forms, of glucose. Even though isomers have the same empirical formula, their atoms are arranged in different ways; that is, their three-dimensional structures are different. These structural differences often account for substantial functional differ- ences between the isomers. Glucose and fructose, for exam- ple, are structural isomers. In fructose, the double-bonded oxygen is attached to an internal carbon rather than to a terminal one. Your taste buds can tell the difference, as fructose tastes much sweeter than glucose, despite the fact that both sugars have the same chemical composition. This structural difference also has an important chemical conse- quence: the two sugars form different polymers. Unlike fructose, galactose has the same bond structure as glucose; the only difference between galactose and glucose is the orientation of one hydroxyl group. Because the hy- droxyl group positions are mirror images of each other, galactose and glucose are called stereoisomers. Again, this seemingly slight difference has important consequences, as this hydroxyl group is often involved in creating polymers with distinct functions, such as starch (energy storage) and cellulose (structural support). Sugars are among the most important energy-storage molecules in organisms, containing many energy-storing C—H bonds. The structural differences among sugar isomers can confer substantial functional differences upon the molecules. 54 Part I The Origin of Living Things Sucrose CH 2 OH CH 2 OH OH HO O CH 2 OH HO OH OH O O Lactose OH OH HO CH 2 OH H H H H H H H H H H H H H OH OH OH O H H O CH 2 OH H H H OH O Galactose Glucose Glucose Fructose FIGURE 3.23 Disaccharides. Sugars like sucrose and lactose are disaccharides, composed of two monosaccharides linked by a covalent bond. C — — — OC — — — O — —— H — C — OH OH — H — C — OH — H HO — C — H C — — O — H — C — OH — H Fructose H — C — OH — —— H — C — OH — H — C — OH — H HO — C — H — H — C — OH — H Glucose HO — C — H — —— H — C — OH — H — C — OH — H HO — C — H — H — C — OH — H Galactose Structural isomer Stereoisomer H — C — FIGURE 3.24 Isomers and stereoisomers. Glucose, fructose, and galactose are isomers with the empirical formula C 6 H 12 O 6 . A structural isomer of glucose, such as fructose, has identical chemical groups bonded to different carbon atoms, while a stereoisomer of glucose, such as galactose, has identical chemical groups bonded to the same carbon atoms but in different orientations. Linking Sugars Together Transport Disaccharides Most organisms transport sugars within their bodies. In humans, the glucose that circulates in the blood does so as a simple monosaccharide. In plants and many other or- ganisms, however, glucose is converted into a transport form before it is moved from place to place within the or- ganism. In such a form it is less readily metabolized (used for energy) during transport. Transport forms of sugars are commonly made by linking two monosaccharides to- gether to form a disaccharide (Greek di, “two”). Disaccha- rides serve as effective reservoirs of glucose because the normal glucose-utilizing enzymes of the organism cannot break the bond linking the two monosaccharide subunits. Enzymes that can do so are typically present only in the tissue where the glucose is to be used. Transport forms differ depending on which monosac- charides link to form the disaccharide. Glucose forms transport disaccharides with itself and many other mono- saccharides, including fructose and galactose. When glu- cose forms a disaccharide with its structural isomer, fruc- tose, the resulting disaccharide is sucrose, or table sugar (figure 3.25a). Sucrose is the form in which most plants transport glucose and the sugar that most humans (and other animals) eat. Sugarcane is rich in sucrose, and so are sugar beets. When glucose is linked to its stereoisomer, galactose, the resulting disaccharide is lactose, or milk sugar. Many mammals supply energy to their young in the form of lac- tose. Adults have greatly reduced levels of lactase, the en- zyme required to cleave lactose into its two monosaccha- ride components, and thus cannot metabolize lactose as efficiently. Most of the energy that is channeled into lac- tose production is therefore reserved for their offspring. Storage Polysaccharides Organisms store the metabolic energy contained in monosaccharides by converting them into disaccharides, such as maltose (figure 3.25b), which are then linked togeth- er into insoluble forms that are deposited in specific storage areas in their bodies. These insoluble polysaccharides are long polymers of monosaccharides formed by dehydration synthesis. Plant polysaccharides formed from glucose are called starches. Plants store starch as granules within chlo- roplasts and other organelles. Because glucose is a key met- abolic fuel, the stored starch provides a reservoir of energy available for future needs. Energy for cellular work can be retrieved by hydrolyzing the links that bind the glucose subunits together. The starch with the simplest structure is amylose, which is composed of many hundreds of glucose molecules linked together in long, unbranched chains. Each linkage occurs between the number 1 carbon of one glucose molecule and the number 4 carbon of another, so that amylose is, in ef- fect, a longer form of maltose. The long chains of amylose tend to coil up in water (figure 3.26a), a property that ren- ders amylose insoluble. Potato starch is about 20% amy- lose. When amylose is digested by a sprouting potato plant (or by an animal that eats a potato), enzymes first break it into fragments of random length, which are more soluble because they are shorter. Baking or boiling potatoes has the same effect, breaking the chains into fragments. Another enzyme then cuts these fragments into molecules of mal- tose. Finally, the maltose is cleaved into two glucose mole- cules, which cells are able to metabolize. Most plant starch, including the remaining 80% of po- tato starch, is a somewhat more complicated variant of amylose called amylopectin (figure 3.26b). Pectins are branched polysaccharides. Amylopectin has short, linear amylose branches consisting of 20 to 30 glucose subunits. Chapter 3 The Chemical Building Blocks of Life 55 CH 2 OH Glucose HO OH OH O CH 2 OH H 2 O + Maltose Glucose HO OH OH OH O CH 2 OH HO OH OH O CH 2 OH O OH OH OH O CH 2 OH Glucose HO OH OH OH OH O CH 2 OH CH 2 OH H 2 O + Sucrose Fructose HO OH HO O CH 2 OH CH 2 OH OH HO O CH 2 OH HO OH OH O O (a) (b) FIGURE 3.25 How disaccharides form. Some disaccharides are used to transport glucose from one part of an organism’s body to another; one example is sucrose (a), which is found in sugarcane. Other disaccharides, such as maltose in grain (b), are used for storage. In some plants these chains are cross- linked. The cross-links create an insol- uble mesh of glucose, which can be de- graded only by another kind of enzyme. The size of the mesh differs from plant to plant; in rice about 100 amylose chains, each with one or two cross-links, forms the mesh. The animal version of starch is gly- cogen. Like amylopectin, glycogen is an insoluble polysaccharide containing branched amylose chains. In glycogen, the average chain length is much greater and there are more branches than in plant starch (figure 3.26c). Hu- mans and other vertebrates store ex- cess food energy as glycogen in the liv- er and in muscle cells; when the demand for energy in a tissue increas- es, glycogen is hydrolyzed to release glucose. Nonfattening Sweets Imagine a kind of table sugar that looks, tastes, and cooks like the real thing, but has no calories or harmful side effects. You could eat mountains of candy made from such sweeteners without gaining weight. As Louis Pas- teur discovered in the late 1800s, most sugars are “right-handed” molecules, in that the hydroxyl group that binds a critical carbon atom is on the right side. However, “left-handed” sugars, in which the hydroxyl group is on the left side, can be made readily in the laboratory. These synthetic sugars are mirror-image chemical twins of the natural form, but the enzymes that break down sugars in the human di- gestive system can tell the difference. To digest a sugar molecule, an enzyme must first grasp it, much like a shoe fitting onto a foot, and all of the body’s enzymes are right-handed! A left-handed sugar doesn’t fit, any more than a shoe for the right foot fits onto a left foot. The Latin word for “left” is levo, and left-handed sugars are called levo-, or 1-sugars. They do not occur in nature except for trace amounts in red algae, snail eggs, and sea- weed. Because they pass through the body without being used, they can let diet-conscious sweet-lovers have their cake and eat it, too. Nor will they contribute to tooth decay because bacteria cannot metabolize them, either. Starches are glucose polymers. Most starches are branched and some are cross-linked. The branching and cross-linking render the polymer insoluble and protect it from degradation. 56 Part I The Origin of Living Things (a) Amylose (b) Amylopectin (c) Glycogen FIGURE 3.26 Storage polysaccharides. Starches are long glucose polymers that store energy in plants. (a) The simplest starches are long chains of maltose called amylose, which tend to coil up in water. (b) Most plants contain more complex starches called amylopectins, which are branched. (c) Animals store glucose in glycogen, which is more extensively branched than amylopectin and contains longer chains of amylose. Structural Carbohydrates While some chains of sugars store en- ergy, others serve as structural material for cells. Cellulose For two glucose molecules to link to- gether, the glucose subunits must be the same form. Glucose can form a ring in two ways, with the hydroxyl group attached to the carbon where the ring closes being locked into place ei- ther below or above the plane of the ring. If below, it is called the alpha form, and if above, the beta form. All of the glucose subunits of the starch chain are alpha-glucose. When a chain of glucose molecules consists of all beta-glucose subunits, a polysaccharide with very different properties results. This structural polysaccharide is cel- lulose, the chief component of plant cell walls (figure 3.27). Cellulose is chemi- cally similar to amylose, with one im- portant difference: the starch-degrading enzymes that occur in most organisms cannot break the bond between two beta-glucose sugars. This is not because the bond is stronger, but rather be- cause its cleavage requires an enzyme most organisms lack. Because cellulose cannot be broken down readily, it works well as a biological structural material and occurs widely in this role in plants. Those few animals able to break down cellulose find it a rich source of energy. Certain vertebrates, such as cows, can digest cellulose by means of bacteria and protists they har- bor in their intestines which provide the necessary enzymes. Chitin The structural building material in insects, many fungi, and certain other organisms is called chitin (figure 3.28). Chitin is a modified form of cellulose with a nitrogen group added to the glucose units. When cross-linked by proteins, it forms a tough, resistant surface material that serves as the hard exoskeleton of arthropods such as insects and crusta- ceans (see chapter 46). Few organisms are able to digest chitin. Structural carbohydrates are chains of sugars that are not easily digested. They include cellulose in plants and chitin in arthropods and fungi. Chapter 3 The Chemical Building Blocks of Life 57 FIGURE 3.27 A journey into wood. This jumble of cellulose fibers (a) is from a yellow pine (Pinus ponderosa) (20×). (b) While starch chains consist of alpha- glucose subunits, (c) cellulose chains consist of beta-glucose subunits. Cellulose fibers can be very strong and are quite resistant to metabolic breakdown, which is one reason why wood is such a good building material. OH CH 2 OH α form of glucose Starch: chain of α-glucose subunits HO OH OH H H HH H OH O OO O O 41 1 1 O O O 4 4 CH 2 OH (b) (c) β form of glucose Cellulose: chain of β-glucose subunits HO OH OH H H H H H O O O O O O O O 41 (a) FIGURE 3.28 Chitin. Chitin, which might be considered to be a modified form of cellulose, is the principal structural element in the external skeletons of many invertebrates, such as this lobster. Chapter 3 Summary Questions Media Resources 3.1 Molecules are the building blocks of life. ? The chemistry of living systems is the chemistry of carbon-containing compounds. ? Carbon’s unique chemical properties allow it to poly- merize into chains by dehydration synthesis, forming the four key biological macromolecules: carbohy- drates, lipids, proteins, and nucleic acids. 58 Part I The Origin of Living Things 1. What types of molecules are formed by dehydration reac- tions? What types of molecules are formed by hydrolysis? ? Organic Chemistry ? Explorations: How Proteins Function ? Proteins ? Student Research: A new Protein in Inserts ? Nucleic Acids ? Lipids ? Experiment: Anfinsen: Amino Acid Sequence Determines Protein Shape ? Carbohydrates 2. How are amino acids linked to form proteins? 3. Explain what is meant by the primary, secondary, tertiary, and quaternary structure of a protein. ? Proteins are polymers of amino acids. ? Because the 20 amino acids that occur in proteins have side groups with different chemical properties, the function and shape of a protein are critically af- fected by its particular sequence of amino acids. 3.2 Proteins perform the chemistry of the cell. 4. What are the three compo- nents of a nucleotide? How are nucleotides linked to form nucle- ic acids? 5. Which of the purines and py- rimidines are capable of forming base-pairs with each other? ? Hereditary information is stored as a sequence of nu- cleotides in a linear polymer called DNA, which ex- ists in cells as a double helix. ? Cells use the information in DNA by producing a complementary single strand of RNA which directs the synthesis of a protein whose amino acid sequence corresponds to the nucleotide sequence of the DNA from which the RNA was transcribed. 3.3 Nucleic acids store and transfer genetic information. 6. What are the two kinds of subunits that make up a fat mole- cule, and how are they arranged in the molecule? 7. Describe the differences be- tween a saturated and an unsat- urated fat. ? Fats are one type of water-insoluble molecules called lipids. ? Fats are molecules that contain many energy-rich C—H bonds and, thus, provide an efficient form of long-term energy storage. ? Types of lipids include phospholipids, fats, terpenes, steroids, and prostaglandins. 3.4 Lipids make membranes and store energy. 3.5 Carbohydrates store energy and provide building materials. ? Carbohydrates store considerable energy in their carbon-hydrogen (C—H) bonds. ? The most metabolically important carbohydrate is glucose, a six-carbon sugar. ? Excess energy resources may be stored in complex sugar polymers called starches (in plants) and glyco- gen (in animals and fungi). 8. What does it mean to say that glucose, fructose, and galactose are isomers? Which two are structural isomers, and how do they differ from each other? Which two are stereoisomers, and how do they differ from each other? http://www.mhhe.com/raven6e http://www.biocourse.com 59 4 The Origin and Early History of Life Concept Outline 4.1 All living things share key characteristics. What Is Life? All known organisms share certain general properties, and to a large degree these properties define what we mean by life. 4.2 There are many ideas about the origin of life. Theories about the Origin of Life. There are both religious and scientific views about the origin of life. This text treats only the latter—only the scientifically testable. Scientists Disagree about Where Life Started. The atmosphere of the early earth was rich in hydrogen, providing a ready supply of energetic electrons with which to build organic molecules. The Miller-Urey Experiment. Experiments attempting to duplicate the conditions of early earth produce many of the key molecules of living organisms. 4.3 The first cells had little internal structure. Theories about the Origin of Cells. The first cells are thought to have arisen spontaneously, but there is little agreement as to the mechanism. The Earliest Cells. The earliest fossils are of bacteria too small to see with the unaided eye. 4.4 The first eukaryotic cells were larger and more complex than bacteria. The First Eukaryotic Cells. Fossils of the first eukaryotic cells do not appear in rocks until 1.5 billion years ago, over 2 billion years after bacteria. Multicellular life is restricted to the four eukaryotic kingdoms of life. Has Life Evolved Elsewhere? It seems probable that life has evolved on other worlds besides our own. The possible presence of life in the warm waters beneath the surface of Europa, a moon of Jupiter, is a source of current speculation. T here are a great many scientists with intriguing ideas that explain how life may have originated on earth, but there is very little that we know for sure. New hypotheses are being proposed constantly, and old ones reevaluated. By the time this text is published, some of the ideas presented here about the origin of life will surely be obsolete. Thus, the contesting ideas are presented in this chapter in an open-ended format, attempting to make clear that there is as yet no one answer to the question of how life originated on earth. Although recent photographs taken by the Hubble Space Telescope have revived controversy about the age of the universe, it seems clear the earth itself was formed about 4.6 billion years ago. The oldest clear evidence of life—mi- crofossils in ancient rock—are 3.5 billion years old. The ori- gin of life seems to have taken just the right combination of physical events and chemical processes (figure 4.1). FIGURE 4.1 The origin of life. The fortuitous mix of physical events and chemical elements at the right place and time created the first living cells on earth. Movement. One of the first things the astronauts might do is observe the blob to see if it moves. Most animals move about (figure 4.2), but movement from one place to another in itself is not diagnostic of life. Most plants and even some animals do not move about, while numerous nonliving objects, such as clouds, do move. The criterion of movement is thus neither neces- sary (possessed by all life) nor sufficient (possessed only by life). Sensitivity. The astronauts might prod the blob to see if it responds. Almost all living things respond to stimuli (figure 4.3). Plants grow toward light, and animals retreat from fire. Not all stimuli produce responses, however. Imagine kicking a redwood tree or singing to a hibernating bear. This criterion, although superior to the first, is still inadequate to define life. Death. The astronauts might attempt to kill the blob. All living things die, while inanimate objects do not. Death is not easily distinguished from disorder, how- ever; a car that breaks down has not died because it was never alive. Death is simply the loss of life, so this is a circular definition at best. Unless one can detect life, death is a meaningless concept, and hence a very inade- quate criterion for defining life. Complexity. Finally, the astronauts might cut up the blob, to see if it is complexly organized. All living things are complex. Even the simplest bacteria 60 Part I The Origin of Living Things The earth formed as a hot mass of molten rock about 4.6 billion years ago. As the earth cooled, much of the water vapor present in its atmosphere condensed into liquid water, which accumulated on the surface in chemically rich oceans. One scenario for the origin of life is that it originated in this dilute, hot smelly soup of ammonia, formaldehyde, formic acid, cyanide, methane, hydrogen sulfide, and organic hy- drocarbons. Whether at the oceans’ edge, in hydrothermal deep-sea vents, or elsewhere, the consensus among re- searchers is that life arose spontaneously from these early waters less than 4 billion years ago. While the way in which this happened remains a puzzle, one cannot escape a certain curiosity about the earliest steps that eventually led to the origin of all living things on earth, including ourselves. How did organisms evolve from the complex molecules that swirled in the early oceans? What Is Life? Before we can address this question, we must first consider what qualifies something as “living.” What is life? This is a difficult question to answer, largely because life itself is not a simple concept. If you try to write a definition of “life,” you will find that it is not an easy task, because of the loose man- ner in which the term is used. Imagine a situation in which two astronauts encounter a large, amorphous blob on the surface of a planet. How would they determine whether it is alive? 4.1 All living things share key characteristics. FIGURE 4.2 Movement. Animals have evolved mechanisms that allow them to move about in their environment. While some animals, like this giraffe, move on land, others move through water or air. FIGURE 4.3 Sensitivity. This father lion is responding to a stimulus: he has just been bitten on the rump by his cub. As far as we know, all organisms respond to stimuli, although not always to the same ones or in the same way. Had the cub bitten a tree instead of its father, the response would not have been as dramatic. contain a bewildering array of molecules, organized into many complex structures. However a computer is also complex, but not alive. Complexity is a necessary criterion of life, but it is not sufficient in itself to iden- tify living things because many complex things are not alive. To determine whether the blob is alive, the astronauts would have to learn more about it. Probably the best thing they could do would be to examine it more carefully and de- termine whether it resembles the organisms we are familiar with, and if so, how. Fundamental Properties of Life As we discussed in chapter 1, all known organisms share cer- tain general properties. To a large degree, these properties define what we mean by life. The following fundamental properties are shared by all organisms on earth. Cellular organization. All organisms consist of one or more cells—complex, organized assemblages of mol- ecules enclosed within membranes (figure 4.4). Sensitivity. All organisms respond to stimuli— though not always to the same stimuli in the same ways. Growth. All living things assimilate energy and use it to grow, a process called metabolism. Plants, algae, and some bacteria use sunlight to create covalent carbon- carbon bonds from CO 2 and H 2 O through photosyn- thesis. This transfer of the energy in covalent bonds is essential to all life on earth. Development. Multicellular organisms undergo systematic gene-directed changes as they grow and mature. Reproduction. All living things reproduce, passing on traits from one generation to the next. Although some organisms live for a very long time, no organism lives forever, as far as we know. Because all organisms die, ongoing life is impossible without reproduction. Regulation. All organisms have regulatory mecha- nisms that coordinate internal processes. Homeostasis. All living things maintain relatively constant internal conditions, different from their envi- ronment. The Key Role of Heredity Are these properties adequate to define life? Is a membrane-enclosed entity that grows and reproduces alive? Not necessarily. Soap bubbles and proteinoid microspheres spontaneously form hollow bubbles that enclose a small volume of water. These spheres can enclose energy-processing molecules, and they may also grow and subdivide. Despite these features, they are certainly not alive. Therefore, the criteria just listed, although necessary for life, are not sufficient to define life. One ingredient is missing—a mechanism for the preserva- tion of improvement. Heredity. All organisms on earth possess a genetic system that is based on the replication of a long, complex molecule called DNA. This mechanism allows for adaptation and evolution over time, also distinguishing characteristics of living things. To understand the role of heredity in our definition of life, let us return for a moment to proteinoid microspheres. When we examine an individual microsphere, we see it at that precise moment in time but learn nothing of its prede- cessors. It is likewise impossible to guess what future droplets will be like. The droplets are the passive prisoners of a changing environment, and it is in this sense that they are not alive. The essence of being alive is the ability to en- compass change and to reproduce the results of change permanently. Heredity, therefore, provides the basis for the great division between the living and the nonliving. Change does not become evolution unless it is passed on to a new generation. A genetic system is the sufficient condi- tion of life. Some changes are preserved because they increase the chances of survival in a hostile world, while others are lost. Not only did life evolve—evolution is the very essence of life. All living things on earth are characterized by cellular organization, heredity, and a handful of other characteristics that serve to define the term life. Chapter 4 The Origin and Early History of Life 61 FIGURE 4.4 Cellular organization (150×). These Paramecia, which are complex, single-celled organisms called protists, have just ingested several yeast cells. The yeasts, stained red in this photograph, are enclosed within membrane-bounded sacs called digestive vacuoles. A variety of other organelles are also visible. Theories about the Origin of Life The question of how life originated is not easy to answer because it is impossible to go back in time and observe life’s beginnings; nor are there any witnesses. There is testi- mony in the rocks of the earth, but it is not easily read, and often it is silent on issues crying out for an- swers. There are, in principle, at least three possibilities: 1. Special creation. Life-forms may have been put on earth by supernatural or divine forces. 2. Extraterrestrial origin. Life may not have originated on earth at all; instead, life may have infected earth from some other planet. 3. Spontaneous origin. Life may have evolved from inanimate matter, as associations among molecules became more and more complex. Special Creation. The theory of special creation, that a divine God created life is at the core of most major religions. The oldest hypothesis about life’s origins, it is also the most widely accepted. Far more Americans, for example, believe that God created life on earth than believe in the other two hypotheses. Many take a more ex- treme position, accepting the biblical account of life’s creation as factually correct. This viewpoint forms the basis for the very unscientific “scientific creationism” viewpoint discussed in chapter 21. Extraterrestrial Origin. The theory of panspermia proposes that meteors or cosmic dust may have carried significant amounts of complex organic molecules to earth, kicking off the evolution of life. Hundreds of thou- sands of meteorites and comets are known to have slammed into the early earth, and recent findings suggest that at least some may have carried organic materials. Nor is life on other planets ruled out. For example, the discovery of liquid water under the surface of Jupiter’s ice-shrouded moon Europa and suggestions of fossils in rocks from Mars lend some credence to this idea. The hypothesis that an early source of carbonaceous material is extraterrestrial is testable, although it has not yet been proven. Indeed, NASA is planning to land on Europa, drill through the surface, and send a probe down to see if there is life. Spontaneous Origin. Most scien- tists tentatively accept the theory of spontaneous origin, that life evolved from inanimate matter. In this view, the force leading to life was selec- tion. As changes in molecules in- creased their stability and caused them to persist longer, these mole- cules could initiate more and more complex associations, culminating in the evolution of cells. Taking a Scientific Viewpoint In this book we will focus on the sec- ond and third possibilities, attempting to understand whether natural forces could have led to the origin of life and, if so, how the process might have occurred. This is not to say that the first possibility is definitely not the correct one. Any one of the three pos- sibilities might be true. Nor do the second and third possibilities preclude religion (a divine agency might have acted via evolution, for example). However, we are limiting the scope of our inquiry to scientific matters, and only the second and third possibilities permit testable hypotheses to be constructed—that is, explanations that can be tested and potentially disproved. In our search for understanding, we must look back to the early times. There are fossils of simple living things, bacteria, in rocks 3.5 billion years old. They tell us that life originated during the first billion years of the history of our planet. As we attempt to determine how this process took place, we will first focus on how organic molecules may have originated (figure 4.5), and then we will consider how those molecules might have become organized into living cells. Panspermia and spontaneous origin are the only testable hypotheses of life’s origin currently available. 62 Part I The Origin of Living Things 4.2 There are many ideas about the origin of life. FIGURE 4.5 Lightning. Before life evolved, the simple molecules in the earth’s atmosphere combined to form more complex molecules. The energy that drove these chemical reactions may have come from lightning and forms of geothermal energy. Scientists Disagree about Where Life Started While most researchers agree that life first appeared as the primitive earth cooled and its rocky crust formed, there is little agreement as to just where this occurred. Did Life Originate at the Ocean’s Edge? The more we learn about earth’s early history, the more likely it seems that earth’s first organisms emerged and lived at very high temperatures. Rubble from the forming solar system slammed into early earth from 4.6 to 3.8 billion years ago, keeping the surface molten hot. As the bombardment slowed down, temperatures dropped. By about 3.8 billion years ago, ocean temperatures are thought to have dropped to a hot 49° to 88°C (120° to 190°F). Between 3.8 and 3.5 billion years ago, life first appeared, promptly after the earth was inhabitable. Thus, as intolerable as early earth’s infernal temperatures seem to us today, they gave birth to life. Very few geochemists agree on the exact composition of the early atmosphere. One popular view is that it contained principally carbon dioxide (CO 2 ) and nitrogen gas (N 2 ), along with significant amounts of water vapor (H 2 O). It is possible that the early atmosphere also contained hydrogen gas (H 2 ) and compounds in which hydrogen atoms were bonded to the other light elements (sulfur, nitrogen, and carbon), producing hydrogen sulfide (H 2 S), ammonia (NH 3 ), and methane (CH 4 ). We refer to such an atmosphere as a reducing atmosphere because of the ample availability of hydrogen atoms and their electrons. In such a reducing atmosphere it would not take as much energy as it would today to form the carbon- rich molecules from which life evolved. The key to this reducing atmosphere hypothesis is the assumption that there was very little oxygen around. In an atmosphere with oxygen, amino acids and sugars react spontaneously with the oxygen to form carbon dioxide and water. Therefore, the building blocks of life, the amino acids, would not last long and the spontaneous formation of complex carbon molecules could not occur. Our atmosphere changed once organisms began to carry out photosynthesis, harnessing the energy in sunlight to split water molecules and form complex carbon molecules, giving off gaseous oxy- gen molecules in the process. The earth’s atmosphere is now approximately 21% oxygen. Critics of the reducing atmosphere hypothesis point out that no carbonates have been found in rocks dating back to the early earth. This suggests that at that time carbon diox- ide was locked up in the atmosphere, and if that was the case, then the prebiotic atmosphere would not have been re- ducing. Another problem for the reducing atmosphere hypothe- sis is that because a prebiotic reducing atmosphere would have been oxygen free, there would have been no ozone. Without the protective ozone layer, any organic compounds that might have formed would have been broken down quickly by ultraviolet radiation. Other Suggestions If life did not originate at the ocean’s edge under the blanket of a reducing atmosphere, where did it originate? Under frozen oceans. One hypothesis proposes that life originated under a frozen ocean, not unlike the one that covers Jupiter’s moon Europa today. All evidence suggests, however, that the early earth was quite warm and frozen oceans quite unlikely. Deep in the earth’s crust. Another hypothesis is that life originated deep in the earth’s crust. In 1988 Gunter Wachtershauser proposed that life might have formed as a by-product of volcanic activity, with iron and nickel sulfide minerals acting as chemical catalysts to recom- bine gases spewing from eruptions into the building blocks of life. In later work he and coworkers were able to use this unusual chemistry to build precursors for amino acids (although they did not actually succeed in making amino acids), and to link amino acids together to form peptides. Critics of this hypothesis point out that the concentration of chemicals used in their experiments greatly exceed what is found in nature. Within clay. Other researchers have proposed the un- usual hypothesis that life is the result of silicate surface chemistry. The surface of clays have positive charges to attract organic molecules, and exclude water, providing a potential catalytic surface on which life’s early chemistry might have occurred. While interesting conceptually, there is little evidence that this sort of process could ac- tually occur. At deep-sea vents. Becoming more popular is the hy- pothesis that life originated at deep-sea hydrothermal vents, with the necessary prebiotic molecules being syn- thesized on metal sulfides in the vents. The positive charge of the sulfides would have acted as a magnet for negatively charged organic molecules. In part, the cur- rent popularity of this hypothesis comes from the new science of genomics, which suggests that the ancestors of today’s prokaryotes are most closely related to the bacte- ria that live on the deep-sea vents. No one is sure whether life originated at the ocean’s edge, under frozen ocean, deep in the earth’s crust, within clay, or at deep-sea vents. Perhaps one of these hypotheses will be proven correct. Perhaps the correct theory has not yet been proposed. When life first appeared on earth, the environment was very hot. All of the spontaneous origin hypotheses assume that the organic chemicals that were the building blocks of life arose spontaneously at that time. How is a matter of considerable disagreement. Chapter 4 The Origin and Early History of Life 63 The Miller-Urey Experiment An early attempt to see what kinds of organic molecules might have been produced on the early earth was carried out in 1953 by Stanley L. Miller and Harold C. Urey. In what has become a classic experiment, they attempted to reproduce the conditions at ocean’s edge under a reducing atmosphere. Even if this assumption proves incorrect— the jury is still out on this—their experiment is critically important, as it ushered in the whole new field of prebi- otic chemistry. To carry out their experiment, they (1) assembled a re- ducing atmosphere rich in hydrogen and excluding gaseous oxygen; (2) placed this atmosphere over liquid water, which would have been present at ocean’s edge; (3) maintained this mixture at a temperature somewhat below 100°C; and (4) simulated lightning by bombarding it with energy in the form of sparks (figure 4.6). They found that within a week, 15% of the carbon origi- nally present as methane gas (CH 4 ) had converted into other simple carbon compounds. Among these compounds were formaldehyde (CH 2 O) and hydrogen cyanide (HCN; figure 4.7). These compounds then combined to form sim- ple molecules, such as formic acid (HCOOH) and urea (NH 2 CONH 2 ), and more complex molecules containing carbon-carbon bonds, including the amino acids glycine and alanine. As we saw in chapter 3, amino acids are the basic build- ing blocks of proteins, and proteins are one of the major kinds of molecules of which organisms are composed. In similar experiments performed later by other scientists, more than 30 different carbon compounds were identified, including the amino acids glycine, alanine, glutamic acid, valine, proline, and aspartic acid. Other biologically impor- tant molecules were also formed in these experiments. For example, hydrogen cyanide contributed to the production of a complex ring-shaped molecule called adenine—one of the bases found in DNA and RNA. Thus, the key mole- cules of life could have formed in the atmosphere of the early earth. The Path of Chemical Evolution A raging debate among biologists who study the origin of life concerns which organic molecules came first, RNA or proteins. Scientists are divided into three camps, those that focus on RNA, protein, or a combination of the two. All three arguments have their strong points. Like the hypothe- ses that try to account for where life originated, these com- peting hypotheses are diverse and speculative. An RNA World. The “RNA world” group feels that with- out a hereditary molecule, other molecules could not have formed consistently. The “RNA world” argument earned support when Thomas Cech at the University of Colorado discovered ribozymes, RNA molecules that can behave as enzymes, catalyzing their own assembly. Recent work has shown that the RNA contained in ribosomes (discussed in chapter 5) catalyzes the chemical reaction that links amino acids to form proteins. Therefore, the RNA in ribosomes also functions as an enzyme. If RNA has the ability to pass on inherited information and the capacity to act like an enzyme, were proteins really needed? A Protein World. The “protein-first” group argues that without enzymes (which are proteins), nothing could replicate at all, heritable or not. The “protein-first” pro- ponents argue that nucleotides, the individual units of nucleic acids such as RNA, are too complex to have formed spontaneously, and certainly too complex to form spontaneously again and again. While there is no doubt that simple proteins are easier to synthesize from abiotic components than nucleotides, both can form in the labo- ratory under the right conditions. Deciding which came first is a chicken-and-egg paradox. In an effort to shed light on this problem, Julius Rebek and a number of 64 Part I The Origin of Living Things Water vapor Condensed liquid with complex molecules Stopcocks for testing samples Condensor Mixture of gases ("primitive atmosphere") Heated water ("ocean") Electrodes discharge sparks (lightning simulation) Water FIGURE 4.6 The Miller-Urey experiment. The apparatus consisted of a closed tube connecting two chambers. The upper chamber contained a mixture of gases thought to resemble the primitive earth’s atmosphere. Electrodes discharged sparks through this mixture, simulating lightning. Condensers then cooled the gases, causing water droplets to form, which passed into the second heated chamber, the “ocean.” Any complex molecules formed in the atmosphere chamber would be dissolved in these droplets and carried to the ocean chamber, from which samples were withdrawn for analysis. other chemists have created synthetic nucleotide-like molecules in the laboratory that are able to replicate. Moving even further, Rebek and his colleagues have cre- ated synthetic molecules that could replicate and “make mistakes.” This simulates mutation, a necessary ingredi- ent for the process of evolution. A Peptide-Nucleic Acid World. Another important and popular theory about the first organic molecules assumes key roles for both peptides and nucleic acids. Because RNA is so complex and unstable, this theory assumes there must have been a pre-RNA world where the peptide-nucleic acid (PNA) was the basis for life. PNA is stable and simple enough to have formed spontaneously, and is also a self- replicator. Molecules that are the building blocks of living organisms form spontaneously under conditions designed to simulate those of the primitive earth. Chapter 4 The Origin and Early History of Life 65 Water Aldehydes Proprionic acid Lactic acid Glycolic acid Succinic acid Glycine Alanine β-Alanine β-Aminobutyric acid α-Aminobutyric acidN-Methylalanine Valine Proline Aspartic acid Glutamic acid Raw materials First group of intermediate products Second group of intermediate products Examples of final products (isomers are boxed) Immunoacetic propionic acid Iminodiacetic acid Norvaline Isovaline Sarcosine Acetic acid Formic acid N-Methylurea Urea Energy Energy Hydrogen cyanide Nitrogen Ammonia Carbon dioxide Carbon monoxide Methane Hydrogen gas Energy FIGURE 4.7 Results of the Miller-Urey experiment. Seven simple molecules, all gases, were included in the original mixture. Note that oxygen was not among them; instead, the atmosphere was rich in hydrogen. At each stage of the experiment, more complex molecules were formed: first aldehydes, then simple acids, then more complex acids. Among the final products, the molecules that are structural isomers of one another are grouped together in boxes. In most cases only one isomer of a compound is found in living systems today, although many may have been produced in the Miller-Urey experiment. Theories about the Origin of Cells The evolution of cells required early organic molecules to assemble into a functional, interdependent unit. Cells, discussed in the next chapter, are essentially little bags of fluid. What the fluid contains depends on the individual cell, but every cell’s contents differ from the environ- ment outside the cell. Therefore, an early cell may have floated along in a dilute “primordial soup,” but its inte- rior would have had a higher concentration of specific organic molecules. Cell Origins: The Importance of Bubbles How did these “bags of fluid” evolve from simple organic molecules? As you can imagine, the answer to this ques- tion is a matter for debate. Scientists favoring an “ocean’s edge” scenario for the origin of life have proposed that bubbles may have played a key role in this evolutionary step. A bubble, such as those produced by soap solutions, is a hollow spherical structure. Certain molecules, partic- ularly those with hydrophobic regions, will spontaneously form bubbles in water. The structure of the bubble shields the hydrophobic regions of the molecules from contact with water. If you have ever watched the ocean surge upon the shore, you may have noticed the foamy froth created by the agitated water. The edges of the primitive oceans were more than likely very frothy places bom- barded by ultraviolet and other ionizing radiation, and ex- posed to an atmosphere that may have contained methane and other simple organic molecules. Oparin’s Bubble Theory The first bubble theory is attributed to Alexander Oparin, a Russian chemist with extraordinary insight. In the mid-1930s, Oparin suggested that the present-day at- mosphere was incompatible with the creation of life. He proposed that life must have arisen from nonliving matter under a set of very different environmental circumstances some time in the distant history of the earth. His was the theory of primary abiogenesis (primary because all liv- ing cells are now known to come from previously living cells, except in that first case). At the same time, J. B. S. Haldane, a British geneticist, was also independently es- pousing the same views. Oparin decided that in order for cells to evolve, they must have had some means of devel- oping chemical complexity, separating their contents from their environment by means of a cell membrane, and concentrating materials within themselves. He termed these early, chemical-concentrating bubblelike structures protobionts. Oparin’s theories were published in English in 1938, and for awhile most scientists ignored them. However, Harold Urey, an astronomer at the University of Chicago, was quite taken with Oparin’s ideas. He con- vinced one of his graduate students, Stanley Miller, to follow Oparin’s rationale and see if he could “create” life. The Urey-Miller experiment has proven to be one of the most significant experiments in the history of science. As a result Oparin’s ideas became better known and more widely accepted. A Host of Bubble Theories Different versions of “bubble theories” have been cham- pioned by numerous scientists since Oparin. The bubbles they propose go by a variety of names; they may be called microspheres, protocells, protobionts, micelles, liposomes, or coacervates, depending on the composition of the bubbles (lipid or protein) and how they form. In all cases, the bubbles are hollow spheres, and they exhibit a variety of cell-like properties. For example, the lipid bubbles called coacervates form an outer boundary with two layers that resembles a biological membrane. They grow by accumu- lating more subunit lipid molecules from the surrounding medium, and they can form budlike projections and divide by pinching in two, like bacteria. They also can contain amino acids and use them to facilitate various acid-base reactions, including the decomposition of glucose. Although they are not alive, they obviously have many of the characteristics of cells. A Bubble Scenario It is not difficult to imagine that a process of chemical evo- lution involving bubbles or microdrops preceded the origin of life (figure 4.8). The early oceans must have contained untold numbers of these microdrops, billions in a spoonful, each one forming spontaneously, persisting for a while, and then dispersing. Some would, by chance, have contained amino acids with side groups able to catalyze growth- promoting reactions. Those microdrops would have survived longer than ones that lacked those amino acids, because the persistence of both proteinoid microspheres and lipid coacervates is greatly increased when they carry out metabolic reactions such as glucose degradation and when they are actively growing. Over millions of years, then, the complex bubbles that were better able to incorporate molecules and energy from the lifeless oceans of the early earth would have tended to persist longer than the others. Also favored would have been the microdrops that could use these molecules to expand in size, growing large enough to divide into “daughter” 66 Part I The Origin of Living Things 4.3 The first cells had little internal structure. microdrops with features similar to those of their “parent” microdrop. The daughter microdrops have the same favor- able combination of characteristics as their parent, and would have grown and divided, too. When a way to facilitate the reliable transfer of new ability from parent to offspring developed, heredity—and life—began. Current Thinking Whether the early bubbles that gave rise to cells were lipid or protein remains an unresolved argument. While it is true that lipid microspheres (coacervates) will form readily in water, there appears to be no mechanism for their heri- table replication. On the other hand, one can imagine a heritable mechanism for protein microspheres. Although protein microspheres do not form readily in water, Sidney Fox and his colleagues at the University of Miami have shown that they can form under dry conditions. The discovery that RNA can act as an enzyme to assem- ble new RNA molecules on an RNA template has raised the interesting possibility that neither coacervates nor pro- tein microspheres were the first step in the evolution of life. Perhaps the first components were RNA molecules, and the initial steps on the evolutionary journey led to increasingly complex and stable RNA molecules. Later, stability might have improved further when a lipid (or possibly protein) microsphere surrounded the RNA. At present, those studying this problem have not arrived at a consensus about whether RNA evolved before or after a bubblelike structure that likely preceded cells. Eventually, DNA took the place of RNA as the replicator in the cell and the storage molecule for genetic information. DNA, because it is a double helix, stores information in a more stable fashion than RNA, which is single-stranded. Little is known about how the first cells originated. Current hypotheses involve chemical evolution within bubbles, but there is no general agreement about their composition, or about how the process occurred. Chapter 4 The Origin and Early History of Life 67 1. Volcanoes erupted under the sea, releasing gases enclosed in bubbles. 2. The gases, concentrated inside the bubbles, reacted to produce simple organic molecules. 3. When the bubbles persisted long enough to rise to the surface, they popped, releasing their contents to the air. 4. Bombarded by the sun's ultraviolet radiation, lightning, and other energy sources, the simple organic molecules released from the bubbles reacted to form more complex organic molecules. 5. The more complex organic molecules fell back into the sea in raindrops. There, they could again be enclosed in bubbles and begin the process again. FIGURE 4.8 A current bubble hypothesis. In 1986 geophysicist Louis Lerman proposed that the chemical processes leading to the evolution of life took place within bubbles on the ocean’s surface. The Earliest Cells What do we know about the earliest life-forms? The fossils found in ancient rocks show an obvious progression from simple to complex organisms, beginning about 3.5 billion years ago. Life may have been present earlier, but rocks of such great antiquity are rare, and fossils have not yet been found in them. Microfossils The earliest evidence of life appears in microfossils, fos- silized forms of microscopic life (figure 4.9). Microfossils were small (1 to 2 micrometers in diameter) and single- celled, lacked external appendages, and had little evidence of internal structure. Thus, they physically resemble present- day bacteria (figure 4.10), although some ancient forms cannot be matched exactly. We call organisms with this sim- ple body plan prokaryotes, from the Greek words meaning “before” and “kernel,” or “nucleus.” The name reflects their lack of a nucleus, a spherical organelle characteristic of the more complex cells of eukaryotes. Judging from the fossil record, eukaryotes did not appear until about 1.5 billion years ago. Therefore, for at least 2 billion years—nearly a half of the age of the earth—bacteria were the only organisms that existed. Ancient Bacteria: Archaebacteria Most organisms living today are adapted to the relatively mild conditions of present-day earth. However, if we look in unusual environments, we encounter organisms that are quite remarkable, differing in form and metabo- lism from other living things. Sheltered from evolution- ary alteration in unchanging habitats that resemble earth’s early environment, these living relics are the sur- viving representatives of the first ages of life on earth. In places such as the oxygen-free depths of the Black Sea or the boiling waters of hot springs and deep-sea vents, we can find bacteria living at very high temperatures without oxygen. These unusual bacteria are called archaebacteria, from the Greek word for “ancient ones.” Among the first to be studied in detail have been the methanogens, or methane-producing bacteria, among the most primitive bacteria that exist today. These organisms are typically simple in form and are able to grow only in an oxygen-free environment; in fact, oxygen poisons them. For this reason they are said to grow “without air,” or anaerobically (Greek an, “without” + aer, “air” + bios, “life”). The methane-producing bacteria convert CO 2 and H 2 into methane gas (CH 4 ). Although primitive, they resemble all other bacteria in having DNA, a lipid cell membrane, an exterior cell wall, and a metabolism based on an energy- carrying molecule called ATP. 68 Part I The Origin of Living Things FIGURE 4.9 Cross-sections of fossil bacteria. These microfossils from the Bitter Springs formation of Australia are of ancient cyanobacteria, far too small to be seen with the unaided eye. In this electron micrograph, the cell walls are clearly evident. FIGURE 4.10 The oldest microfossil. This ancient bacterial fossil, discovered by J. William Schopf of UCLA in 3.5-billion-year-old rocks in western Australia, is similar to present-day cyanobacteria, as you can see by comparing it to figure 4.11. Unusual Cell Structures When the details of cell wall and membrane structure of the methane-producing bacteria were examined, they proved to be different from those of all other bacteria. Ar- chaebacteria are characterized by a conspicuous lack of a protein cross-linked carbohydrate material called peptido- glycan in their cell walls, a key compound in the cell walls of most modern bacteria. Archaebacteria also have unusual lipids in their cell membranes that are not found in any other group of organisms. There are also major differences in some of the fundamental biochemical processes of me- tabolism, different from those of all other bacteria. The methane-producing bacteria are survivors from an earlier time when oxygen gas was absent. Earth’s First Organisms? Other archaebacteria that fall into this classification are some of those that live in very salty environments like the Dead Sea (extreme halophiles—“salt lovers”) or very hot environments like hydrothermal volcanic vents under the ocean (extreme thermophiles—“heat lovers”). Ther- mophiles have been found living comfortably in boiling water. Indeed, many kinds of thermophilic archaebacteria thrive at temperatures of 110°C (230°F). Because these thermophiles live at high temperatures similar to those that may have existed when life first evolved, microbiologists speculate that thermophilic archaebacteria may be relics of earth’s first organisms. Just how different are extreme thermophiles from other organisms? A methane-producing archaebacteria called Methanococcus isolated from deep-sea vents provides a star- tling picture. These bacteria thrive at temperatures of 88°C (185°F) and crushing pressures 245 times greater than at sea level. In 1996 molecular biologists announced that they had succeeded in determining the full nucleotide sequence of Methanococcus. This was possible because archaebacterial DNA is relatively small—it has only 1700 genes, coded in a DNA molecule only 1,739,933 nucleotides long (a human cell has 2000 times more!). The thermophile nucleotide se- quence proved to be astonishingly different from the DNA sequence of any other organism ever studied; fully two- thirds of its genes are unlike any ever known to science be- fore! Clearly these archaebacteria separated from other life on earth a long time ago. Preliminary comparisons to the gene sequences of other bacteria suggest that archaebacte- ria split from other types of bacteria over 3 billion years ago, soon after life began. Eubacteria The second major group of bacteria, the eubacteria, have very strong cell walls and a simpler gene architecture. Most bacteria living today are eubacteria. Included in this group are bacteria that have evolved the ability to capture the en- ergy of light and transform it into the energy of chemical bonds within cells. These organisms are photosynthetic, as are plants and algae. One type of photosynthetic eubacteria that has been im- portant in the history of life on earth is the cyanobacteria, sometimes called “blue-green algae” (figure 4.11). They have the same kind of chlorophyll pigment that is most abundant in plants and algae, as well as other pigments that are blue or red. Cyanobacteria produce oxygen as a result of their photosynthetic activities, and when they appeared at least 3 billion years ago, they played a decisive role in increasing the concentration of free oxygen in the earth’s atmosphere from below 1% to the current level of 21%. As the concentration of oxygen increased, so did the amount of ozone in the upper layers of the atmosphere. The thickening ozone layer afforded protection from most of the ultraviolet radiation from the sun, radiation that is highly destructive to proteins and nucleic acids. Certain cyanobacteria are also responsible for the accu- mulation of massive limestone deposits. All bacteria now living are members of either Archaebacteria or Eubacteria. Chapter 4 The Origin and Early History of Life 69 FIGURE 4.11 Living cyanobacteria. Although not multicellular, these bacteria often aggregate into chains such as those seen here. All fossils more than 1.5 billion years old are generally sim- ilar to one another structurally. They are small, simple cells; most measure 0.5 to 2 micrometers in diameter, and none are more than about 6 micrometers in diameter. These simple cells eventually evolved into larger, more complex forms—the first eukaryotic cells. The First Eukaryotic Cells In rocks about 1.5 billion years old, we begin to see the first microfossils that are noticeably different in appearance from the earlier, simpler forms (figure 4.12). These cells are much larger than bacteria and have internal membranes and thicker walls. Cells more than 10 micrometers in diam- eter rapidly increased in abundance. Some fossilized cells 1.4 billion years old are as much as 60 micrometers in di- ameter; others, 1.5 billion years old, contain what appear to be small, membrane-bound structures. Indirect chemical traces hint that eukaryotes may go as far back as 2.7 billion years, although no fossils as yet support such an early ap- pearance of eukaryotes. These early fossils mark a major event in the evolution of life: a new kind of organism had appeared (figure 4.13). These new cells are called eukaryotes, from the Greek words for “true” and “nucleus,” because they possess an in- ternal structure called a nucleus. All organisms other than the bacteria are eukaryotes. Origin of the Nucleus and ER Many bacteria have infoldings of their outer membranes extending into the cytoplasm and serving as passageways to the surface. The network of internal membranes in 70 Part I The Origin of Living Things 4.4 The first eukaryotic cells were larger and more complex than bacteria. 100 μm FIGURE 4.12 Microfossil of a primitive eukaryote. This multicellular alga is between 900 million and 1 billion years old. Geological evidence Life forms PRECAMBRIAN CAMBRIAN PHANEROZOIC PROTEROZOIC ARCHEAN Appearance of first multicellular organisms Appearance of first eukaryotes Appearance of aerobic (oxygen-using) respiration Appearance of oxygen-forming photosynthesis (cyanobacteria) Appearance of chemoautotrophs (sulfate respiration) Appearance of life (prokaryotes): anaerobic (methane-producing) bacteria and anaerobic (hydrogen sulfide–forming) photosynthesis Formation of the earth Oldest multicellular fossils Oldest compartmentalized fossil cells Disappearance of iron from oceans and formation of iron oxides Oldest definite fossils Oldest dated rocks Millions of years ago 570 600 1500 2500 3500 4500 FIGURE 4.13 The geological timescale. The periods refer to different stages in the evolution of life on earth. The timescale is calibrated by examining rocks containing particular kinds of fossils; the fossils are dated by determining the degree of spontaneous decay of radioactive isotopes locked within rock when it was formed. eukaryotes called endoplasmic reticulum (ER) is thought to have evolved from such infoldings, as is the nuclear en- velope, an extension of the ER network that isolates and protects the nucleus (figure 4.14). Origin of Mitochondria and Chloroplasts Bacteria that live within other cells and perform specific functions for their host cells are called endosymbiotic bacte- ria. Their widespread presence in nature led Lynn Margulis to champion the endosymbiotic theory in the early 1970s. This theory, now widely accepted, suggests that a critical stage in the evolution of eukaryotic cells in- volved endosymbiotic relationships with prokaryotic or- ganisms. According to this theory, energy-producing bacteria may have come to reside within larger bacteria, eventually evolving into what we now know as mitochon- dria. Similarly, photosynthetic bacteria may have come to live within other larger bacteria, leading to the evolution of chloroplasts, the photosynthetic organelles of plants and algae. Bacteria with flagella, long whiplike cellular appendages used for propulsion, may have become sym- biotically involved with nonflagellated bacteria to pro- duce larger, motile cells. The fact that we now witness so many symbiotic relationships lends general support to this theory. Even stronger support comes from the obser- vation that present-day organelles such as mitochondria, chloroplasts, and centrioles contain their own DNA, which is remarkably similar to the DNA of bacteria in size and character. Sexual Reproduction Eukaryotic cells also possess the ability to reproduce sexu- ally, something prokaryotes cannot do effectively. Sexual reproduction is the process of producing offspring, with two copies of each chromosome, by fertilization, the union of two cells that each have one copy of each chromosome. The great advantage of sexual reproduction is that it allows for frequent genetic recombination, which generates the variation that is the raw material for evolution. Not all eu- karyotes reproduce sexually, but most have the capacity to do so. The evolution of meiosis and sexual reproduction (discussed in chapter 12) led to the tremendous explosion of diversity among the eukaryotes. Multicellularity Diversity was also promoted by the development of multi- cellularity. Some single eukaryotic cells began living in as- sociation with others, in colonies. Eventually individual members of the colony began to assume different duties, and the colony began to take on the characteristics of a sin- gle individual. Multicellularity has arisen many times among the eukaryotes. Practically every organism big enough to see with the unaided eye is multicellular, includ- ing all animals and plants. The great advantage of multicel- lularity is that it fosters specialization; some cells devote all of their energies to one task, other cells to another. Few in- novations have had as great an impact on the history of life as the specialization made possible by multicellularity. Chapter 4 The Origin and Early History of Life 71 Infolding of the plasma membrane DNA Cell wall Bacterial cell Prokaryotic ancestor of eukaryotic cells Eukaryotic cell Endoplasmic reticulum (ER) Nuclear envelope Nucleus FIGURE 4.14 Origin of the nucleus and endoplasmic reticulum. Many bacteria today have infoldings of the plasma membrane (see also figure 34.7). The eukaryotic internal membrane system called the endoplasmic reticulum (ER) and the nuclear envelope may have evolved from such infoldings of the plasma membrane encasing prokaryotic cells that gave rise to eukaryotic cells. The Kingdoms of Life Confronted with the great diversity of life on earth today, biologists have attempted to categorize similar organisms in order to better understand them, giving rise to the sci- ence of taxonomy. In later chapters, we will discuss tax- onomy and classification in detail, but for now we can generalize that all living things fall into one of three domains which include six kingdoms (figure 4.15): Kingdom Archaebacteria: Prokaryotes that lack a peptidoglycan cell wall, including the methanogens and extreme halophiles and thermophiles. Kingdom Eubacteria: Prokaryotic organisms with a peptidoglycan cell wall, including cyanobacteria, soil bacteria, nitrogen-fixing bacteria, and pathogenic (disease-causing) bacteria. Kingdom Protista: Eukaryotic, primarily unicellu- lar (although algae are multicellular), photosynthetic or heterotrophic organisms, such as amoebas and paramecia. Kingdom Fungi: Eukaryotic, mostly multicellular (al- though yeasts are unicellular), heterotrophic, usually nonmotile organisms, with cell walls of chitin, such as mushrooms. Kingdom Plantae: Eukaryotic, multicellular, non- motile, usually terrestrial, photosynthetic organisms, such as trees, grasses, and mosses. Kingdom Animalia: Eukaryotic, multicellular, motile, heterotrophic organisms, such as sponges, spiders, newts, penguins, and humans. As more is learned about living things, particularly from the newer evidence that DNA studies provide, scientists will continue to reevaluate the relationships among the king- doms of life. For at least the first 1 billion years of life on earth, all organisms were bacteria. About 1.5 billion years ago, the first eukaryotes appeared. Biologists place living organisms into six general categories called kingdoms. 72 Part I The Origin of Living Things Microsporidia Animals Plants Ciliates Slime molds S. cerevisiae Euglena Diplomonads (Lamblia) EUKARYA E. coli B. subtilus Thermotoga Synechocystis sp. Flavobacteria Green sulfur bacteria Borrelia burgdorferi Aquifex BACTERIA Methano- pyrus Methanococcus jannaschii Halobacterium Halococcus Archaeoglobus Methanobacterium Thermococcus Sulfolobus ARCHAEA (b) (a) FIGURE 4.15 The three domains of life. The kingdoms Archaebacteria and Eubacteria are as different from each other as from eukaryotes, so biologists have assigned them a higher category, a “domain.” (a) A three-domain tree of life based on ribosomal RNA consists of the Eukarya, Bacteria, and Archaea. (b) New analyses of complete genome sequences contradict the rRNA tree, and suggest other arrangements such as this one, which splits the Archaea. Apparently genes hopped from branch to branch as early organisms either stole genes from their food or swapped DNA with their neighbors, even distantly related ones. Has Life Evolved Elsewhere? We should not overlook the possibility that life processes might have evolved in different ways on other planets. A functional genetic system, capable of accumulating and replicating changes and thus of adaptation and evolution, could theoretically evolve from molecules other than car- bon, hydrogen, nitrogen, and oxygen in a different environ- ment. Silicon, like carbon, needs four electrons to fill its outer energy level, and ammonia is even more polar than water. Perhaps under radically different temperatures and pressures, these elements might form molecules as diverse and flexible as those carbon has formed on earth. The universe has 10 20 (100,000,000,000,000,000,000) stars similar to our sun. We don’t know how many of these stars have planets, but it seems increasingly likely that many do. Since 1996, astronomers have been detect- ing planets orbiting distant stars. At least 10% of stars are thought to have planetary systems. If only 1 in 10,000 of these planets is the right size and at the right distance from its star to duplicate the conditions in which life orig- inated on earth, the “life experiment” will have been re- peated 10 15 times (that is, a million billion times). It does not seem likely that we are alone. Ancient Bacteria on Mars? A dull gray chunk of rock collected in 1984 in Antarctica ignited an uproar about ancient life on Mars with the report that the rock contains evidence of possible life. Analysis of gases trapped within small pockets of the rock indicate it is a meteorite from Mars. It is, in fact, the oldest rock known to science—fully 4.5 billion years old. Back then, when this rock formed on Mars, that cold, arid planet was much warmer, flowed with water, and had a carbon dioxide atmosphere—conditions not too different from those that spawned life on earth. When examined with powerful electron microscopes, carbonate patches within the meteorite exhibit what look like microfossils, some 20 to 100 nanometers in length. One hundred times smaller than any known bacteria, it is not clear they actually are fossils, but the resemblance to bacte- ria is striking. Viewed as a whole, the evidence of bacterial life associ- ated with the Mars meteorite is not compelling. Clearly, more painstaking research remains to be done before the discovery can claim a scientific consensus. However, while there is no conclusive evidence of bacterial life associated with this meteorite, it seems very possible that life has evolved on other worlds in addition to our own. Deep-Sea Vents The possibility that life on earth actually originated in the vicinity of deep-sea hydrothermal vents is gaining popular- ity. At the bottom of the ocean, where these vents spewed out a rich froth of molecules, the geological turbulence and radioactive energy battering the land was absent, and things were comparatively calm. The thermophilic archaebacteria found near these vents today are the most ancient group of organisms living on earth. Perhaps the gentler environment of the ocean depths was the actual cradle of life. Other Planets Has life evolved on other worlds within our solar system? There are planets other than ancient Mars with conditions not unlike those on earth. Europa, a large moon of Jupiter, is a promising candidate (figure 4.16). Europa is covered with ice, and photos taken in close orbit in the winter of 1998 re- veal seas of liquid water beneath a thin skin of ice. Additional satellite photos taken in 1999 suggest that a few miles under the ice lies a liquid ocean of water larger than earth’s, warmed by the push and pull of the gravitational attraction of Jupiter’s many large satellite moons. The conditions on Europa now are far less hostile to life than the conditions that existed in the oceans of the primitive earth. In coming decades satellite missions are scheduled to explore this ocean for life. There are so many stars that life may have evolved many times. Although evidence for life on Mars is not compelling, the seas of Europa offer a promising candidate which scientists are eager to investigate. Chapter 4 The Origin and Early History of Life 73 FIGURE 4.16 Is there life elsewhere? Currently the most likely candidate for life elsewhere within the solar system is Europa, one of the many moons of the large planet Jupiter. Chapter 4 Summary Questions Media Resources 4.1 All living things share key characteristics. 74 Part I The Origin of Living Things ?All living things are characterized by cellular organization, growth, reproduction, and heredity. ?Other properties commonly exhibited by living things include movement and sensitivity to stimuli. 1. What characteristics of living things are necessary characteristics (possessed by all living things), and which are sufficient characteristics (possessed only by living things)? ?Of the many explanations of how life might have originated, only the theories of spontaneous and extraterrestrial origins provide scientifically testable explanations. ?Experiments recreating the atmosphere of primitive earth, with the energy sources and temperatures thought to be prevalent at that time, have led to the spontaneous formation of amino acids and other biologically significant molecules. 2. What molecules are thought to have been present in the atmosphere of the early earth? Which molecule that was notably absent then is now a major component of the atmosphere? 4.2 There are many ideas about the origin of life. ? The first cells are thought to have arisen from aggregations of molecules that were more stable and, therefore, persisted longer. ? It has been suggested that RNA may have arisen before cells did, and subsequently became packaged within a membrane. ? Bacteria were the only life-forms on earth for about 1 billion years. At least three kinds of bacteria were present in ancient times: methane utilizers, anaerobic photosynthesizers, and eventually O 2 -forming photosynthesizers. 3. What evidence supports the argument that RNA evolved first on the early earth? What evidence supports the argument that proteins evolved first? 4. What are coacervates, and what characteristics do they have in common with organisms? Are they alive? Why or why not? 5. What were the earliest known organisms like, and when did they appear? What present-day organisms do they resemble? 4.3 The first cells had little internal structure. ? The first eukaryotes can be seen in the fossil record about 1.5 billion years ago. All organisms other than bacteria are their descendants. ? Biologists group all living organisms into six “kingdoms,” each profoundly different from the others. ? The two most ancient kingdoms contain prokaryotes (bacteria); the other four contain eukaryotes. ?There are approximately 10 20 stars in the universe similar to our sun. It is almost certain that life has evolved on planets circling some of them. 6. When did the first eukaryotes appear? By what mechanism are they thought to have evolved from the earlier prokaryotes? 7. What sorts of organisms are contained in each of the six kingdoms of life recognized by biologists? 4.4 The first eukaryotic cells were larger and more complex than bacteria. ? Origin of Life ? Art Quizzes: -Miller-Urey Experiment -Miller-Urey Experiment Results ? Key Events in Earth’s History BIOLOGY RAVEN JOHNSON SIX TH EDITION www.mhhe.com/raven6/resources4.mhtml ? Art Quiz: Current Bubble Hypothesis 75 How Do the Cells of a Growing Plant Know in Which Direction to Elongate? Sometimes questions that seem simple can be devilishly dif- ficult to answer. Imagine, for example, that you are holding a green blade of grass in your hand. The grass blade has been actively growing, its cells dividing and then stretching and elongating as the blade lengthens. Did you ever wonder how the individual cells within the blade of grass know in what direction to grow? To answer this deceptively simple question, we will first need to provide answers to several others. Like Sherlock Holmes following a trail of clues, we must approach the an- swer we seek in stages. Question One. First, we need to ask how a blade of grass is able to grow at all. Plant cells are very different from ani- mal cells in one key respect: every plant cell is encased within a tough cell wall made of cellulose and other tough building materials. This wall provides structural strength and protection to the plant cell, just as armor plate does for a battle tank. But battle tanks can’t stretch into longer shapes! How is a plant cell able to elongate? It works like this. A growing cell first performs a little chemistry to make its wall slightly acidic. The acidity acti- vates enzymes that attack the cell wall from the inside, rear- ranging cellulose cross-links until the wall loses its rigidity. The cell wall is now able to stretch. The cell then sucks in water, creating pressure. Like blowing up a long balloon, the now-stretchable cell elongates. Question Two. In a growing plant organ, like the blade of grass, each growing cell balloons out lengthwise. Stating this more formally, a botanist would say the cell elongates parallel to the axis along which the blade of grass is extend- ing. This observation leads to the second question we must answer: How does an individual plant cell control the di- rection in which it elongates? It works like this. Before the stretchable cell balloons out, tiny microfibrils of cellulose are laid down along its inside sur- face. On a per weight basis, these tiny fibrils have the tensile strength of steel! Arrays of these cellulose microfibrils are organized in bands perpendicular to the axis of elongation, like steel belts. These tough bands reinforce the plant cell wall laterally, so that when the cell sucks in water, there is only one way for the cell to expand—lengthwise, along the axis. Question Three. Now we’re getting somewhere. How are the newly made cellulose microfibrils laid down so that they are oriented correctly, perpendicular to the axis of elongation? It works like this. The complicated enzymic machine that makes the cellulose microfibrils is guided by special guiderails that run like railroad tracks along the interior sur- face. The enzyme complex travels along these guiderails, laying down microfibrils as it goes. The guiderails are con- structed of chainlike protein molecules called microtubules, assembled into overlapping arrays. Botanists call these ar- rays of microtubules associated with the interior of the cell surface “cortical microtubules.” Question Four. But we have only traded one puzzle for another. How are the cortical microtubules positioned cor- rectly, perpendicular to the axis of elongation? It works like this. In newly made cells, the microtubule assemblies are already present, but are not organized. They simply lie about in random disarray. As the cell prepares to elongate by lessening the rigidity of its cell wall, the micro- tubule assemblies become organized into the orderly trans- verse arrays we call cortical microtubules. Question Five. Finally, we arrive at the question we had initially set out to answer. How are microtubule assemblies aligned properly? What sort of signal directs them to ori- ent perpendicular to the axis of elongation? THAT is the question we need to answer. Part II Biology of the Cell Seeing cortical microtubules. Cortical microtubules in epidermal cells of a fava bean are tagged with a flourescent protein so that their ordered array can be seen. Real People Doing Real Science The Experiment This question has been addressed experimentally in a sim- ple and direct way in the laboratory of Richard Cyr at Pennsylvania State University. Rigid plant cells conduct mechanical force well from one cell to another, and Carol Wymer (then a graduate student in the Cyr lab) suspected some sort of mechanical force is the signal guiding cortical microtubule alignment Wymer set out to test this hypothesis using centrifuga- tion. If cortical microtubules are obtaining their positional information from an applied force, then their alignment should be affected by centrifugal force, and should be im- possible if the integrity of the cell wall (which is supposedly transmitting the mechanical force) is perturbed with chem- icals that prevent cell wall formation. Wymer, along with others in the Cyr lab, started out with cells that were not elongated. She isolated protoplasts (cells without walls) from the tobacco plant, Nicotiana tabacum, by exposing the plant cells to enzymes that break down the cell wall, creating a spherical plant cell. If allowed to grow in cul- ture, these protoplasts will eventually re-form their cell walls. In order to examine the effects of directional force on the elongation patterns of plant cells, Wymer and co-workers exposed the tobacco protoplasts to a directional force gener- ated by a centrifuge. Prior experiments had determined that centrifugation at the low speeds used in these experi- ments does not disrupt the integrity or shape of the proto- plasts. The protoplasts were immobilized for centrifugation by embedding them in an agar medium supported in a mold. The embedded protoplasts were spun in a centrifuge at 450 rpm for 15 minutes. Following centrifugation, the embedded cells were cultured for 72 hours, allowing for cell elongation to occur. Following centrifugation, fluorescently tagged micro- tubule antibody was applied to the protoplasts, which were then examined with immunofluorescence microscopy for microtubule orientation. To confirm the involvement of microtubules as sensors of directional force in cell elongation, some protoplasts were incubated prior to centrifugation with a chemical her- bicide, APM, which disrupts microtubules. The Results The biophysical force of centrifugation had significant ef- fects on the pattern of elongation in the protoplasts follow- ing the 72-hour culturing period. The microtubules were randomly arranged in protoplasts that were not centrifuged but were more ordered in protoplasts that had been cen- trifuged. The microtubules in these cells were oriented parallel to the direction of the force, in a direction approxi- mately perpendicular to the axis of elongation (graph a above). These results support the hypothesis that plant cell growth responds to an external biophysical force. It is true that plant cells are not usually exposed to the type of mechanical force generated by centrifugation but this manipulation demonstrates how a physical force can af- fect cell growth, assumably by influencing the orientation of cortical microtubules. These could be small, transient bio- physical forces acting at the subcellular level. In preparations exposed to the microtubule disrupting chemical amiprophos-methyl (APM), directed elongation was blocked (graph b above). This suggests that reorienta- tion of microtubules is indeed necessary to direct the elon- gation axis of the plant Taken together, these experiments support the hypothesis that the microtubule reorientation that directs cell elongation may be oriented by a mechanical force. Just what the natural force might be is an open question, providing an opportunity for lots of interesting future experiments that are being pur- sued in the Cyr lab. Axis of elongation relative to centrifugal force (degrees) 6 9 Number of cells (percentage) 12 0306090 3 0 Axis of elongation relative to centrifugal force (degrees) 6 9 12 0306090 3 0 Not centrifuged Centrifuged Not centrifuged Centrifuged (b)(a) APM treated Effects of centrifugation on cell elongation. (a) Protoplasts (plant cells without cell walls) that were centrifuged showed preferential elongation in a direction approximately perpendicular to the direction of the force. (b) Protoplasts exposed to APM, a microtubule dis- rupting chemical, exhibited random cell elongation with or without centrifugation. To explore this experiment further, go to the Virtual Lab at www.mhhe.com/raven6/vlab2.mhtml 77 5 Cell Structure Concept Outline 5.1 All organisms are composed of cells. Cells. A cell is a membrane-bounded unit that contains DNA and cytoplasm. All organisms are cells or aggregates of cells, descendants of the first cells. Cells Are Small. The greater relative surface area of small cells enables more rapid communication between the cell interior and the environment. 5.2 Eukaryotic cells are far more complex than bacterial cells. Bacteria Are Simple Cells. Bacterial cells are small and lack membrane-bounded organelles. Eukaryotic Cells Have Complex Interiors. Eukaryotic cells are compartmentalized by membranes. 5.3 Take a tour of a eukaryotic cell. The Nucleus: Information Center for the Cell. The nucleus of a eukaryotic cell isolates the cell’s DNA. The Endoplasmic Reticulum: Compartmentalizing the Cell. An extensive system of membranes subdivides the cell interior. The Golgi Apparatus: Delivery System of the Cell. A system of membrane channels collects, modifies, packages, and distributes molecules within the cell. Vesicles: Enzyme Storehouses. Sacs that contain enzymes digest or modify particles in the cell, while other vesicles transport substances in and out of cells. Ribosomes: Sites of Protein Synthesis. An RNA-protein complex directs the production of proteins. Organelles That Contain DNA. Some organelles with very different functions contain their own DNA. The Cytoskeleton: Interior Framework of the Cell. A network of protein fibers supports the shape of the cell and anchors organelles. Cell Movement. Eukaryotic cell movement utilizes cytoskeletal elements. Special Things about Plant Cells. Plant cells have a large central vacuole and strong, multilayered cell walls. 5.4 Symbiosis played a key role in the origin of some eukaryotic organelles. Endosymbiosis. Mitochondria and chloroplasts may have arisen from prokaryotes engulfed by other prokaryotes. A ll organisms are composed of cells. The gossamer wing of a butterfly is a thin sheet of cells, and so is the glistening outer layer of your eyes. The hamburger or tomato you eat is composed of cells, and its contents soon become part of your cells. Some organisms consist of a sin- gle cell too small to see with the unaided eye (figure 5.1), while others, like us, are composed of many cells. Cells are so much a part of life as we know it that we cannot imagine an organism that is not cellular in nature. In this chapter we will take a close look at the internal structure of cells. In the following chapters, we will focus on cells in action—on how they communicate with their environment, grow, and reproduce. FIGURE. 5.1 The single-celled organism Dileptus. The hairlike projections that cover its surface are cilia, which it undulates to propel itself through the water (1000×). 78 Part II Biology of the Cell 5.1 All organisms are composed of cells. 2 X 10 -4 mm2 X 10 -2 mm 2 X 10 1 mm 2 X 10 0 mm 2 X 10 -1 mm 2 X 10 -3 mm Cells What is a typical cell like, and what would we find inside it? The general plan of cellular organization varies in the cells of different organisms, but despite these modifications, all cells resemble each other in certain fundamental ways. Be- fore we begin our detailed examination of cell structure, let’s first summarize three major features all cells have in common: a plasma membrane, a nucleoid or nucleus, and cytoplasm. The Plasma Membrane Surrounds the Cell The plasma membrane encloses a cell and separates its contents from its surroundings. The plasma membrane is a phospholipid bilayer about 5 to 10 nanometers (5 to 10 bil- lionths of a meter) thick with proteins embedded in it. Viewed in cross-section with the electron microscope, such membranes appear as two dark lines separated by a lighter area. This distinctive appearance arises from the tail-to-tail packing of the phospholipid molecules that make up the membrane (see figure 3.18). The proteins of a membrane may have large hydrophobic domains, which associate with and become embedded in the phospholipid bilayer. The proteins of the plasma membrane are in large part responsible for a cell’s ability to interact with its en- vironment. Transport proteins help molecules and ions move across the plasma membrane, either from the envi- ronment to the interior of the cell or vice versa. Receptor proteins induce changes within the cell when they come in contact with specific molecules in the environment, such as hormones. Markers identify the cell as a particu- lar type. This is especially important in multicellular FIGURE 5.2 The size of cells and their contents. This diagram shows the size of human skin cells, organelles, and molecules. In general, the diameter of a human skin cell is 20 micrometers (μm) or 2 × 10 -2 mm, of a mitochondrion is 2 μm or 2 × 10 -3 mm, of a ribosome is 20 nanometers (nm) or 2 × 10 -5 mm, of a protein molecule is 2 nm or 2 × 10 -6 mm, and of an atom is 0.2 nm or 2 × 10 -7 mm. Chapter 5 Cell Structure 79 2 X 10 -7 mm 2 X 10 -5 mm 2 X 10 -6 mm organisms, whose cells must be able to recognize each other as they form tissues. We’ll examine the structure and function of cell mem- branes more thoroughly in chapter 6. The Central Portion of the Cell Contains the Genetic Material Every cell contains DNA, the hereditary molecule. In prokaryotes (bacteria), most of the genetic material lies in a single circular molecule of DNA. It typically resides near the center of the cell in an area called the nucleoid, but this area is not segregated from the rest of the cell’s interior by membranes. By contrast, the DNA of eukaryotes is contained in the nucleus, which is surrounded by two membranes. In both types of organisms, the DNA contains the genes that code for the proteins synthesized by the cell. The Cytoplasm Comprises the Rest of the Cell’s Interior A semifluid matrix called the cytoplasm fills the interior of the cell, exclusive of the nucleus (nucleoid in prokaryotes) lying within it. The cytoplasm contains the chemical wealth of the cell: the sugars, amino acids, and proteins the cell uses to carry out its everyday activities. In eukaryotic cells, the cytoplasm also contains specialized membrane-bounded compartments called organelles. The Cell Theory A general characteristic of cells is their microscopic size. While there are a few exceptions—the marine alga Acetabu- laria can be up to 5 centimeters long—a typical eukaryotic cell is 10 to 100 micrometers (10 to 100 millionths of a meter) in diameter (figure 5.2); most bacterial cells are only 1 to 10 micrometers in diameter. Because cells are so small, no one observed them until microscopes were invented in the mid-seventeenth century. Robert Hooke first described cells in 1665, when he used a microscope he had built to examine a thin slice of cork, a nonliving tissue found in the bark of certain trees. Hooke observed a honeycomb of tiny, empty (because the cells were dead) compartments. He called the compartments in the cork cellulae (Latin, “small rooms”), and the term has come down to us as cells. The first living cells were observed a few years later by the Dutch naturalist Antonie van Leeuwenhoek, who called the tiny organisms that he ob- served “animalcules,” meaning little animals. For another century and a half, however, biologists failed to recognize the importance of cells. In 1838, botanist Matthias Schlei- den made a careful study of plant tissues and developed the first statement of the cell theory. He stated that all plants “are aggregates of fully individualized, independent, sepa- rate beings, namely the cells themselves.” In 1839, Theodor Schwann reported that all animal tissues also con- sist of individual cells. The cell theory, in its modern form, includes the fol- lowing three principles: 1. All organisms are composed of one or more cells, and the life processes of metabolism and heredity occur within these cells. 2. Cells are the smallest living things, the basic units of organization of all organisms. 3. Cells arise only by division of a previously existing cell. Although life likely evolved spontaneously in the environment of the early earth, biologists have con- cluded that no additional cells are originating sponta- neously at present. Rather, life on earth represents a continuous line of descent from those early cells. A cell is a membrane-bounded unit that contains the DNA hereditary machinery and cytoplasm. All organisms are cells or aggregates of cells. Cells Are Small How many cells are big enough to see with the unaided eye? Other than egg cells, not many. Most are less than 50 micrometers in diameter, far smaller than the period at the end of this sen- tence. The Resolution Problem How do we study cells if they are too small to see? The key is to understand why we can’t see them. The reason we can’t see such small objects is the limited resolution of the human eye. Resolution is defined as the minimum distance two points can be apart and still be distinguished as two separated points. When two objects are closer together than about 100 micrometers, the light reflected from each strikes the same “detector” cell at the rear of the eye. Only when the objects are farther than 100 micrometers apart will the light from each strike different cells, allowing your eye to resolve them as two objects rather than one. Microscopes One way to increase resolution is to increase magnification, so that small objects appear larger. Robert Hooke and Antonie van Leeuwenhoek were able to see small cells by magnifying their size, so that the cells appeared larger than the 100-micrometer limit imposed by the human eye. Hooke and van Leeuwenhoek accomplished this feat with microscopes that magnified images of cells by bending light through a glass lens. The size of the image that falls on the sheet of detector cells lining the back of your eye depends on how close the object is to your eye—the closer the object, the bigger the image. Your eye, however, is incapable of focusing comfortably on an object closer than about 25 centimeters, because the eye is limited by the size and thickness of its lens. Hooke and van Leeuwenhoek assisted the eye by interposing a glass lens between object and eye. The glass lens adds additional focusing power. Because the glass lens makes the object appear closer, the image on the back of the eye is bigger than it would be without the lens. Modern light microscopes use two magnifying lenses (and a variety of correcting lenses) that act like back-to-back eyes. The first lens focuses the image of the object on the second lens, which magnifies it again and focuses it on the back of the eye. Microscopes that magnify in stages using several lenses are called compound microscopes. They can resolve structures that are separated by more than 200 nm. An image from a compound microscope is shown in figure 5.3a. Increasing Resolution Light microscopes, even compound ones, are not powerful enough to resolve many structures within cells. For exam- ple, a membrane is only 5 nanometers thick. Why not just add another magnifying stage to the microscope and so in- crease its resolving power? Because when two objects are closer than a few hundred nanometers, the light beams re- flecting from the two images start to overlap. The only way two light beams can get closer together and still be resolved is if their “wavelengths” are shorter. One way to avoid overlap is by using a beam of electrons rather than a beam of light. Electrons have a much shorter wavelength, and a microscope employing electron beams has 1000 times the resolving power of a light microscope. Transmission electron microscopes, so called because the electrons used to visualize the specimens are transmitted through the material, are capable of resolving objects only 0.2 nanometer apart—just twice the diameter of a hydrogen atom! Figure 5.3b shows a transmission electron micro- graph. A second kind of electron microscope, the scanning electron microscope, beams the electrons onto the surface of the specimen from a fine probe that passes rapidly back and forth. The electrons reflected back from the surface of the specimen, together with other electrons that the speci- men itself emits as a result of the bombardment, are ampli- fied and transmitted to a television screen, where the image can be viewed and photographed. Scanning electron mi- croscopy yields striking three-dimensional images and has improved our understanding of many biological and physi- cal phenomena (figure 5.3c). 80 Part II Biology of the Cell (a) (b) (c) FIGURE 5.3 Human sperm cells viewed with three different microscopes. (a) Image of sperm taken with a light microscope. (b)Transmission electron micrograph of a sperm cell. (c) Scanning electron micrograph of sperm cells. Why Aren’t Cells Larger? Most cells are not large for practical reasons. The most im- portant of these is communication. The different regions of a cell need to communicate with one another in order for the cell as a whole to function effectively. Proteins and or- ganelles are being synthesized, and materials are continually entering and leaving the cell. All of these processes involve the diffusion of substances at some point, and the larger a cell is, the longer it takes for substances to diffuse from the plasma membrane to the center of the cell. For this reason, an organism made up of many relatively small cells has an advantage over one composed of fewer, larger cells. The advantage of small cell size is readily visualized in terms of the surface area-to-volume ratio. As a cell’s size increases, its volume increases much more rapidly than its surface area. For a spherical cell, the increase in surface area is equal to the square of the increase in di- ameter, while the increase in volume is equal to the cube of the increase in diameter. Thus, if two cells differ by a factor of 10 cm in diameter, the larger cell will have 10 2 , or 100 times, the surface area, but 10 3 , or 1000 times, the volume, of the smaller cell (figure 5.4). A cell’s sur- face provides its only opportunity for interaction with the environment, as all substances enter and exit a cell via the plasma membrane. This membrane plays a key role in controlling cell function, and because small cells have more surface area per unit of volume than large ones, the control is more effective when cells are rela- tively small. Although most cells are small, some cells are nonetheless quite large and have apparently overcome the surface area- to-volume problem by one or more adaptive mechanisms. For example, some cells have more than one nucleus, allow- ing genetic information to be spread around a large cell. Also, some large cells actively move material around their cytoplasm so that diffusion is not a limiting factor. Lastly, some large cells, like your own neurons, are long and skinny so that any given point in the cytoplasm is close to the plasma membrane, and thus diffusion between the inside and outside of the cell can still be rapid. Multicellular organisms usually consist of many small cells rather than a few large ones because small cells function more efficiently. They have a greater relative surface area, enabling more rapid communication between the center of the cell and the environment. Chapter 5 Cell Structure 81 Cell radius (r) Surface area (4H9266r 2 ) Volume ( 4 – 3 H9266r 3 ) 1 cm 12.57 cm 2 4.189 cm 3 10 cm 1257 cm 2 4189 cm 3 FIGURE 5.4 Surface area-to-volume ratio. As a cell gets larger, its volume increases at a faster rate than its surface area. If the cell radius increases by 10 times, the surface area increases by 100 times, but the volume increases by 1000 times. A cell’s surface area must be large enough to meet the needs of its volume. Bacteria Are Simple Cells Prokaryotes, the bacteria, are the sim- plest organisms. Prokaryotic cells are small, consisting of cytoplasm sur- rounded by a plasma membrane and en- cased within a rigid cell wall, with no distinct interior compartments (figure 5.5). A prokaryotic cell is like a one- room cabin in which eating, sleeping, and watching TV all occur in the same room. Bacteria are very important in the economy of living organisms. They harvest light in photosynthesis, break down dead organisms and recycle their components, cause disease, and are in- volved in many important industrial processes. Bacteria are the subject of chapter 34. Strong Cell Walls Most bacteria are encased by a strong cell wall composed of peptidoglycan, which consists of a carbohydrate matrix (polymers of sugars) that is cross-linked by short polypeptide units. No eukaryotes possess cell walls with this type of chemical composition. With a few exceptions like TB and leprosy-causing bacteria, all bacteria may be classified into two types based on differences in their cell walls detected by the Gram staining procedure. The name refers to the Danish microbiologist Hans Christian Gram, who developed the procedure to detect the pres- ence of certain disease-causing bacteria. Gram-positive bacteria have a thick, single-layered cell wall that retains a violet dye from the Gram stain procedure, causing the stained cells to appear purple under a microscope. More complex cell walls have evolved in other groups of bacte- ria. In them, the wall is multilayered and does not retain the purple dye after Gram staining; such bacteria exhibit the background red dye and are characterized as gram- negative. The susceptibility of bacteria to antibiotics often depends on the structure of their cell walls. Penicillin and van- comycin, for example, interfere with the ability of bacteria to cross-link the peptide units that hold the carbohydrate chains of the wall together. Like removing all the nails from a wooden house, this destroys the integrity of the ma- trix, which can no longer prevent water from rushing in, swelling the cell to bursting. Cell walls protect the cell, maintain its shape, and prevent excessive uptake of water. Plants, fungi, and most protists also have cell walls of a different chemical structure, which we will discuss in later chapters. Long chains of sugars called polysaccharides cover the cell walls of many bacteria. They enable a bacterium to ad- here to teeth, skin, food—practically any surface that will support their growth. Many disease-causing bacteria secrete a jellylike protective capsule of polysaccharide around the cell. Rotating Flagella Some bacteria use a flagellum (plural, flagella) to move. Flagella are long, threadlike structures protruding from the surface of a cell that are used in locomotion and feeding. Bacterial flagella are protein fibers that extend out from a bacterial cell. There may be one or more per cell, or none, depending on the species. Bacteria can swim at speeds up to 20 cell diameters per second by rotating their flagella like screws (figure 5.6). A “motor” unique to bacteria that is em- bedded within their cell walls and membranes powers the rotation. Only a few eukaryotic cells have structures that truly rotate. Simple Interior Organization If you were to look at an electron micrograph of a bacterial cell, you would be struck by the cell’s simple organiza- tion. There are few, if any, internal compartments, and while they contain simple structures like ribosomes, most have no membrane-bounded organelles, the kinds so characteristic of eukaryotic cells. Nor do bacteria have a true nucleus. The entire cytoplasm of a bacterial cell is one unit with no internal support structure. Consequently, 82 Part II Biology of the Cell 5.2 Eukaryotic cells are far more complex than bacterial cells. Flagellum Cell wall Plasma membrane Capsule Ribosomes DNAPili FIGURE 5.5 Structure of a bacterial cell. Generalized cell organization of a bacterium. Some bacteria have hairlike growths on the outside of the cell called pili. the strength of the cell comes primarily from its rigid wall (see figure 5.5). The plasma membrane of a bacterial cell carries out some of the functions organelles perform in eukaryotic cells. When a bacterial cell divides, for example, the bacterial chromosome, a simple circle of DNA, replicates before the cell divides. The two DNA molecules that result from the replication attach to the plasma membrane at different points, ensuring that each daughter cell will contain one of the identical units of DNA. Moreover, some photosynthetic bacteria, such as cyanobacte- ria and Prochloron (figure 5.7), have an extensively folded plasma membrane, with the folds extending into the cell’s interior. These membrane folds contain the bacterial pigments connected with photosynthesis. Because a bacterial cell contains no membrane-bounded organelles, the DNA, enzymes, and other cytoplasmic con- stituents have access to all parts of the cell. Reactions are not compartmentalized as they are in eukaryotic cells, and the whole bacterium operates as a single unit. Bacteria are small cells that lack interior organization. They are encased by an exterior wall composed of carbohydrates cross-linked by short polypeptides, and some are propelled by rotating flagella. Chapter 5 Cell Structure 83 Bacterial cell wall Flagellin Rotary motor (b) Sheath (a) (c) FIGURE 5.6 Bacteria swim by rotating their flagella. (a) The photograph is of Vibrio cholerae, the microbe that causes the serious disease cholera. The unsheathed core visible at the top of the photograph is composed of a single crystal of the protein flagellin. (b) In intact flagella, the core is surrounded by a flexible sheath. Imagine that you are standing inside the Vibrio cell, turning the flagellum like a crank. (c) You would create a spiral wave that travels down the flagellum, just as if you were turning a wire within a flexible tube. The bacterium creates this kind of rotary motion when it swims. FIGURE 5.7 Electron micrograph of a photosynthetic bacterial cell. Extensive folded photosynthetic membranes are visible in this Prochloron cell (14,500×). The single, circular DNA molecule is located in the clear area in the central region of the cell. Eukaryotic Cells Have Complex Interiors Eukaryotic cells (figures 5.8 and 5.9) are far more complex than prokaryotic cells. The hallmark of the eukaryotic cell is compartmentalization. The interiors of eukaryotic cells contain numerous organelles, membrane-bounded struc- tures that close off compartments within which multiple biochemical processes can proceed simultaneously and indepen- dently. Plant cells often have a large membrane-bounded sac called a central vacuole, which stores proteins, pigments, and waste materials. Both plant and ani- mal cells contain vesicles, smaller sacs that store and transport a variety of mate- rials. Inside the nucleus, the DNA is 84 Part II Biology of the Cell Centriole Lysosome Mitochondrion Ribosomes Rough endoplasmic reticulum Cytoplasm Nucleus Nucleolus Nuclear envelope Smooth endoplasmic reticulum Cytoskeleton Golgi apparatus Plasma membrane Microvilli (a) Plasma membrane Nucleolus Nucleus Lysosome Ribosomes Rough endoplasmic reticulum Golgi apparatus Mitochondrion Smooth endoplasmic reticulum (b) FIGURE 5.8 Structure of an animal cell. (a) A generalized diagram of an animal cell. (b) Micrograph of a human white blood cell (40,500H11003) with drawings detailing organelles. wound tightly around proteins and packaged into compact units called chromosomes. All eukaryotic cells are supported by an internal protein scaffold, the cytoskeleton. While the cells of animals and some protists lack cell walls, the cells of fungi, plants, and many protists have strong cell walls composed of cel- lulose or chitin fibers embedded in a matrix of other polysaccharides and proteins. This composition is very different from the peptidoglycan that makes up bacterial cell walls. Let’s now examine the structure and function of the internal components of eukaryotic cells in more detail. Eukaryotic cells contain membrane-bounded organelles that carry out specialized functions. Chapter 5 Cell Structure 85 Plasma membrane Mitochondrion Nucleus Chloroplast Central vacuole (b) Cell wall Plasmodesma FIGURE 5.9 Structure of a plant cell. A generalized illustration (a) and micrograph (b) of a plant cell. Most mature plant cells contain large central vacuoles which occupy a major portion of the internal volume of the cell (14,000H11003). Cell wall Plasma membrane Central vacuole Mitochondrion Ribosomes Golgi apparatus Nucleus Nucleolus Chloroplasts Nuclear envelope Rough endoplasmic reticulum Cytoplasm Smooth endoplasmic reticulum Plasmo- desmata Lysosome (a) The Nucleus: Information Center for the Cell The largest and most easily seen organelle within a eukary- otic cell is the nucleus (Latin, for kernel or nut), first described by the English botanist Robert Brown in 1831. Nuclei are roughly spherical in shape and, in animal cells, they are typically located in the central region of the cell (figure 5.10). In some cells, a network of fine cytoplasmic filaments seems to cradle the nucleus in this position. The nucleus is the repository of the genetic information that directs all of the activities of a living eukaryotic cell. Most eukaryotic cells possess a single nucleus, although the cells of fungi and some other groups may have several to many nuclei. Mammalian erythrocytes (red blood cells) lose their nuclei when they mature. Many nuclei exhibit a dark- staining zone called the nucleolus, which is a region where intensive synthesis of ribosomal RNA is taking place. 86 Part II Biology of the Cell 5.3 Take a tour of a eukaryotic cell. (c) Cytoplasm Pore Nucleus FIGURE 5.10 The nucleus. (a) The nucleus is composed of a double membrane, called a nuclear envelope, enclosing a fluid-filled interior containing the chromosomes. In cross-section, the individual nuclear pores are seen to extend through the two membrane layers of the envelope; the dark material within the pore is protein, which acts to control access through the pore. (b) A freeze-fracture scanning electron micrograph of a cell nucleus showing nuclear pores (9500×). (c) A transmission electron micrograph (see figure 6.6) of the nuclear membrane showing a nuclear pore. Nuclear pores Nuclear pore Nuclear envelope Nucleoplasm Outer membrane Inner membrane Nucleolus (a) Pore (b) The Nuclear Envelope: Getting In and Out The surface of the nucleus is bounded by two phospho- lipid bilayer membranes, which together make up the nuclear envelope (see figure 5.10). The outer mem- brane of the nuclear envelope is continuous with the cytoplasm’s interior membrane system, called the endo- plasmic reticulum. Scattered over the surface of the nuclear envelope, like craters on the moon, are shallow depressions called nuclear pores. These pores form 50 to 80 nanometers apart at locations where the two mem- brane layers of the nuclear envelope pinch together. Rather than being empty, nuclear pores are filled with proteins that act as molecular channels, permitting certain molecules to pass into and out of the nucleus. Passage is restricted primarily to two kinds of molecules: (1) proteins moving into the nucleus to be incorporated into nuclear structures or to catalyze nuclear activities; and (2) RNA and protein-RNA complexes formed in the nucleus and exported to the cytoplasm. The Chromosomes: Packaging the DNA In both bacteria and eukaryotes, DNA contains the hered- itary information specifying cell structure and function. However, unlike the circular DNA of bacteria, the DNA of eukaryotes is divided into several linear chromosomes. Except when a cell is dividing, its chromosomes are fully extended into threadlike strands, called chromatin, of DNA complexed with protein. This open arrangement allows proteins to attach to specific nucleotide sequences along the DNA. Without this access, DNA could not direct the day-to-day activities of the cell. The chromo- somes are associated with packaging proteins called histones. When a cell prepares to divide, the DNA coils up around the histones into a highly condensed form. In the initial stages of this condensation, units of histone can be seen with DNA wrapped around like a sash. Called nucleosomes, these initial aggregations resemble beads on a string (figure 5.11). Coiling continues until the DNA is in a compact mass. Under a light micro- scope, these fully condensed chromosomes are readily seen in dividing cells as densely staining rods (figure 5.12). After cell division, eukaryotic chromosomes uncoil and can no longer be individually distinguished with a light microscope. Uncoiling the chromosomes into a more extended form permits enzymes to makes RNA copies of DNA. Only by means of these RNA copies can the information in the DNA be used to direct the synthesis of proteins. The nucleus of a eukaryotic cell contains the cell’s hereditary apparatus and isolates it from the rest of the cell. A distinctive feature of eukaryotes is the organization of their DNA into complex chromosomes. Chapter 5 Cell Structure 87 Central histone Spacer histone Nucleosome Chromosome DNA FIGURE 5.11 Nucleosomes. Each nucleosome is a region in which the DNA is wrapped tightly around a cluster of histone proteins. FIGURE 5.12 Eukaryotic chromosomes. These condensed chromosomes within an onion root tip are visible under the light microscope (500×). The Endoplasmic Reticulum: Compartmentalizing the Cell The interior of a eukaryotic cell is packed with membranes (table 5.1). So thin that they are invisible under the low re- solving power of light microscopes, this endomembrane system fills the cell, dividing it into compartments, channel- ing the passage of molecules through the interior of the cell, and providing surfaces for the synthesis of lipids and some proteins. The presence of these membranes in eukaryotic cells constitutes one of the most fundamental distinctions between eukaryotes and prokaryotes. The largest of the internal membranes is called the endoplasmic reticulum (ER). The term endoplasmic means “within the cytoplasm,” and the term reticulum is Latin for “a little net.” Like the plasma membrane, the ER is composed of a lipid bilayer embedded with proteins. It weaves in sheets through the interior of the cell, creating a series of channels between its folds (figure 5.13). Of the many compartments in eukaryotic cells, the two largest are the inner region of the ER, called the cisternal space, and the region exterior to it, the cytosol. Rough ER: Manufacturing Proteins for Export The ER surface regions that are devoted to protein synthe- sis are heavily studded with ribosomes, large molecular aggregates of protein and ribonucleic acid (RNA) that trans- late RNA copies of genes into protein (we will examine ribosomes in detail later in this chapter). Through the elec- tron microscope, these ribosome-rich regions of the ER appear pebbly, like the surface of sandpaper, and they are therefore called rough ER (see figure 5.13). The proteins synthesized on the surface of the rough ER are destined to be exported from the cell. Proteins to be ex- ported contain special amino acid sequences called signal sequences. As a new protein is made by a free ribosome (one not attached to a membrane), the signal sequence of the growing polypeptide attaches to a recognition factor that carries the ribosome and its partially completed protein to a “docking site” on the surface of the ER. As the protein is assembled it passes through the ER membrane into the interior ER compartment, the cisternal space, from which it is transported by vesicles to the Golgi apparatus (figure 5.14). The protein then travels within vesicles to the inner surface of the plasma membrane, where it is released to the outside of the cell. 88 Part II Biology of the Cell Table 5.1 Eukaryotic Cell Structures and Their Functions Structure Description Function Cell wall Cytoskeleton Flagella (cilia) Plasma membrane Endoplasmic reticulum Nucleus Golgi apparatus Lysosomes Microbodies Mitochondria Chloroplasts Chromosomes Nucleolus Ribosomes Outer layer of cellulose or chitin; or absent Network of protein filaments Cellular extensions with 9 + 2 arrangement of pairs of microtubules Lipid bilayer with embedded proteins Network of internal membranes Structure (usually spherical) surrounded by double membrane that contains chromosomes Stacks of flattened vesicles Vesicles derived from Golgi apparatus that contain hydrolytic digestive enzymes Vesicles formed from incorporation of lipids and proteins containing oxidative and other enzymes Bacteria-like elements with double membrane Bacteria-like elements with membranes containing chlorophyll, a photosynthetic pigment Long threads of DNA that form a complex with protein Site of genes for rRNA synthesis Small, complex assemblies of protein and RNA, often bound to endoplasmic reticulum Protection; support Structural support; cell movement Motility or moving fluids over surfaces Regulates what passes into and out of cell; cell-to-cell recognition Forms compartments and vesicles; participates in protein and lipid synthesis Control center of cell; directs protein synthesis and cell reproduction Packages proteins for export from cell; forms secretory vesicles Digest worn-out organelles and cell debris; play role in cell death Isolate particular chemical activities from rest of cell “Power plants” of the cell; sites of oxidative metabolism Sites of photosynthesis Contain hereditary information Assembles ribosomes Sites of protein synthesis Smooth ER: Organizing Internal Activities Regions of the ER with relatively few bound ribosomes are referred to as smooth ER. The membranes of the smooth ER contain many embedded enzymes, most of them active only when associated with a membrane. Enzymes anchored within the ER, for example, catalyze the synthesis of a vari- ety of carbohydrates and lipids. In cells that carry out exten- sive lipid synthesis, such as those in the testes, intestine, and brain, smooth ER is particularly abundant. In the liver, the enzymes of the smooth ER are involved in the detoxification of drugs including amphetamines, morphine, codeine, and phenobarbital. Some vesicles form at the plasma membrane by budding inward, a process called endocytosis. Some then move into the cytoplasm and fuse with the smooth endoplasmic reticu- lum. Others form secondary lysosomes or other interior vesicles. The endoplasmic reticulum (ER) is an extensive system of folded membranes that divides the interior of eukaryotic cells into compartments and channels. Rough ER synthesizes proteins, while smooth ER organizes the synthesis of lipids and other biosynthetic activities. Chapter 5 Cell Structure 89 0.08 μm Ribosomes Rough endoplasmic reticulum Smooth endoplasmic reticulum FIGURE 5.13 The endoplasmic reticulum. Ribosomes are associated with only one side of the rough ER; the other side is the boundary of a separate compartment within the cell into which the ribosomes extrude newly made proteins destined for secretion. Smooth endoplasmic reticulum has few to no bound ribosomes. Cytoplasm Lumen mRNA Ribosome Membrane of endoplasmic reticulum Polypeptide Signal sequence FIGURE 5.14 Signal sequences direct proteins to their destinations in the cell. In this example, a sequence of hydrophobic amino acids (the signal sequence) on a secretory protein attaches them (and the ribosomes making them) to the membrane of the ER. As the protein is synthesized, it passes into the lumen (internal chamber) of the ER. The signal sequence is clipped off after the leading edge of the protein enters the lumen. The Golgi Apparatus: Delivery System of the Cell At various locations within the endomembrane system, flattened stacks of membranes called Golgi bodies occur, often interconnected with one another. These structures are named for Camillo Golgi, the nineteenth-century Italian physician who first called attention to them. The numbers of Golgi bodies a cell contains ranges from 1 or a few in protists, to 20 or more in animal cells and several hundred in plant cells. They are especially abundant in glandular cells, which manufacture and secrete sub- stances. Collectively the Golgi bodies are referred to as the Golgi apparatus (figure 5.15). The Golgi apparatus functions in the collection, packag- ing, and distribution of molecules synthesized at one place in the cell and utilized at another location in the cell. A Golgi body has a front and a back, with distinctly different membrane compositions at the opposite ends. The front, or receiving end, is called the cis face, and is usually located near ER. Materials move to the cis face in transport vesicles that bud off of the ER. These vesicles fuse with the cis face, emptying their contents into the interior, or lumen, of the Golgi apparatus. These ER-synthesized molecules then pass through the channels of the Golgi apparatus until they reach the back, or discharging end, called the trans face, where they are discharged in secretory vesicles (figure 5.16). Proteins and lipids manufactured on the rough and smooth ER membranes are transported into the Golgi ap- paratus and modified as they pass through it. The most common alteration is the addition or modification of short sugar chains, forming a glycoprotein when sugars are com- plexed to a protein and a glycolipid when sugars are bound to a lipid. In many instances, enzymes in the Golgi appara- tus modify existing glycoproteins and glycolipids made in the ER by cleaving a sugar from their sugar chain or modi- fying one or more of the sugars. The newly formed or altered glycoproteins and glycol- ipids collect at the ends of the Golgi bodies, in flattened stacked membrane folds called cisternae (Latin, “collecting vessels”). Periodically, the membranes of the cisternae push together, pinching off small, membrane- bounded secretory vesicles containing the glycoprotein and glycolipid molecules. These vesicles then move to other locations in the cell, distributing the newly synthesized molecules to their appropriate destinations. Liposomes are synthetically manufactured vesicles that contain any variety of desirable substances (such as drugs), and can be injected into the body. Because the membrane of liposomes is similar to plasma and organellar membranes, these liposomes serve as an effective and natural delivery system to cells and may prove to be of great therapeutic value. The Golgi apparatus is the delivery system of the eukaryotic cell. It collects, packages, modifies, and distributes molecules that are synthesized at one location within the cell and used at another. 90 Part II Biology of the Cell Secretory vesicles Vesicle 0.57 μm FIGURE 5.15 The Golgi apparatus. The Golgi apparatus is a smooth, concave membranous structure located near the middle of the cell. It receives material for processing on one surface and sends the material packaged in vesicles off the other. The substance in a vesicle could be for export out of the cell or for distribution to another region within the same cell. Chapter 5 Cell Structure 91 Budding vesicle Fusion of vesicle with Golgi apparatus Migrating transport vesicle Protein Proteins Transport vesicle Golgi apparatus Secretory vesicle Smooth endoplasmic reticulum Rough endoplasmic reticulum Nuclear pore Nucleus Cisternae Ribosome Trans face Cis face Cell membrane Protein expelled Cytoplasm Extracellular fluid FIGURE 5.16 How proteins are transported within the cell. Proteins are manufactured at the ribosome and then released into the internal compartments of the rough ER. If the newly synthesized proteins are to be used at a distant location in or outside of the cell, they are transported within vesicles that bud off the rough ER and travel to the cis face, or receiving end, of the Golgi apparatus. There they are modified and packaged into secretory vesicles. The secretory vesicles then migrate from the trans face, or discharging end, of the Golgi apparatus to other locations in the cell, or they fuse with the cell membrane, releasing their contents to the external cellular environment. Vesicles: Enzyme Storehouses Lysosomes: Intracellular Digestion Centers Lysosomes, membrane-bounded diges- tive vesicles, are also components of the endomembrane system that arise from the Golgi apparatus. They contain high levels of degrading enzymes, which cat- alyze the rapid breakdown of proteins, nucleic acids, lipids, and carbohydrates. Throughout the lives of eukaryotic cells, lysosomal enzymes break down old or- ganelles, recycling their component mol- ecules and making room for newly formed organelles. For example, mito- chondria are replaced in some tissues every 10 days. The digestive enzymes in lysosomes function best in an acidic environment. Lysosomes actively engaged in digestion keep their battery of hydrolytic enzymes (enzymes that catalyze the hydrolysis of molecules) fully active by pumping protons into their inte- riors and thereby maintaining a low internal pH. Lyso- somes that are not functioning actively do not maintain an acidic internal pH and are called primary lysosomes. When a primary lysosome fuses with a food vesicle or other or- ganelle, its pH falls and its arsenal of hydrolytic enzymes is activated; it is then called a secondary lysosome. In addition to breaking down organelles and other struc- tures within cells, lysosomes also eliminate other cells that the cell has engulfed in a process called phagocytosis, a spe- cific type of endocytosis (see chapter 6). When a white blood cell, for example, phagocytizes a passing pathogen, lysosomes fuse with the resulting “food vesicle,” releasing their enzymes into the vesicle and degrading the material within (figure 5.17). Microbodies Eukaryotic cells contain a variety of enzyme-bearing, membrane-enclosed vesicles called microbodies. Micro- bodies are found in the cells of plants, animals, fungi, and protists. The distribution of enzymes into microbodies is one of the principal ways in which eukaryotic cells orga- nize their metabolism. While lysosomes bud from the endomembrane system, microbodies grow by incorporating lipids and protein, then dividing. Plant cells have a special type of microbody called a glyoxysome that contains enzymes that convert fats into carbohydrates. Another type of microbody, a peroxisome, contains enzymes that catalyze the removal of electrons and associated hydrogen atoms (figure 5.18). If these oxidative enzymes were not isolated within micro- bodies, they would tend to short-circuit the metabolism of the cytoplasm, which often involves adding hydrogen atoms to oxygen. The name peroxisome refers to the hydrogen peroxide produced as a by-product of the activities of the oxidative enzymes in the microbody. Hydrogen peroxide is dangerous to cells because of its violent chemical reactivity. However, peroxisomes also contain the enzyme catalase, which breaks down hydrogen peroxide into harmless water and oxygen. Lysosomes and peroxisomes are vesicles that contain digestive and detoxifying enzymes. The isolation of these enzymes in vesicles protects the rest of the cell from inappropriate digestive activity. 92 Part II Biology of the Cell Cytoplasm Phagocytosis Food vesicle Golgi apparatus Lysosomes Plasma membrane Digestion of phagocytized food particles or cells Endoplasmic reticulum Transport vesicle Old or damaged organelle Breakdown of old organelle Extracellular fluid FIGURE 5.17 Lysosomes. Lysosomes contain hydrolytic enzymes that digest particles or cells taken into the cell by phagocytosis and break down old organelles. 0.21 μm FIGURE 5.18 A peroxisome. Peroxisomes are spherical organelles that may contain a large diamond-shaped crystal composed of protein. Peroxisomes contain digestive and detoxifying enzymes that produce hydrogen peroxide as a by- product. Ribosomes: Sites of Protein Synthesis Although the DNA in a cell’s nucleus encodes the amino acid sequence of each protein in the cell, the proteins are not assembled there. A simple experiment demonstrates this: if a brief pulse of radioactive amino acid is administered to a cell, the radioactivity shows up associated with newly made protein, not in the nucleus, but in the cytoplasm. When investigators first carried out these experiments, they found that protein synthesis was associated with large RNA- protein complexes they called ribosomes. Ribosomes are made up of several molecules of a special form of RNA called ribosomal RNA, or rRNA, bound within a complex of several dozen different proteins. Ribosomes are among the most complex molecular assemblies found in cells. Each ribosome is composed of two subunits (figure 5.19). The subunits join to form a functional ribosome only when they attach to another kind of RNA, called messenger RNA (mRNA) in the cytoplasm. To make proteins, the ribosome attaches to the mRNA, which is a transcribed copy of a portion of DNA, and uses the information to direct the synthesis of a protein. Bacterial ribosomes are smaller than eukaryotic ribo- somes. Also, a bacterial cell typically has only a few thou- sand ribosomes, while a metabolically active eukaryotic cell, such as a human liver cell, contains several million. Proteins that function in the cytoplasm are made by free ribosomes suspended there, while proteins bound within membranes or destined for export from the cell are assembled by ribo- somes bound to rough ER. The Nucleolus Manufactures Ribosomal Subunits When cells are synthesizing a large number of proteins, they must first make a large number of ribosomes. To facili- tate this, many hundreds of copies of the portion of the DNA encoding the rRNA are clustered together on the chromosome. By transcribing RNA molecules from this cluster, the cell rapidly generates large numbers of the mol- ecules needed to produce ribosomes. At any given moment, many rRNA molecules dangle from the chromosome at the sites of these clusters of genes that encode rRNA. Proteins associate with the dangling rRNA molecules. These areas where ribosomes are being assembled are easily visible within the nucleus as one or more dark-staining regions, called nucleoli (singular, nucle- olus; figure 5.20). Nucleoli can be seen under the light mi- croscope even when the chromosomes are extended, unlike the rest of the chromosomes, which are visible only when condensed. Ribosomes are the sites of protein synthesis in the cytoplasm. Chapter 5 Cell Structure 93 Small subunit Large subunit Ribosome FIGURE 5.19 A ribosome. Ribosomes consist of a large and a small subunit composed of rRNA and protein. The individual subunits are synthesized in the nucleolus and then move through the nuclear pores to the cytoplasm, where they assemble. Ribosomes serve as sites of protein synthesis. FIGURE 5.20 The nucleolus. This is the interior of a rat liver cell, magnified about 6000 times. A single large nucleus occupies the center of the micrograph. The electron-dense area in the lower left of the nucleus is the nucleolus, the area where the major components of the ribosomes are produced. Partly formed ribosomes can be seen around the nucleolus. Organelles That Contain DNA Among the most interesting cell organelles are those in addition to the nucleus that contain DNA. Mitochondria: The Cell’s Chemical Furnaces Mitochondria (singular, mito- chondrion) are typically tubular or sausage-shaped organelles about the size of bacteria and found in all types of eukaryotic cells (figure 5.21). Mitochondria are bounded by two membranes: a smooth outer membrane and an inner one folded into numerous contiguous layers called cristae (singular, crista). The cristae partition the mitochondrion into two compartments: a matrix, lying inside the inner membrane; and an outer compartment, or intermem- brane space, lying between the two mitochondrial mem- branes. On the surface of the inner membrane, and also embedded within it, are proteins that carry out oxidative metabolism, the oxygen-requiring process by which en- ergy in macromolecules is stored in ATP. Mitochondria have their own DNA; this DNA contains several genes that produce proteins essential to the mito- chondrion’s role in oxidative metabolism. All of these genes are copied into RNA and used to make proteins within the mitochondrion. In this process, the mitochondria employ small RNA molecules and ribosomal components that the mitochondrial DNA also encodes. However, most of the genes that produce the enzymes used in oxidative metabo- lism are located in the nucleus. A eukaryotic cell does not produce brand new mito- chondria each time the cell divides. Instead, the mito- chondria themselves divide in two, doubling in number, and these are partitioned between the new cells. Most of the components required for mitochondrial division are encoded by genes in the nucleus and translated into pro- teins by cytoplasmic ribosomes. Mitochondrial replica- tion is, therefore, impossible without nuclear participa- tion, and mitochondria thus cannot be grown in a cell-free culture. Chloroplasts: Where Photosynthesis Takes Place Plants and other eukaryotic organisms that carry out photosynthesis typically contain from one to several hundred chloroplasts. Chloroplasts bestow an obvious advantage on the organisms that possess them: they can manufacture their own food. Chloroplasts contain the photosynthetic pigment chlorophyll that gives most plants their green color. The chloroplast body is enclosed, like the mitochon- drion, within two membranes that resemble those of mito- chondria (figure 5.22). However, chloroplasts are larger and more complex than mitochondria. In addition to the outer and inner membranes, which lie in close association with each other, chloroplasts have a closed compartment of stacked membranes called grana (singular, granum), which lie internal to the inner membrane. A chloroplast may con- tain a hundred or more grana, and each granum may con- tain from a few to several dozen disk-shaped structures called thylakoids. On the surface of the thylakoids are the light-capturing photosynthetic pigments, to be discussed in depth in chapter 10. Surrounding the thylakoid is a fluid matrix called the stroma. Like mitochondria, chloroplasts contain DNA, but many of the genes that specify chloroplast components are also located in the nucleus. Some of the elements used in the photosynthetic process, including the specific protein components necessary to accomplish the reaction, are syn- thesized entirely within the chloroplast. 94 Part II Biology of the Cell Intermembrane space Inner membrane Outer membrane Matrix Crista membrane e (a) (b) FIGURE 5.21 Mitochondria. (a) The inner membrane of a mitochondrion is shaped into folds called cristae, which greatly increase the surface area for oxidative metabolism. (b) Mitochondria in cross-section and cut lengthwise (70,000×). Other DNA-containing organelles in plants are called leucoplasts, which lack pigment and a complex internal structure. In root cells and some other plant cells, leu- coplasts may serve as starch storage sites. A leucoplast that stores starch (amylose) is sometimes termed an amyloplast. These organelles—chloroplasts, leucoplasts, and amylo- plasts—are collectively called plastids. All plastids come from the division of existing plastids. Centrioles: Microtubule Assembly Centers Centrioles are barrel-shaped organelles found in the cells of animals and most protists. They occur in pairs, usually located at right angles to each other near the nuclear membranes (figure 5.23); the region surrounding the pair in almost all animal cells is referred to as a centrosome. Although the matter is in some dispute, at least some centrioles seem to contain DNA, which apparently is in- volved in producing their structural proteins. Centrioles help to assemble microtubules, long, hollow cylinders of the protein tubulin. Microtubules influence cell shape, move the chromosomes in cell division, and provide the functional internal structure of flagella and cilia, as we will discuss later. Centrioles may be contained in areas called microtubule-organizing centers (MTOCs). The cells of plants and fungi lack centrioles, and cell biolo- gists are still in the process of characterizing their MTOCs. Both mitochondria and chloroplasts contain specific genes related to some of their functions, but both depend on nuclear genes for other functions. Some centrioles also contain DNA, which apparently helps control the synthesis of their structural proteins. Chapter 5 Cell Structure 95 Outer membrane Inner membrane Granum Thylakoid Stroma FIGURE 5.22 Chloroplast structure. The inner membrane of a chloroplast is fused to form stacks of closed vesicles called thylakoids. Within these thylakoids, photosynthesis takes place. Thylakoids are typically stacked one on top of the other in columns called grana. 0.09 μm Microtubule triplet FIGURE 5.23 Centrioles. (a) This electron micrograph shows a pair of centrioles (75,000×). The round shape is a centriole in cross- section; the rectangular shape is a centriole in longitudinal section. (b) Each centriole is composed of nine triplets of microtubules. (a) (b) The Cytoskeleton: Interior Framework of the Cell The cytoplasm of all eukaryotic cells is crisscrossed by a network of protein fibers that supports the shape of the cell and anchors organelles to fixed locations. This net- work, called the cytoskeleton (figure 5.24), is a dynamic system, constantly forming and disassembling. Individual fibers form by polymerization, as identical protein sub- units attract one another chemically and spontaneously assemble into long chains. Fibers disassemble in the same way, as one subunit after another breaks away from one end of the chain. Eukaryotic cells may contain three types of cytoskeletal fibers, each formed from a different kind of subunit: 1. Actin filaments. Actin filaments are long fibers about 7 nanometers in diameter. Each filament is composed of two protein chains loosely twined to- gether like two strands of pearls (figure 5.25a). Each “pearl,” or subunit, on the chains is the globular pro- tein actin. Actin molecules spontaneously form these filaments, even in a test tube; a cell regulates the rate of their formation through other proteins that act as switches, turning on polymerization when appropriate. Actin filaments are responsible for cellular movements such as contraction, crawling, “pinching” during divi- sion, and formation of cellular extensions. 2. Microtubules. Microtubules are hollow tubes about 25 nanometers in diameter, each composed of a ring of 13 protein protofilaments (figure 5.25b). Globular proteins consisting of dimers of alpha and beta tubulin subunits polymerize to form the 13 protofilaments. The protofilaments are arrayed side by side around a central core, giving the microtubule its characteristic tube shape. In many cells, micro- tubules form from MTOC nucleation centers near the center of the cell and radiate toward the periph- ery. They are in a constant state of flux, continually polymerizing and depolymerizing (the average half- life of a microtubule ranges from 10 minutes in a nondividing animal cell to as short as 20 seconds in a dividing animal cell), unless stabilized by the binding of guanosine triphosphate (GTP) to the ends, which inhibits depolymerization. The ends of the micro- tubule are designated as “+” (away from the nucle- ation center) or “?” (toward the nucleation center). Along with allowing for cellular movement, micro- tubules are responsible for moving materials within the cell itself. Special motor proteins, discussed later in this chapter, move cellular organelles around the cell on microtubular “tracks.” Kinesin proteins move organelles toward the “+” end (toward the cell pe- riphery), and dyneins move them toward the “?” end. 96 Part II Biology of the Cell Nuclear envelope Nucleolus Ribosomes Mitochondrion Rough endoplasmic reticulum Smooth endoplasmic reticulum Cytoskeleton FIGURE 5.24 The cytoskeleton. In this diagrammatic cross-section of a eukaryotic cell, the cytoskeleton, a network of fibers, supports organelles such as mitochondria. 3. Intermediate filaments. The most durable ele- ment of the cytoskeleton in animal cells is a system of tough, fibrous protein molecules twined together in an overlapping arrangement (figure 5.25c). These fibers are characteristically 8 to 10 nanometers in di- ameter, intermediate in size between actin filaments and microtubules (which is why they are called in- termediate filaments). Once formed, intermediate filaments are stable and usually do not break down. Intermediate filaments constitute a heterogeneous group of cytoskeletal fibers. The most common type, composed of protein subunits called vimentin, provides structural stability for many kinds of cells. Keratin, another class of intermediate filament, is found in epithelial cells (cells that line organs and body cavities) and associated structures such as hair and fingernails. The intermediate filaments of nerve cells are called neurofilaments. As we will discuss in the next section, the cytoskeleton provides an interior framework that supports the shape of the cell, stretching the plasma membrane much as the poles of a circus tent. Changing the relative length of cytoskeleton filaments allows cells to rapidly alter their shape, extending projections out or folding inward. Within the cell, the framework of filaments provides a molecular highway along which molecules can be transported. Elements of the cytoskeleton crisscross the cytoplasm, supporting the cell shape and anchoring organelles in place. There are three principal types of fibers: actin filaments, microtubules, and intermediate filaments. Chapter 5 Cell Structure 97 (b) Microtubule(a) Actin filament (c) Intermediate filament Mitochondrion Microtubule Intermediate filament Ribosome Rough endoplasmic reticulum Actin filament Cell membrane FIGURE 5.25 Molecules that make up the cytoskeleton. (a) Actin filaments. Actin filaments are made of two strands of the fibrous protein actin twisted together and usually occur in bundles. Actin filaments are ubiquitous, although they are concentrated below the plasma membrane in bundles known as stress fibers, which may have a contractile function. (b) Microtubules. Microtubules are composed of 13 stacks of tubulin protein subunits arranged side by side to form a tube. Microtubules are comparatively stiff cytoskeletal elements that serve to organize metabolism and intracellular transport in the nondividing cell. (c) Intermediate filaments. Intermediate filaments are composed of overlapping staggered tetramers of protein. This molecular arrangement allows for a ropelike structure that imparts tremendous mechanical strength to the cell. Cell Movement Essentially all cell motion is tied to the movement of actin filaments, microtubules, or both. Intermediate filaments act as intracellular tendons, preventing excessive stretching of cells, and actin filaments play a major role in determining the shape of cells. Because actin filaments can form and dis- solve so readily, they enable some cells to change shape quickly. If you look at the surfaces of such cells under a mi- croscope, you will find them alive with motion, as projec- tions, called microvilli in animal cells, shoot outward from the surface and then retract, only to shoot out elsewhere moments later (figure 5.26). Some Cells Crawl It is the arrangement of actin filaments within the cell cy- toplasm that allows cells to “crawl,” literally! Crawling is a significant cellular phenomenon, essential to inflamma- tion, clotting, wound healing, and the spread of cancer. White blood cells in particular exhibit this ability. Pro- duced in the bone marrow, these cells are released into the circulatory system and then eventually crawl out of capillaries and into the tissues to destroy potential pathogens. Cells exist in a gel-sol state; that is, at any given time, some regions of the cell are rigid (gel) and some are more fluid (sol). The cell is typically more sol-like in its interior, and more gel-like at its perimeter. To crawl, the cell cre- ates a weak area in the gel perimeter, and then forces the fluid (sol) interior through the weak area, forming a pseudopod (“false foot”). As a result a large section of cy- toplasm oozes off in a different direction, but still remains within the plasma membrane. Once extended, the pseudo- pod stabilizes into a gel state, assembling actin filaments. Specific membrane proteins in the pseudopod stick to the surface the cell is crawling on, and the rest of the cell is dragged in that direction. The pressure required to force out a developing pseudopod is created when actin filaments in the trailing end of the cell contract, just as squeezing a water balloon at one end forces the balloon to bulge out at the other end. Moving Material within the Cell Actin filaments and microtubules often orchestrate their ac- tivities to affect cellular processes. For example, during cell reproduction (see chapter 11), newly replicated chromo- somes move to opposite sides of a dividing cell because they are attached to shortening microtubules. Then, in animal cells, a belt of actin pinches the cell in two by contracting like a purse string. Muscle cells also use actin filaments to contract their cytoskeletons. The fluttering of an eyelash, the flight of an eagle, and the awkward crawling of a baby all de- pend on these cytoskeletal movements within muscle cells. Not only is the cytoskeleton responsible for the cell’s shape and movement, but it also provides a scaffold that holds certain enzymes and other macromolecules in defined areas of the cytoplasm. Many of the enzymes involved in cell metabolism, for example, bind to actin filaments; so do ribosomes. By moving and anchoring particular enzymes near one another, the cytoskeleton, like the endoplasmic reticulum, organizes the cell’s activities. Intracellular Molecular Motors Certain eukaryotic cells must move materials from one place to another in the cytoplasm. Most cells use the endomem- brane system as an intracellular highway; the Golgi appara- tus packages materials into vesicles that move through the channels of the endoplasmic reticulum to the far reaches of the cell. However, this highway is only effective over short distances. When a cell has to transport materials through long extensions like the axon of a nerve cell, the ER high- ways are too slow. For these situations, eukaryotic cells have developed high-speed locomotives that run along micro- tubular tracks. Four components are required: (1) a vesicle or or- ganelle that is to be transported, (2) a motor molecule that provides the energy-driven motion, (3) a connector molecule that connects the vesicle to the motor mole- cule, and (4) microtubules on which the vesicle will ride like a train on a rail. For example, embedded within the membranes of endoplasmic reticulum is a protein called kinectin that bind the ER vesicles to the motor protein ki- nesin. As nature’s tiniest motors, these motor proteins lit- erally pull the transport vesicles along the microtubular tracks. Kinesin uses ATP to power its movement toward 98 Part II Biology of the Cell FIGURE 5.26 The surfaces of some cells are in constant motion. This amoeba, a single-celled protist, is advancing toward you, its advancing edges extending projections outward. The moving edges have been said to resemble the ruffled edges of a skirt. the cell periphery, dragging the vesicle with it as it travels along the microtubule. Another vesicle protein (or per- haps a slight modification of kinesin—further research is needed to determine which) binds vesicles to the motor protein dynein, which directs movement in the opposite direction, inward toward the cell’s center. (Dynein is also involved in the movement of eukaryotic flagella, as dis- cussed below.) The destination of a particular transport vesicle and its contents is thus determined by the nature of the linking protein embedded within the vesicle’s membrane. Swimming with Flagella and Cilia Earlier in this chapter, we described the structure of bacterial flagella. Eukaryotic cells have a completely different kind of flagellum, consisting of a circle of nine microtubule pairs surrounding two central microtubules; this arrangement is referred to as the 9 + 2 structure (figure 5.27). As pairs of microtubules move past one another using arms composed of the motor protein dynein, the eukaryotic flagellum undu- lates rather than rotates. When examined carefully, each flagellum proves to be an outward projection of the cell’s interior, containing cytoplasm and enclosed by the plasma membrane. The microtubules of the flagellum are derived from a basal body, situated just below the point where the flagellum protrudes from the surface of the cell. The flagellum’s complex microtubular apparatus evolved early in the history of eukaryotes. Although the cells of many multicellular and some unicellular eukaryotes today no longer possess flagella and are nonmotile, an organization similar to the 9 + 2 arrangement of microtubules can still be found within them, in structures called cilia (singular, cilium). Cilia are short cellular projections that are often organized in rows (see figure 5.1). They are more numerous than flagella on the cell surface, but have the same internal structure. In many multi- cellular organisms, cilia carry out tasks far removed from their original function of propelling cells through water. In several kinds of vertebrate tissues, for example, the beating of rows of cilia moves water over the tissue surface. The sensory cells of the vertebrate ear also contain cilia; sound waves bend these cilia, the initial sensory input of hearing. Thus, the 9 + 2 structure of flagella and cilia appears to be a fundamental component of eukaryotic cells. Some eukaryotic cells use pseudopodia to crawl about within multicellular organisms, while many protists swim using flagella and cilia. Materials are transported within cells by special motor proteins. Chapter 5 Cell Structure 99 Outer microtubule pair Microtubules Flagellum Basal body Plasma membrane Dynein arm Radial spoke Central microtubule pair (a) (b) (c) (d) 4.36 μm FIGURE 5.27 Flagella and cilia. (a) A eukaryotic flagellum originates directly from a basal body. (b) The flagellum has two microtubules in its core connected by radial spokes to an outer ring of nine paired microtubules with dynein arms. (c) The basal body consists of nine microtubule triplets connected by short protein segments. The structure of cilia is similar to that of flagella, but cilia are usually shorter. (d) The surface of this Paramecium is covered with a dense forest of cilia. Special Things about Plant Cells Vacuoles: A Central Storage Compartment The center of a plant cell usually contains a large, appar- ently empty space, called the central vacuole (figure 5.28). This vacuole is not really empty; it contains large amounts of water and other materials, such as sugars, ions, and pigments. The central vacuole functions as a storage center for these important substances and also helps to in- crease the surface-to-volume ratio of the plant cell by ap- plying pressure to the cell membrane. The cell membrane expands outward under this pressure, thereby increasing its surface area. Cell Walls: Protection and Support Plant cells share a characteristic with bacteria that is not shared with animal cells—that is, plants have cell walls, which protect and support the plant cell. Although bacteria also have cell walls, plant cell walls are chemically and struc- turally different from bacterial cell walls. Cell walls are also present in fungi and some protists. In plants, cell walls are composed of fibers of the polysaccharide cellulose. Primary walls are laid down when the cell is still growing, and be- tween the walls of adjacent cells is a sticky substance called the middle lamella, which glues the cells together (figure 5.29). Some plant cells produce strong secondary walls, which are deposited inside the primary walls of fully ex- panded cells. Plant cells store substances in a large central vacuole, and encase themselves within a strong cellulose cell wall. 100 Part II Biology of the Cell Cell Middle lamella Primary wall Secondary wall FIGURE 5.29 Cell walls in plants. As shown in this drawing (a) and transmission electron micrograph (b), plant cell walls are thicker, stronger, and more rigid than those of bacteria. Primary cell walls are laid down when the cell is young. Thicker secondary cell walls may be added later when the cell is fully grown. Primary walls Cell 1 Secondary wall Cell 2 (b) Middle lamella (a) 1.83 μm FIGURE 5.28 The central vacuole. A plant’s central vacuole stores dissolved substances and can increase in size to increase the surface area of a plant cell. Chapter 5 Cell Structure 101 Endosymbiosis Symbiosis is a close relationship between organisms of differ- ent species that live together. The theory of endosymbiosis proposes that some of today’s eukaryotic organelles evolved by a symbiosis in which one species of prokaryote was en- gulfed by and lived inside another species of prokaryote that was a precursor to eukaryotes (figure 5.30). According to the endosymbiont theory, the engulfed prokaryotes provided their hosts with certain advantages associated with their spe- cial metabolic abilities. Two key eukaryotic organelles are believed to be the descendants of these endosymbiotic prokaryotes: mitochondria, which are thought to have origi- nated as bacteria capable of carrying out oxidative metabo- lism; and chloroplasts, which apparently arose from photo- synthetic bacteria. The endosymbiont theory is supported by a wealth of evidence. Both mitochondria and chloroplasts are sur- rounded by two membranes; the inner membrane proba- bly evolved from the plasma membrane of the engulfed bacterium, while the outer membrane is probably derived from the plasma membrane or endoplasmic reticulum of the host cell. Mitochondria are about the same size as most bacteria, and the cristae formed by their inner membranes resemble the folded membranes in various groups of bac- teria. Mitochondrial ribosomes are also similar to bacterial ribosomes in size and structure. Both mitochondria and chloroplasts contain circular molecules of DNA similar to those in bacteria. Finally, mitochondria divide by simple fission, splitting in two just as bacterial cells do, and they apparently replicate and partition their DNA in much the same way as bacteria. Table 5.2 compares and reviews the features of three types of cells. Some eukaryotic organelles are thought to have arisen by endosymbiosis. 5.4 Symbiosis played a key role in the origin of some eukaryotic organelles. FIGURE 5.30 Endosymbiosis. This figure shows how a double membrane may have been created during the symbiotic origin of mitochondria or chloroplasts. Table 5.2 A Comparison of Bacterial, Animal, and Plant Cells Bacterium Animal Plant EXTERIOR STRUCTURES Cell wall Cell membrane Flagella INTERIOR STRUCTURES ER Ribosomes Microtubules Centrioles Golgi apparatus Nucleus Mitochondria Chloroplasts Chromosomes Lysosomes Vacuoles Present (protein-polysaccharide) Present May be present (single strand) Absent Present Absent Absent Absent Absent Absent Absent A single circle of DNA Absent Absent Absent Present May be present Usually present Present Present Present Present Present Present Absent Multiple; DNA-protein complex Usually present Absent or small Present (cellulose) Present Absent except in sperm of a few species Usually present Present Present Absent Present Present Present Present Multiple; DNA-protein complex Present Usually a large single vacuole Chapter 5 Summary Questions Media Resources 5.1 All organisms are composed of cells. ? The cell is the smallest unit of life. All living things are made of cells. ? The cell is composed of a nuclear region, which holds the hereditary apparatus, enclosed within the cytoplasm. ? In all cells, the cytoplasm is bounded by a membrane composed of phospholipid and protein. 102 Part II Biology of the Cell 1. What are the three principles of the cell theory? 2. How does the surface area- to-volume ratio of cells limit the size that cells can attain? 5.2 Eukaryotic cells are far more complex than bacterial cells. ? Bacteria, which have prokaryotic cell structure, do not have membrane-bounded organelles within their cells. Their DNA molecule is circular. ?The eukaryotic cell is larger and more complex, with many internal compartments. 3. How are prokaryotes different from eukaryotes in terms of their cell walls, interior organization, and flagella? ?A eukaryotic cell is organized into three principal zones: the nucleus, the cytoplasm, and the plasma membrane. Located in the cytoplasm are numerous organelles, which perform specific functions for the cell. ? Many of these organelles, such as the endoplasmic reticulum, Golgi apparatus (which gives rise to lysosomes), and nucleus, are part of a complex endomembrane system. ? Mitochondria and chloroplasts are part of the energy- processing system of the cell. ? The cytoskeleton encompasses a variety of fibrous proteins that provide structural support and perform other functions for the cell. ? Many eukaryotic cells possess flagella or cilia having a 9 + 2 arrangement of microtubules; sliding of the microtubules past one another bends these cellular appendages. ?Cells transport materials long distances within the cytoplasm by packaging them into vesicles that are pulled by motor proteins along microtubule tracks. 4. What is the endoplasmic reticulum? What is its function? How does rough ER differ from smooth ER? 5. What is the function of the Golgi apparatus? How do the substances released by the Golgi apparatus make their way to other locations in the cell? 6. What types of eukaryotic cells contain mitochondria? What function do mitochondria perform? 7. What unique metabolic activity occurs in chloroplasts? 8. What cellular functions do centrioles participate in? 9. What kinds of cytoskeleton fibers are stable and which are changeable? 10. How do cilia compare with eukaryotic flagella? 5.3 Take a tour of a eukaryotic cell. ? Present-day mitochondria and chloroplasts probably evolved as a consequence of early endosymbiosis: the ancestor of the eukaryotic cell engulfed a bacterium, and the bacterium continued to function within the host cell. 11. What is the endosymbiont theory? What is the evidence supporting this theory? 5.4 Symbiosis played a key role in the origin of some eukaryotic organelles. ? Exploration: Cell Size ? Surface to Volume ? Art Activities: -Animal Cell Structure -Plant Cell Structure -Nonphotosynthetic Bacterium -Cyanobacterium ? Scientists on Science: The Joy of Discovery ? Art Quizzes: -Nucleosomes -Rough ER and Protein Synthesis -Protein Transport ? Art Activities: -Anatomy of the Nucleus -Golgi Apparatus Structure -Mitochondrion Structure -Organization of Cristae -Chloroplast Structure -The Cytoskeleton -Plant Cell ? Endomembrane ?Energy Organelles ? Cytoskeleton BIOLOGY RAVEN JOHNSON SIX TH EDITION www.mhhe.com/raven6/resources5.mhtml 103 6 Membranes Concept Outline 6.1 Biological membranes are fluid layers of lipid. The Phospholipid Bilayer. Cells are encased by membranes composed of a bilayer of phospholipid. The Lipid Bilayer Is Fluid. Because individual phospholipid molecules do not bind to one another, the lipid bilayer of membranes is a fluid. 6.2 Proteins embedded within the plasma membrane determine its character. The Fluid Mosaic Model. A varied collection of proteins float within the lipid bilayer. Examining Cell Membranes. Visualizing a plasma membrane requires a powerful electron microscope. Kinds of Membrane Proteins. The proteins in a membrane function in support, transport, recognition, and reactions. Structure of Membrane Proteins. Membrane proteins are anchored into the lipid bilayer by their nonpolar regions. 6.3 Passive transport across membranes moves down the concentration gradient. Diffusion. Random molecular motion results in a net movement of molecules to regions of lower concentration. Facilitated Diffusion. Passive movement across a membrane is often through specific carrier proteins. Osmosis. Polar solutes interact with water and can affect the movement of water across semipermeable membranes. 6.4 Bulk transport utilizes endocytosis. Bulk Passage Into and Out of the Cell. To transport large particles, membranes form vesicles. 6.5 Active transport across membranes is powered by energy from ATP. Active Transport. Cells transport molecules up a concentration gradient using ATP-powered carrier proteins. Coupled Transport. Active transport of ions drives coupled uptake of other molecules up their concentration gradients. A mong a cell’s most important activities are its interac- tions with the environment, a give and take that never ceases. Without it, life could not persist. While living cells and eukaryotic organelles (figure 6.1) are encased within a lipid membrane through which few water-soluble sub- stances can pass, the membrane contains protein passage- ways that permit specific substances to move in and out of the cell and allow the cell to exchange information with its environment. We call this delicate skin of protein mole- cules embedded in a thin sheet of lipid a plasma mem- brane. This chapter will examine the structure and func- tion of this remarkable membrane. FIGURE 6.1 Membranes within a human cell. Sheets of endoplasmic reticulum weave through the cell interior. The large oval is a mitochondrion, itself filled with extensive internal membranes. just as a layer of oil impedes the passage of a drop of water (“oil and water do not mix”). This barrier to the passage of water-soluble substances is the key biological property of the lipid bilayer. In addition to the phospholipid molecules that make up the lipid bilayer, the membranes of every cell also contain proteins that extend through the lipid bilayer, providing passageways across the membrane. The basic foundation of biological membranes is a lipid bilayer, which forms spontaneously. In such a layer, the nonpolar hydrophobic tails of phospholipid molecules point inward, forming a nonpolar barrier to water-soluble molecules. 104 Part II Biology of the Cell The Phospholipid Bilayer The membranes that encase all living cells are sheets of lipid only two molecules thick; more than 10,000 of these sheets piled on one another would just equal the thickness of this sheet of paper. The lipid layer that forms the foun- dation of a cell membrane is composed of molecules called phospholipids (figure 6.2). Phospholipids Like the fat molecules you studied in chapter 3, a phos- pholipid has a backbone derived from a three-carbon molecule called glycerol. Attached to this backbone are fatty acids, long chains of carbon atoms ending in a car- boxyl (—COOH) group. A fat molecule has three such chains, one attached to each carbon in the backbone; be- cause these chains are nonpolar, they do not form hydro- gen bonds with water, and the fat molecule is not water- soluble. A phospholipid, by contrast, has only two fatty acid chains attached to its backbone. The third carbon on the backbone is attached instead to a highly polar organic alcohol that readily forms hydrogen bonds with water. Because this alcohol is attached by a phosphate group, the molecule is called a phospholipid. One end of a phospholipid molecule is, therefore, strongly nonpolar (water-insoluble), while the other end is strongly polar (water-soluble). The two nonpolar fatty acids extend in one direction, roughly parallel to each other, and the polar alcohol group points in the other di- rection. Because of this structure, phospholipids are often diagrammed as a polar head with two dangling nonpolar tails (as in figure 6.2b). Phospholipids Form Bilayer Sheets What happens when a collection of phospholipid molecules is placed in water? The polar water molecules repel the long nonpolar tails of the phospholipids as the water mole- cules seek partners for hydrogen bonding. Due to the polar nature of the water molecules, the nonpolar tails of the phospholipids end up packed closely together, sequestered as far as possible from water. Every phospholipid molecule orients to face its polar head toward water and its nonpolar tails away. When two layers form with the tails facing each other, no tails ever come in contact with water. The result- ing structure is called a lipid bilayer (figure 6.3). Lipid bi- layers form spontaneously, driven by the tendency of water molecules to form the maximum number of hydrogen bonds. The nonpolar interior of a lipid bilayer impedes the pas- sage of any water-soluble substances through the bilayer, 6.1 Biological membranes are f luid layers of lipid. Fatty acid Phosphorylated alcohol (a) (b) Polar (hydrophilic) region Nonpolar (hydrophobic) region Fatty acid G L Y C E R O L FIGURE 6.2 Phospholipid structure. (a) A phospholipid is a composite molecule similar to a triacylglycerol, except that only two fatty acids are bound to the glycerol backbone; a phosphorylated alcohol occupies the third position on the backbone. (b) Because the phosphorylated alcohol usually extends from one end of the molecule and the two fatty acid chains extend from the other, phospholipids are often diagrammed as a polar head with two nonpolar hydrophobic tails. The Lipid Bilayer Is Fluid A lipid bilayer is stable because water’s affinity for hydro- gen bonding never stops. Just as surface tension holds a soap bubble together, even though it is made of a liquid, so the hydrogen bonding of water holds a membrane to- gether. But while water continually drives phospholipid molecules into this configuration, it does not locate specific phospholipid molecules relative to their neighbors in the bilayer. As a result, individual phospholipids and unan- chored proteins are free to move about within the mem- brane. This can be demonstrated vividly by fusing cells and watching their proteins reassort (figure 6.4). Phospholipid bilayers are fluid, with the viscosity of olive oil (and like oil, their viscosity increases as the tem- perature decreases). Some membranes are more fluid than others, however. The tails of individual phospholipid mole- cules are attracted to one another when they line up close together. This causes the membrane to become less fluid, because aligned molecules must pull apart from one an- other before they can move about in the membrane. The greater the degree of alignment, the less fluid the mem- brane. Some phospholipid tails do not align well because they contain one or more double bonds between carbon atoms, introducing kinks in the tail. Membranes containing such phospholipids are more fluid than membranes that lack them. Most membranes also contain steroid lipids like cholesterol, which can either increase or decrease mem- brane fluidity, depending on temperature. The lipid bilayer is liquid like a soap bubble, rather than solid like a rubber balloon. Chapter 6 Membranes 105 Polar hydrophilic heads Nonpolar hydrophobic tails Polar hydrophilic heads FIGURE 6.3 A phospholipid bilayer. The basic structure of every plasma membrane is a double layer of lipid, in which phospholipids aggregate to form a bilayer with a nonpolar interior. The phospholipid tails do not align perfectly and the membrane is “fluid.” Individual phospholipid molecules can move from one place to another in the membrane. Mouse cell Fusion of cells Intermixed membrane proteins Human cell FIGURE 6.4 Proteins move about in membranes. Protein movement within membranes can be demonstrated easily by labeling the plasma membrane proteins of a mouse cell with fluorescent antibodies and then fusing that cell with a human cell. At first, all of the mouse proteins are located on the mouse side of the fused cell and all of the human proteins are located on the human side of the fused cell. However, within an hour, the labeled and unlabeled proteins are intermixed throughout the hybrid cell’s plasma membrane. The Fluid Mosaic Model A plasma membrane is composed of both lipids and glob- ular proteins. For many years, biologists thought the pro- tein covered the inner and outer surfaces of the phospho- lipid bilayer like a coat of paint. The widely accepted Davson-Danielli model, proposed in 1935, portrayed the membrane as a sandwich: a phospholipid bilayer between two layers of globular protein. This model, however, was not consistent with what researchers were learning in the 1960s about the structure of membrane proteins. Unlike most proteins found within cells, membrane proteins are not very soluble in water—they possess long stretches of nonpolar hydrophobic amino acids. If such proteins in- deed coated the surface of the lipid bilayer, as the Davson-Danielli model suggests, then their nonpolar por- tions would separate the polar portions of the phospho- lipids from water, causing the bilayer to dissolve! Because this doesn’t happen, there is clearly something wrong with the model. In 1972, S. Singer and G. Nicolson revised the model in a simple but profound way: they proposed that the globular proteins are inserted into the lipid bilayer, with their nonpo- lar segments in contact with the nonpolar interior of the bilayer and their polar portions protruding out from the membrane surface. In this model, called the fluid mosaic model, a mosaic of proteins float in the fluid lipid bilayer like boats on a pond (figure 6.5). Components of the Cell Membrane A eukaryotic cell contains many membranes. While they are not all identical, they share the same fundamental ar- chitecture. Cell membranes are assembled from four com- ponents (table 6.1): 1. Lipid bilayer. Every cell membrane is composed of a phospholipid bilayer. The other components of the membrane are enmeshed within the bilayer, which provides a flexible matrix and, at the same time, im- poses a barrier to permeability. 106 Part II Biology of the Cell 6.2 Proteins embedded within the plasma membrane determine its character. Extracellular fluid Carbohydrate Glycolipid Transmembrane protein Glycoprotein Peripheral protein Cholesterol Filaments of cytoskeleton Cytoplasm FIGURE 6.5 The fluid mosaic model of the plasma membrane. A variety of proteins protrude through the plasma membrane of animal cells, and nonpolar regions of the proteins tether them to the membrane’s nonpolar interior. The three principal classes of membrane proteins are transport proteins, receptors, and cell surface markers. Carbohydrate chains are often bound to the extracellular portion of these proteins, as well as to the membrane phospholipids. These chains serve as distinctive identification tags, unique to particular cells. 2. Transmembrane proteins. A major component of every membrane is a collection of proteins that float on or in the lipid bilayer. These proteins provide pas- sageways that allow substances and information to cross the membrane. Many membrane proteins are not fixed in position; they can move about, as the phospholipid molecules do. Some membranes are crowded with proteins, while in others, the proteins are more sparsely distributed. 3. Network of supporting fibers. Membranes are structurally supported by intracellular proteins that reinforce the membrane’s shape. For example, a red blood cell has a characteristic biconcave shape because a scaffold of proteins called spectrin links proteins in the plasma membrane with actin filaments in the cell’s cytoskeleton. Membranes use networks of other pro- teins to control the lateral movements of some key membrane proteins, anchoring them to specific sites. 4. Exterior proteins and glycolipids. Membrane sections assemble in the endoplasmic reticulum, transfer to the Golgi complex, and then are trans- ported to the plasma membrane. The endoplasmic reticulum adds chains of sugar molecules to mem- brane proteins and lipids, creating a “sugar coating” called the glycocalyx that extends from the membrane on the outside of the cell only. Different cell types ex- hibit different varieties of these glycoproteins and glycolipids on their surfaces, which act as cell identity markers. The fluid mosaic model proposes that membrane proteins are embedded within the lipid bilayer. Membranes are composed of a lipid bilayer within which proteins are anchored. Plasma membranes are supported by a network of fibers and coated on the exterior with cell identity markers. Chapter 6 Membranes 107 Table 6.1 Components of the Cell Membrane Component Composition Function How It Works Example Phospholipid bilayer Carriers Channels Receptors Spectrins Clathrins Glycoproteins Glycolipid Provides permeability barrier, matrix for proteins Transport molecules across membrane against gradient Passively transport molecules across membrane Transmit information into cell Determine shape of cell Anchor certain proteins to specific sites, especially on the exterior cell membrane in receptor-mediated endocytosis “Self”-recognition Tissue recognition Excludes water-soluble molecules from nonpolar interior of bilayer “Escort” molecules through the membrane in a series of conformational changes Create a tunnel that acts as a passage through membrane Signal molecules bind to cell- surface portion of the receptor protein; this alters the portion of the receptor protein within the cell, inducing activity Form supporting scaffold beneath membrane, anchored to both membrane and cytoskeleton Proteins line coated pits and facilitate binding to specific molecules Create a protein/carbohydrate chain shape characteristic of individual Create a lipid/carbohydrate chain shape characteristic of tissue Phospholipid molecules Transmembrane proteins Interior protein network Cell surface markers Bilayer of cell is impermeable to water- soluble molecules, like glucose Glycophorin carrier for sugar transport Sodium and potassium channels in nerve cells Specific receptors bind peptide hormones and neurotransmitters Red blood cell Localization of low- density lipoprotein receptor within coated pits Major histocompatibility complex protein recognized by immune system A, B, O blood group markers Examining Cell Membranes Biologists examine the delicate, filmy struc- ture of a cell membrane using electron mi- croscopes that provide clear magnification to several thousand times. We discussed two types of electron microscopes in chap- ter 5: the transmission electron microscope (TEM) and the scanning electron micro- scope (SEM). When examining cell mem- branes with electron microscopy, speci- mens must be prepared for viewing. In one method of preparing a specimen, the tissue of choice is embedded in a hard matrix, usually some sort of epoxy (figure 6.6). The epoxy block is then cut with a microtome, a machine with a very sharp blade that makes incredibly thin slices. The knife moves up and down as the spec- imen advances toward it, causing transpar- ent “epoxy shavings” less than 1 microme- ter thick to peel away from the block of tissue. These shavings are placed on a grid and a beam of electrons is directed through the grid with the TEM. At the high magnification an electron microscope provides, resolution is good enough to re- veal the double layers of a membrane. Freeze-fracturing a specimen is another way to visualize the inside of the mem- brane. The tissue is embedded in a medium and quick-frozen with liquid ni- trogen. The frozen tissue is then “tapped” with a knife, causing a crack between the phospholipid layers of membranes. Pro- teins, carbohydrates, pits, pores, channels, or any other structure affiliated with the membrane will pull apart (whole, usually) and stick with one side of the split mem- brane. A very thin coating of platinum is then evaporated onto the fractured surface forming a replica of “cast” of the surface. Once the topography of the membrane has been preserved in the “cast,” the actual tis- sue is dissolved away, and the “cast” is ex- amined with electron microscopy, creating a strikingly different view of the mem- brane (see figure 5.10b). Visualizing a plasma membrane requires a very powerful electron microscope. Electrons can either be passed through a sample or bounced off it. 108 Part II Biology of the Cell 1. A small chunk of tissue containing cells of interest is preserved chemically. 3. A diamond knife sections the tissue-epoxy block like a loaf of bread, creating slices 25 nm thick. 2. The tissue is embedded in epoxy and allowed to harden. Knife Forceps Grid Section Tissue Wax paper Grid Section Lead "stain" Tissue Epoxy 4. A tissue section is mounted on a small grid. 5. The section on the grid is "stained" with an electron- dense element (such as lead). 6. The section is examined by directing a beam of electrons through the grid in the transmission electron microscope (TEM). 7. The high resolution of the TEM allows detailed examination of ultrathin sections of tissues and cells. FIGURE 6.6 Thin section preparation for viewing membranes with electron microscopy. Kinds of Membrane Proteins As we’ve seen, the plasma membrane is a complex assem- bly of proteins enmeshed in a fluid array of phospholipid molecules. This enormously flexible design permits a broad range of interactions with the environment, some directly involving membrane proteins (figure 6.7). Though cells interact with their environment through their plasma membranes in many ways, we will focus on six key classes of membrane protein in this and the following chapter (chapter 7). 1. Transporters. Membranes are very selective, al- lowing only certain substances to enter or leave the cell, either through channels or carriers. In some in- stances, they take up molecules already present in the cell in high concentration. 2. Enzymes. Cells carry out many chemical reactions on the interior surface of the plasma membrane, using enzymes attached to the membrane. 3. Cell surface receptors. Membranes are exquisitely sensitive to chemical messages, detecting them with re- ceptor proteins on their surfaces that act as antennae. 4. Cell surface identity markers. Membranes carry cell surface markers that identify them to other cells. Most cell types carry their own ID tags, specific com- binations of cell surface proteins characteristic of that cell type. 5. Cell adhesion proteins. Cells use specific proteins to glue themselves to one another. Some act like Vel- cro, while others form a more permanent bond. 6. Attachments to the cytoskeleton. Surface pro- teins that interact with other cells are often anchored to the cytoskeleton by linking proteins. The many proteins embedded within a membrane carry out a host of functions, many of which are associated with transport of materials or information across the membrane. Chapter 6 Membranes 109 Outside Plasma membrane Inside Transporter Cell surface receptorEnzyme Cell surface identity marker Attachment to the cytoskeleton Cell adhesion Figure 6.7 Functions of plasma membrane proteins. Membrane proteins act as transporters, enzymes, cell surface receptors, and cell surface markers, as well as aiding in cell-to-cell adhesion and securing the cytoskeleton. Structure of Membrane Proteins If proteins float on lipid bilayers like ships on the sea, how do they manage to extend through the membrane to create channels, and how can certain proteins be anchored into particular positions on the cell membrane? Anchoring Proteins in the Bilayer Many membrane proteins are attached to the surface of the membrane by special molecules that associate with phos- pholipids and thereby anchor the protein to the membrane. Like a ship tied up to a floating dock, these proteins are free to move about on the surface of the membrane teth- ered to a phospholipid. In contrast, other proteins actually traverse the lipid bi- layer. The part of the protein that extends through the lipid bilayer, in contact with the nonpolar interior, consists of one or more nonpolar helices or several β-pleated sheets of nonpolar amino acids (figure 6.8). Because water avoids nonpolar amino acids much as it does nonpolar lipid chains, the nonpolar portions of the protein are held within the interior of the lipid bilayer. Although the polar ends of the protein protrude from both sides of the membrane, the protein itself is locked into the membrane by its nonpolar segments. Any movement of the protein out of the mem- brane, in either direction, brings the nonpolar regions of the protein into contact with water, which “shoves” the protein back into the interior. Extending Proteins across the Bilayer Cells contain a variety of different transmembrane pro- teins, which differ in the way they traverse the bilayer, de- pending on their functions. Anchors. A single nonpolar segment is adequate to an- chor a protein in the membrane. Anchoring proteins of this sort attach the spectrin network of the cytoskeleton to the interior of the plasma membrane (figure 6.9). Many pro- teins that function as receptors for extracellular signals are also “single-pass” anchors that pass through the membrane only once. The portion of the receptor that extends out from the cell surface binds to specific hormones or other molecules when the cell encounters them; the binding in- duces changes at the other end of the protein, in the cell’s interior. In this way, information outside the cell is trans- lated into action within the cell. The mechanisms of cell signaling will be addressed in detail in chapter 7. Channels. Other proteins have several helical segments that thread their way back and forth through the mem- brane, forming a channel like the hole in a doughnut. For example, bacteriorhodopsin is one of the key transmem- brane proteins that carries out photosynthesis in bacteria. It contains seven nonpolar helical segments that traverse the membrane, forming a circular pore through which protons pass during the light-driven pumping of protons (figure 6.10). Other transmembrane proteins do not create chan- nels but rather act as carriers to transport molecules across the membrane. All water-soluble molecules or ions that enter or leave the cell are either transported by carriers or pass through channels. Pores. Some transmembrane proteins have extensive nonpolar regions with secondary configurations of β- pleated sheets instead of α helices. The β sheets form a characteristic motif, folding back and forth in a circle so the sheets come to be arranged like the staves of a barrel. This so-called β barrel, open on both ends, is a common feature of the porin class of proteins that are found within the outer membrane of some bacteria (figure 6.11). Transmembrane proteins are anchored into the bilayer by their nonpolar segments. While anchor proteins may pass through the bilayer only once, many channels and pores are created by proteins that pass back and forth through the bilayer repeatedly, creating a circular hole in the bilayer. 110 Part II Biology of the Cell Phospholipids Polar areas of protein Cholesterol Nonpolar areas of protein FIGURE 6.8 How nonpolar regions lock proteins into membranes. A spiral helix of nonpolar amino acids (red) extends across the nonpolar lipid interior, while polar (purple) portions of the protein protrude out from the bilayer. The protein cannot move in or out because such a movement would drag nonpolar segments of the protein into contact with water. Chapter 6 Membranes 111 Cytoplasmic side of cell membrane Cytoskeletal proteins Junctional complex 100 nm Ankyrin Actin Glycophorin Spectrin Linker protein FIGURE 6.9 Anchoring proteins. Spectrin extends as a mesh anchored to the cytoplasmic side of a red blood cell plasma membrane. The spectrin protein is represented as a twisted dimer, attached to the membrane by special proteins such as junctional complexes and ankyrin; glycophorins can also be involved in attachments. This cytoskeletal protein network confers resiliency to cells like the red blood cell. NH 2 H + H + COOH Cytoplasm Retinal chromophore Nonpolar (hydrophobic) H9251-helices in the cell membrane FIGURE 6.10 A channel protein. This transmembrane protein mediates photosynthesis in the bacterium Halobacterium halobium. The protein traverses the membrane seven times with hydrophobic helical strands that are within the hydrophobic center of the lipid bilayer. The helical regions form a channel across the bilayer through which protons are pumped by the retinal chromophore (green). Bacterial outer membrane Porin monomer H9252-pleated sheets FIGURE 6.11 A pore protein. The bacterial transmembrane protein porin creates large open tunnels called pores in the outer membrane of a bacterium. Sixteen strands of β-pleated sheets run antiparallel to each other, creating a β barrel in the bacterial outer cell membrane. The tunnel allows water and other materials to pass through the membrane. 112 Part II Biology of the Cell Diffusion Molecules and ions dissolved in water are in constant mo- tion, moving about randomly. This random motion causes a net movement of these substances from regions where their concentration is high to regions where their concen- tration is lower, a process called diffusion (figure 6.12). Net movement driven by diffusion will continue until the concentrations in all regions are the same. You can demon- strate diffusion by filling a jar to the brim with ink, capping it, placing it at the bottom of a bucket of water, and then carefully removing the cap. The ink molecules will slowly diffuse out from the jar until there is a uniform concentra- tion in the bucket and the jar. This uniformity in the con- centration of molecules is a type of equilibrium. Facilitated Transport Many molecules that cells require, including glucose and other energy sources, are polar and cannot pass through the nonpolar interior of the phospholipid bilayer. These molecules enter the cell through specific channels in the plasma membrane. The inside of the channel is polar and thus “friendly” to the polar molecules, facilitating their transport across the membrane. Each type of biomolecule that is transported across the plasma membrane has its own type of transporter (that is, it has its own channel which fits it like a glove and cannot be used by other molecules). Each channel is said to be selective for that type of molecule, and thus to be selectively permeable, as only molecules admit- ted by the channels it possesses can enter it. The plasma membrane of a cell has many types of channels, each selec- tive for a different type of molecule. Diffusion of Ions through Channels One of the simplest ways for a substance to diffuse across a cell membrane is through a channel, as ions do. Ions are solutes (substances dissolved in water) with an unequal number of protons and electrons. Those with an excess of protons are positively charged and called cations. Ions with more electrons are negatively charged and called anions. Because they are charged, ions interact well with polar molecules like water but are repelled by the nonpolar inte- rior of a phospholipid bilayer. Therefore, ions cannot move between the cytoplasm of a cell and the extracellular fluid without the assistance of membrane transport proteins. Ion channels possess a hydrated interior that spans the mem- brane. Ions can diffuse through the channel in either direc- tion without coming into contact with the hydrophobic tails of the phospholipids in the membrane, and the trans- ported ions do not bind to or otherwise interact with the channel proteins. Two conditions determine the direction of net movement of the ions: their relative concentrations on either side of the membrane, and the voltage across the membrane (a topic we’ll explore in chapter 54). Each type of channel is specific for a particular ion, such as calcium (Ca ++ ) or chloride (Cl – ), or in some cases for a few kinds of ions. Ion channels play an essential role in signaling by the nervous system. Diffusion is the net movement of substances to regions of lower concentration as a result of random spontaneous motion. It tends to distribute substances uniformly. Membrane transport proteins allow only certain molecules and ions to diffuse through the plasma membrane. 6.3 Passive transport across membranes moves down the concentration gradient. Lump of sugar Sugar molecule FIGURE 6.12 Diffusion. If a lump of sugar is dropped into a beaker of water (a), its molecules dissolve (b) and diffuse (c). Eventually, diffusion results in an even distribution of sugar molecules throughout the water (d). (a) (b) (c) (d) Facilitated Diffusion Carriers, another class of membrane proteins, transport ions as well as other solutes like sugars and amino acids across the membrane. Like channels, carriers are specific for a certain type of solute and can trans- port substances in either direction across the membrane. Unlike chan- nels, however, they facilitate the movement of solutes across the mem- brane by physically binding to them on one side of the membrane and re- leasing them on the other. Again, the direction of the solute’s net movement simply depends on its concentration gradient across the membrane. If the concentration is greater in the cyto- plasm, the solute is more likely to bind to the carrier on the cytoplasmic side of the membrane and be released on the extracellular side. This will cause a net movement from inside to outside. If the concentration is greater in the extracellular fluid, the net movement will be from out- side to inside. Thus, the net movement always occurs from areas of high concentration to low, just as it does in simple diffusion, but carriers facilitate the process. For this rea- son, this mechanism of transport is sometimes called facil- itated diffusion (figure 6.13). Facilitated Diffusion in Red Blood Cells Several examples of facilitated diffusion by carrier proteins can be found in the membranes of vertebrate red blood cells (RBCs). One RBC carrier protein, for example, trans- ports a different molecule in each direction: Cl – in one di- rection and bicarbonate ion (HCO 3 – ) in the opposite direc- tion. As you will learn in chapter 52, this carrier is important in transporting carbon dioxide in the blood. A second important facilitated diffusion carrier in RBCs is the glucose transporter. Red blood cells keep their inter- nal concentration of glucose low through a chemical trick: they immediately add a phosphate group to any entering glucose molecule, converting it to a highly charged glucose phosphate that cannot pass back across the membrane. This maintains a steep concentration gradient for glucose, favoring its entry into the cell. The glucose transporter that carries glucose into the cell does not appear to form a channel in the membrane for the glucose to pass through. Instead, the transmembrane protein appears to bind the glucose and then flip its shape, dragging the glucose through the bilayer and releasing it on the inside of the plasma membrane. Once it releases the glucose, the glucose transporter reverts to its original shape. It is then available to bind the next glucose molecule that approaches the out- side of the cell. Transport through Selective Channels Saturates A characteristic feature of transport through selective chan- nels is that its rate is saturable. In other words, if the con- centration gradient of a substance is progressively in- creased, its rate of transport will also increase to a certain point and then level off. Further increases in the gradient will produce no additional increase in rate. The explanation for this observation is that there are a limited number of carriers in the membrane. When the concentration of the transported substance rises high enough, all of the carriers will be in use and the capacity of the transport system will be saturated. In contrast, substances that move across the membrane by simple diffusion (diffusion through channels in the bilayer without the assistance of carriers) do not show saturation. Facilitated diffusion provides the cell with a ready way to prevent the buildup of unwanted molecules within the cell or to take up needed molecules, such as sugars, that may be present outside the cell in high concentrations. Fa- cilitated diffusion has three essential characteristics: 1. It is specific. Any given carrier transports only cer- tain molecules or ions. 2. It is passive. The direction of net movement is de- termined by the relative concentrations of the trans- ported substance inside and outside the cell. 3. It saturates. If all relevant protein carriers are in use, increases in the concentration gradient do not in- crease the transport rate. Facilitated diffusion is the transport of molecules and ions across a membrane by specific carriers in the direction of lower concentration of those molecules or ions. Chapter 6 Membranes 113 Outside of cell Inside of cell FIGURE 6.13 Facilitated diffusion is a carrier-mediated transport process. Molecules bind to a receptor on the extracellular side of the cell and are conducted through the plasma membrane by a membrane protein. Osmosis The cytoplasm of a cell contains ions and molecules, such as sugars and amino acids, dissolved in water. The mixture of these substances and water is called an aqueous solu- tion. Water, the most common of the molecules in the mixture, is the solvent, and the substances dissolved in the water are solutes. The ability of water and solutes to dif- fuse across membranes has important consequences. Molecules Diffuse down a Concentration Gradient Both water and solutes diffuse from regions of high con- centration to regions of low concentration; that is, they dif- fuse down their concentration gradients. When two re- gions are separated by a membrane, what happens depends on whether or not the solutes can pass freely through that membrane. Most solutes, including ions and sugars, are not lipid-soluble and, therefore, are unable to cross the lipid bi- layer of the membrane. Even water molecules, which are very polar, cannot cross a lipid bilayer. Water flows through aquaporins, which are specialized channels for water. A simple experi- ment demonstrates this. If you place an amphibian egg in hypotonic spring water, it does not swell. If you then inject aquaporin mRNA into the egg, the channel proteins are ex- pressed and the egg then swells. Dissolved solutes interact with water molecules, which form hydration shells about the charged solute. When there is a concentration gradient of solutes, the solutes will move from a high to a low concentration, dragging with them their hydration shells of water molecules. When a membrane sepa- rates two solutions, hydration shell water molecules move with the diffusing ions, creating a net movement of water to- wards the low solute. This net water movement across a membrane by diffusion is called osmosis (figure 6.14). The concentration of all solutes in a solution determines the osmotic concentration of the solution. If two solu- tions have unequal osmotic concentrations, the solution with the higher concentration is hyperosmotic (Greek hyper, “more than”), and the solution with the lower con- centration is hypoosmotic (Greek hypo, “less than”). If the osmotic concentrations of two solutions are equal, the solu- tions are isosmotic (Greek iso, “the same”). In cells, a plasma membrane separates two aqueous solu- tions, one inside the cell (the cytoplasm) and one outside 114 Part II Biology of the Cell 3% salt solution Selectively permeable membrane Distilled water Salt solution rising Solution stops rising when weight of column equals osmotic pressure (a) (b) (c) FIGURE 6.14 An experiment demonstrating osmosis. (a) The end of a tube containing a salt solution is closed by stretching a selectively permeable membrane across its face; the membrane allows the passage of water molecules but not salt ions. (b) When this tube is immersed in a beaker of distilled water, the salt cannot cross the membrane, but water can. The water entering the tube causes the salt solution to rise in the tube. (c) Water will continue to enter the tube from the beaker until the weight of the column of water in the tube exerts a downward force equal to the force drawing water molecules upward into the tube. This force is referred to as osmotic pressure. Shriveled cells Normal cells Cells swell and eventually burst Cell body shrinks from cell wall Flaccid cell Normal turgid cell Human red blood cells Plant cells Hyperosmotic solution Isosmotic solution Hypoosmotic solution FIGURE 6.15 Osmosis. In a hyperosmotic solution water moves out of the cell toward the higher concentration of solutes, causing the cell to shrivel. In an isosmotic solution, the concentration of solutes on either side of the membrane is the same. Osmosis still occurs, but water diffuses into and out of the cell at the same rate, and the cell doesn’t change size. In a hypoosmotic solution the concentration of solutes is higher within the cell than without, so the net movement of water is into the cell. (the extracellular fluid). The direction of the net diffusion of water across this membrane is determined by the os- motic concentrations of the solutions on either side (figure 6.15). For example, if the cytoplasm of a cell were hypoos- motic to the extracellular fluid, water would diffuse out of the cell, toward the solution with the higher concentration of solutes (and, therefore, the lower concentration of un- bound water molecules). This loss of water from the cyto- plasm would cause the cell to shrink until the osmotic con- centrations of the cytoplasm and the extracellular fluid become equal. Osmotic Pressure What would happen if the cell’s cytoplasm were hyperos- motic to the extracellular fluid? In this situation, water would diffuse into the cell from the extracellular fluid, causing the cell to swell. The pressure of the cytoplasm pushing out against the cell membrane, or hydrostatic pressure, would increase. On the other hand, the osmotic pressure (figure 6.16), defined as the pressure that must be applied to stop the osmotic movement of water across a membrane, would also be at work. If the membrane were strong enough, the cell would reach an equilibrium, at which the osmotic pressure, which tends to drive water into the cell, is exactly counterbalanced by the hydrostatic pres- sure, which tends to drive water back out of the cell. How- ever, a plasma membrane by itself cannot withstand large internal pressures, and an isolated cell under such condi- tions would burst like an overinflated balloon. Accordingly, it is important for animal cells to maintain isosmotic condi- tions. The cells of bacteria, fungi, plants, and many pro- tists, in contrast, are surrounded by strong cell walls. The cells of these organisms can withstand high internal pres- sures without bursting. Maintaining Osmotic Balance Organisms have developed many solutions to the osmotic dilemma posed by being hyperosmotic to their environment. Extrusion. Some single-celled eukaryotes like the protist Paramecium use organelles called contractile vacuoles to re- move water. Each vacuole collects water from various parts of the cytoplasm and transports it to the central part of the vacuole, near the cell surface. The vacuole possesses a small pore that opens to the outside of the cell. By contracting rhythmically, the vacuole pumps the water out of the cell through the pore. Isosmotic Solutions. Some organisms that live in the ocean adjust their internal concentration of solutes to match that of the surrounding seawater. Isosmotic with re- spect to their environment, there is no net flow of water into or out of these cells. Many terrestrial animals solve the problem in a similar way, by circulating a fluid through their bodies that bathes cells in an isosmotic solution. The blood in your body, for example, contains a high concen- tration of the protein albumin, which elevates the solute concentration of the blood to match your cells. Turgor. Most plant cells are hyperosmotic to their im- mediate environment, containing a high concentration of solutes in their central vacuoles. The resulting internal hy- drostatic pressure, known as turgor pressure, presses the plasma membrane firmly against the interior of the cell wall, making the cell rigid. The newer, softer portions of trees and shrubs depend on turgor pressure to maintain their shape, and wilt when they lack sufficient water. Osmosis is the diffusion of water, but not solutes, across a membrane. Chapter 6 Membranes 115 Urea molecule Water molecules Semipermeable membrane FIGURE 6.16 How solutes create osmotic pressure. Charged or polar substances are soluble in water because they form hydrogen bonds with water molecules clustered around them. When a polar solute (illustrated here with urea) is added to the solution on one side of a membrane, the water molecules that gather around each urea molecule are no longer free to diffuse across the membrane; in effect, the polar solute has reduced the number of free water molecules on that side of the membrane increasing the osmotic pressure. Because the hypoosmotic side of the membrane (on the right, with less solute) has more unbound water molecules than the hyperosmotic side (on the left, with more solute), water moves by diffusion from the right to the left. Bulk Passage Into and Out of the Cell Endocytosis The lipid nature of their biological membranes raises a second problem for cells. The substances cells use as fuel are for the most part large, polar molecules that cannot cross the hydrophobic barrier a lipid bilayer creates. How do organisms get these substances into their cells? One process many single-celled eukaryotes employ is endocy- tosis (figure 6.17). In this process the plasma membrane extends outward and envelops food particles. Cells use three major types of endocytosis: phagocytosis, pinocyto- sis, and receptor-mediated endocytosis. Phagocytosis and Pinocytosis. If the material the cell takes in is particulate (made up of discrete particles), such as an organism or some other fragment of organic matter (figure 6.17a), the process is called phagocytosis (Greek phagein, “to eat” + cytos, “cell”). If the material the cell takes in is liquid (figure 6.17b), it is called pinocytosis (Greek pinein, “to drink”). Pinocytosis is common among animal cells. Mammalian egg cells, for example, “nurse” from sur- rounding cells; the nearby cells secrete nutrients that the maturing egg cell takes up by pinocytosis. Virtually all eu- karyotic cells constantly carry out these kinds of endocyto- sis, trapping particles and extracellular fluid in vesicles and ingesting them. Endocytosis rates vary from one cell type to another. They can be surprisingly high: some types of white blood cells ingest 25% of their cell volume each hour! Receptor-Mediated Endocytosis. Specific molecules are often transported into eukaryotic cells through receptor-mediated endocytosis. Molecules to be trans- ported first bind to specific receptors on the plasma mem- brane. The transport process is specific because only that molecule has a shape that fits snugly into the receptor. The plasma membrane of a particular kind of cell contains a characteristic battery of receptor types, each for a different kind of molecule. The interior portion of the receptor molecule resembles a hook that is trapped in an indented pit coated with the protein clathrin. The pits act like molecular mousetraps, closing over to form an internal vesicle when the right mol- ecule enters the pit (figure 6.18). The trigger that releases the trap is a receptor protein embedded in the membrane of the pit, which detects the presence of a particular target molecule and reacts by initiating endocytosis. The process is highly specific and very fast. One type of molecule that is taken up by receptor- mediated endocytosis is called a low density lipoprotein (LDL). The LDL molecules bring cholesterol into the cell where it can be incorporated into membranes. Cholesterol plays a key role in determining the stiffness of the body’s membranes. In the human genetic disease called hyper- cholesteremia, the receptors lack tails and so are never caught in the clathrin-coated pits and, thus, are never taken up by the cells. The cholesterol stays in the blood- stream of affected individuals, coating their arteries and leading to heart attacks. Fluid-phase endocytosis is the receptor-mediated pinocytosis of fluids. It is important to understand that en- docytosis in itself does not bring substances directly into the cytoplasm of a cell. The material taken in is still sepa- rated from the cytoplasm by the membrane of the vesicle. 116 Part II Biology of the Cell 6.4 Bulk transport utilizes endocytosis. Cytoplasm Phagocytosis Pinocytosis Plasma membrane Plasma membrane Nucleus Cytoplasm Nucleus FIGURE 6.17 Endocytosis. Both phagocytosis (a) and pinocytosis (b) are forms of endocytosis. (a) (b) Exocytosis The reverse of endocytosis is exocytosis, the discharge of material from vesicles at the cell surface (figure 6.19). In plant cells, exocytosis is an important means of exporting the materials needed to construct the cell wall through the plasma membrane. Among protists, contractile vacuole dis- charge is a form of exocytosis. In animal cells, exocytosis provides a mechanism for secreting many hormones, neuro- transmitters, digestive enzymes, and other substances. Cells import bulk materials by engulfing them with their plasma membranes in a process called endocytosis; similarly, they extrude or secrete material through exocytosis. Chapter 6 Membranes 117 Coated pit Target molecule Clathrin Receptor protein Coated vesicle (a) FIGURE 6.18 Receptor-mediated endocytosis. (a) Cells that undergo receptor-mediated endocytosis have pits coated with the protein clathrin that initiate endocytosis when target molecules bind to receptor proteins in the plasma membrane. (b) A coated pit appears in the plasma membrane of a developing egg cell, covered with a layer of proteins (80,000×). When an appropriate collection of molecules gathers in the coated pit, the pit deepens (c) and seals off (d) to form a coated vesicle, which carries the molecules into the cell. (b) (c) (d) Cytoplasm Secretory vesicle Secretory product Plasma membrane (a) (b) FIGURE 6.19 Exocytosis. (a) Proteins and other molecules are secreted from cells in small packets called vesicles, whose membranes fuse with the plasma membrane, releasing their contents to the cell surface. (b) A transmission electron micrograph showing exocytosis. Active Transport While diffusion, facilitated diffusion, and osmosis are pas- sive transport processes that move materials down their concentration gradients, cells can also move substances across the membrane up their concentration gradients. This process requires the expenditure of energy, typically ATP, and is therefore called active transport. Like facili- tated diffusion, active transport involves highly selective protein carriers within the membrane. These carriers bind to the transported substance, which could be an ion or a simple molecule like a sugar (figure 6.20), an amino acid, or a nucleotide to be used in the synthesis of DNA. Active transport is one of the most important functions of any cell. It enables a cell to take up additional molecules of a substance that is already present in its cytoplasm in concentrations higher than in the extracellular fluid. With- out active transport, for example, liver cells would be un- able to accumulate glucose molecules from the blood plasma, as the glucose concentration is often higher inside the liver cells than it is in the plasma. Active transport also enables a cell to move substances from its cytoplasm to the extracellular fluid despite higher external concentrations. The Sodium-Potassium Pump The use of ATP in active transport may be direct or indi- rect. Lets first consider how ATP is used directly to move ions against their concentration gradient. More than one- third of all of the energy expended by an animal cell that is not actively dividing is used in the active transport of sodium (Na + ) and potassium (K + ) ions. Most animal cells have a low internal concentration of Na + , relative to their surroundings, and a high internal concentration of K + . They maintain these concentration differences by actively pumping Na + out of the cell and K + in. The remarkable protein that transports these two ions across the cell mem- brane is known as the sodium-potassium pump (figure 6.21). The cell obtains the energy it needs to operate the pump from adenosine triphosphate (ATP), a molecule we’ll learn more about in chapter 8. The important characteristic of the sodium-potassium pump is that it is an active transport process, transporting Na + and K + from areas of low concentration to areas of high concentration. This transport up their concentration gradients is the opposite of the passive transport in diffu- sion; it is achieved only by the constant expenditure of metabolic energy. The sodium-potassium pump works through a series of conformational changes in the trans- membrane protein: Step 1. Three sodium ions bind to the cytoplasmic side of the protein, causing the protein to change its conformation. Step 2. In its new conformation, the protein binds a molecule of ATP and cleaves it into adenosine diphos- phate and phosphate (ADP + P i ). ADP is released, but the phosphate group remains bound to the protein. The protein is now phosphorylated. Step 3. The phosphorylation of the protein induces a second conformational change in the protein. This change translocates the three Na + across the membrane, 118 Part II Biology of the Cell 6.5 Active transport across membranes is powered by energy from ATP. Exterior Cytoplasm Glucose-binding site Hydrophobic Hydrophilic Charged amino acids + + + + + + + + + + + + + + + + + + + + + + + + + – – – – – – – – – – – – – –– – , FIGURE 6.20 A glucose transport channel. The molecular structure of this particular glucose transport channel is known in considerable detail. The protein’s 492 amino acids form a folded chain that traverses the lipid membrane 12 times. Amino acids with charged groups are less stable in the hydrophobic region of the lipid bilayer and are thus exposed to the cytoplasm or the extracellular fluid. Researchers think the center of the protein consists of five helical segments with glucose-binding sites (in red) facing inward. A conformational change in the protein transports glucose across the membrane by shifting the position of the glucose-binding sites. so they now face the exterior. In this new conformation, the protein has a low affinity for Na + , and the three bound Na + dissociate from the protein and diffuse into the extracellular fluid. Step 4. The new conformation has a high affinity for K + , two of which bind to the extracellular side of the protein as soon as it is free of the Na + . Step 5. The binding of the K + causes another confor- mational change in the protein, this time resulting in the dissociation of the bound phosphate group. Step 6. Freed of the phosphate group, the protein re- verts to its original conformation, exposing the two K + to the cytoplasm. This conformation has a low affinity for K + , so the two bound K + dissociate from the protein and diffuse into the interior of the cell. The original conformation has a high affinity for Na + ; when these ions bind, they initiate another cycle. Three Na + leave the cell and two K + enter in every cycle. The changes in protein conformation that occur during the cycle are rapid, enabling each carrier to transport as many as 300 Na + per second. The sodium- potassium pump appears to be ubiquitous in animal cells, although cells vary widely in the number of pump pro- teins they contain. Active transport moves a solute across a membrane up its concentration gradient, using protein carriers driven by the expenditure of chemical energy. Chapter 6 Membranes 119 P P P A P P P A Na + Extracellular Intracellular ATP ATP P P P A ATP P P A P ADP 1. Protein in membrane binds intracellular sodium. 2. ATP phosphorylates protein with bound sodium. 3. Phosphorylation causes conformational change in protein, allowing sodium to leave. P P A P ADP 4. Extracellular potassium binds to exposed sites. K + P P A P ADP+P i 5. Binding of potassium causes dephos- phorylation of protein. 6. Dephosphorylation of protein triggers change back to original conformation, potassium moves into cell, and the cycle repeats. FIGURE 6.21 The sodium-potassium pump. The protein channel known as the sodium-potassium pump transports sodium (Na + ) and potassium (K + ) ions across the cell membrane. For every three Na + that are transported out of the cell, two K + are transported into the cell. The sodium- potassium pump is fueled by ATP. Coupled Transport Many molecules are transported into cells up a concentration gradient through a process that uses ATP indirectly. The molecules move hand-in-hand with sodium ions or protons that are moving down their concentration gradients. This type of active transport, called cotransport, has two components: 1. Establishing the down gradient. ATP is used to establish the sodium ion or proton down gradient, which is greater than the up gradient of the molecule to be transported. 2. Traversing the up gradient. Cotransport proteins (also called coupled transport proteins) carry the mol- ecule and either a sodium ion or a proton together across the membrane. Because the down gradient of the sodium ion or proton is greater than the up gradient of the molecule to be trans- ported, the net movement across the membrane is in the direction of the down gradient, typically into the cell. Establishing the Down Gradient Either the sodium-potassium pump or the proton pump es- tablishes the down gradient that powers most active trans- port processes of the cell. The Sodium-Potassium Pump. The sodium-potassium pump actively pumps sodium ions out of the cell, powered by energy from ATP. This establishes a sodium ion con- centration gradient that is lower inside the cell. The Proton Pump. The proton pump pumps protons (H + ions) across a membrane using energy derived from energy-rich molecules or from photosynthesis. This cre- ates a proton gradient, in which the concentration of pro- tons is higher on one side of the membrane than the other. Membranes are impermeable to protons, so the only way protons can diffuse back down their concentration gradi- ent is through a second cotransport protein. Traversing the Up Gradient Animal cells accumulate many amino acids and sugars against a concentration gradient: the molecules are transported into the cell from the extracellular fluid, even though their con- centrations are higher inside the cell. These molecules couple with sodium ions to enter the cell down the Na + concentra- tion gradient established by the sodium-potassium pump. In this cotransport process, Na + and a specific sugar or amino acid simultaneously bind to the same transmembrane protein on the outside of the cell, called a symport (figure 6.22). Both are then translocated to the inside of the cell, but in the process Na + moves down its concentration gradient while the sugar or amino acid moves up its concentration gradient. In effect, the cell uses some of the energy stored in the Na + con- centration gradient to accumulate sugars and amino acids. In a related process, called countertransport, the in- ward movement of Na + is coupled with the outward move- ment of another substance, such as Ca ++ or H + . As in co- transport, both Na + and the other substance bind to the same transport protein, in this case called an antiport, but in this case they bind on opposite sides of the membrane and are moved in opposite directions. In countertransport, the cell uses the energy released as Na + moves down its concentration gradient into the cell to extrude a substance up its concentration gradient. The cell uses the proton down gradient established by the proton pump (figure 6.23) in ATP production. The movement of protons through their cotransport protein is coupled to the production of ATP, the energy-storing mol- ecule we mentioned earlier. Thus, the cell expends energy to produce ATP, which provides it with a convenient en- ergy storage form that it can employ in its many activities. The coupling of the proton pump to ATP synthesis, called chemiosmosis, is responsible for almost all of the ATP produced from food (see chapter 9) and all of the ATP pro- duced by photosynthesis (see chapter 10). We know that proton pump proteins are ancient because they are present in bacteria as well as in eukaryotes. The mechanisms for transport across plasma membranes are summarized in table 6.2. Many molecules are cotransported into cells up their concentration gradients by coupling their movement to that of sodium ions or protons moving down their concentration gradients. 120 Part II Biology of the Cell Outside of cell Inside of cell Na + Coupled transport protein Sugar K + Na/K pump FIGURE 6.22 Cotransport through a coupled transport protein. A membrane protein transports sodium ions into the cell, down their concentration gradient, at the same time it transports a sugar molecule into the cell. The gradient driving the Na + entry is so great that sugar molecules can be brought in against their concentration gradient. Chapter 6 Membranes 121 Conformation A Extracellular fluid Cytoplasm H + Conformation AConformation B H + H + H + H + H + ATP ADP+P i FIGURE 6.23 The proton pump. In this general model of energy-driven proton pumping, the transmembrane protein that acts as a proton pump is driven through a cycle of two conformations: A and B. The cycle A→B→A goes only one way, causing protons to be pumped from the inside to the outside of the membrane. ATP powers the pump. Table 6.2 Mechanisms for Transport across Cell Membranes Passage through Process Membrane How It Works Example PASSIVE PROCESSES Diffusion Facilitated diffusion Osmosis ACTIVE PROCESSES Endocytosis Phagocytosis Pinocytosis Carrier-mediated endocytosis Exocytosis Active transport Na + /K + pump Proton pump Direct Protein carrier Direct Membrane vesicle Membrane vesicle Membrane vesicle Membrane vesicle Protein carrier Protein carrier Random molecular motion produces net migration of molecules toward region of lower concentration Molecule binds to carrier protein in membrane and is transported across; net movement is toward region of lower concentration Diffusion of water across differentially permeable membrane Particle is engulfed by membrane, which folds around it and forms a vesicle Fluid droplets are engulfed by membrane, which forms vesicles around them Endocytosis triggered by a specific receptor Vesicles fuse with plasma membrane and eject contents Carrier expends energy to export Na + against a concentration gradient Carrier expends energy to export protons against a concentration gradient Movement of oxygen into cells Movement of glucose into cells Movement of water into cells placed in a hypotonic solution Ingestion of bacteria by white blood cells “Nursing” of human egg cells Cholesterol uptake Secretion of mucus Coupled uptake of glucose into cells against its concentration gradient Chemiosmotic generation of ATP 122 Part II Biology of the Cell Chapter 6 Summary Questions Media Resources 6.1 Biological membranes are fluid layers of lipid. ? Every cell is encased within a fluid bilayer sheet of phospholipid molecules called the plasma membrane. 1. How would increasing the number of phospholipids with double bonds between carbon atoms in their tails affect the fluidity of a membrane? ? Proteins that are embedded within the plasma membrane have their hydrophobic regions exposed to the hydrophobic interior of the bilayer, and their hydrophilic regions exposed to the cytoplasm or the extracellular fluid. ? Membrane proteins can transport materials into or out of the cell, they can mark the identity of the cell, or they can receive extracellular information. 2. Describe the two basic types of structures that are characteristic of proteins that span membranes. 6.2 Proteins embedded within the plasma membrane determine its character. ? Diffusion is the kinetic movement of molecules or ions from an area of high concentration to an area of low concentration. ? Osmosis is the diffusion of water. Because all organisms are composed of mostly water, maintaining osmotic balance is essential to life. 3. If a cell’s cytoplasm were hyperosmotic to the extracellular fluid, how would the concentration of solutes in the cytoplasm compare with that in the extracellular fluid? 6.3 Passive transport across membranes moves down the concentration gradient. ? Materials or volumes of fluid that are too large to pass directly through the cell membrane can move into or out of cells through endocytosis or exocytosis, respectively. ? In these processes, the cell expends energy to change the shape of its plasma membrane, allowing the cell to engulf materials into a temporary vesicle (endocytosis), or eject materials by fusing a filled vesicle with the plasma membrane (exocytosis). 4. How do phagocytosis and pinocytosis differ? 5. Describe the mechanism of receptor-mediated endocytosis. 6.4 Bulk transport utilizes endocytosis. ? Cells use active transport to move substances across the plasma membrane against their concentration gradients, either accumulating them within the cell or extruding them from the cell. Active transport requires energy from ATP, either directly or indirectly. 6. In what two ways does facilitated diffusion differ from simple diffusion across a membrane? 7. How does active transport differ from facilitated diffusion? How is it similar to facilitated diffusion? 6.5 Active transport across membranes is powered by energy from ATP. ? Membrane Structure ? Art Activity: Fluid Mosaic Model ? Art Activity: Membrane Protein Diversity ? Diffusion ? Osmosis ? Diffusion ? Diffusion ? Osmosis ? Student Research: Understanding Membrane Transport ? Exocystosis/ endocytosis ? Exocystosis/ endocytosis ? Exploration: Active Transport ? Active Transport ? Active Transport http://www.mhhe.com/raven6e http://www.biocourse.com 123 7 Cell-Cell Interactions Concept Outline 7.1 Cells signal one another with chemicals. Receptor Proteins and Signaling between Cells. Receptor proteins embedded in the plasma membrane change shape when they bind specific signal molecules, triggering a chain of events within the cell. Types of Cell Signaling. Cell signaling can occur between adjacent cells, although chemical signals called hormones act over long distances. 7.2 Proteins in the cell and on its surface receive signals from other cells. Intracellular Receptors. Some receptors are located within the cell cytoplasm. These receptors respond to lipid- soluble signals, such as steroid hormones. Cell Surface Receptors. Many cell-to-cell signals are water-soluble and cannot penetrate membranes. Instead, the signals are received by transmembrane proteins protruding out from the cell surface. 7.3 Follow the journey of information into the cell. Initiating the Intracellular Signal. Cell surface receptors often use “second messengers” to transmit a signal to the cytoplasm. Amplifying the Signal: Protein Kinase Cascades. Surface receptors and second messengers amplify signals as they travel into the cell, often toward the cell nucleus. 7.4 Cell surface proteins mediate cell-cell interactions. The Expression of Cell Identity. Cells possess on their surfaces a variety of tissue-specific identity markers that identify both the tissue and the individual. Intercellular Adhesion. Cells attach themselves to one another with protein links. Some of the links are very strong, others more transient. Tight Junctions. Adjacent cells form a sheet when connected by tight junctions, and molecules are encouraged to flow through the cells, not between them. Anchoring Junctions. The cytoskeleton of a cell is connected by an anchoring junction to the cytoskeleton of another cell or to the extracellular matrix. Communicating Junctions. Many adjacent cells have direct passages that link their cytoplasms, permitting the passage of ions and small molecules. D id you know that each of the 100 trillion cells of your body shares one key feature with the cells of tigers, bumblebees, and persimmons (figure 7.1)—a feature that most bacteria and protists lack? Your cells touch and com- municate with one another. Sending and receiving a variety of chemical signals, they coordinate their behavior so that your body functions as an integrated whole, rather than as a massive collection of individual cells acting independently. The ability of cells to communicate with one another is the hallmark of multicellular organisms. In this chapter we will look in detail at how the cells of multicellular organisms in- teract with one another, first exploring how they signal one another with chemicals and then examining the ways in which their cell surfaces interact to organize tissues and body structures. FIGURE 7.1 Persimmon cells in close contact with one another. These plant cells and all cells, no matter what their function, interact with their environment, including the cells around them. Monoclonal antibodies. The first method uses mono- clonal antibodies. An antibody is an immune system pro- tein that, like a receptor, binds specifically to another molecule. Each individual immune system cell can make only one particular type of antibody, which can bind to only one specific target molecule. Thus, a cell-line de- rived from a single immune system cell (a clone) makes one specific antibody (a monoclonal antibody). Mono- clonal antibodies that bind to particular receptor pro- teins can be used to isolate those proteins from the thousands of others in the cell. Gene analysis. The study of mutants and isolation of gene sequences has had a tremendous impact on the field of receptor analysis. In chapter 19 we will present a detailed account of how this is done. These advances make it feasible to identify and isolate the many genes that encode for various receptor proteins. Remarkably, these techniques have revealed that the enormous number of receptor proteins can be grouped into just a handful of “families” containing many related recep- tors. Later in this chapter we will meet some of the mem- bers of these receptor families. Cells in a multicellular organism communicate with others by releasing signal molecules that bind to receptor proteins on the surface of the other cells. Recent advances in protein isolation have yielded a wealth of information about the structure and function of these proteins. 124 Part II Biology of the Cell Receptor Proteins and Signaling between Cells Communication between cells is com- mon in nature. Cell signaling occurs in all multicellular organisms, provid- ing an indispensable mechanism for cells to influence one another. The cells of multicellular organisms use a variety of molecules as signals, includ- ing not only peptides, but also large proteins, individual amino acids, nu- cleotides, steroids and other lipids. Even dissolved gases are used as signals. Nitric oxide (NO) plays a role in mediating male erections (Viagra functions by stimulating NO release). Some of these molecules are at- tached to the surface of the signaling cell; others are secreted through the plasma membrane or released by exocytosis. Cell Surface Receptors Any given cell of a multicellular organism is exposed to a constant stream of signals. At any time, hundreds of differ- ent chemical signals may be in the environment surround- ing the cell. However, each cell responds only to certain signals and ignores the rest (figure 7.2), like a person fol- lowing the conversation of one or two individuals in a noisy, crowded room. How does a cell “choose” which sig- nals to respond to? Located on or within the cell are re- ceptor proteins, each with a three-dimensional shape that fits the shape of a specific signal molecule. When a signal molecule approaches a receptor protein of the right shape, the two can bind. This binding induces a change in the re- ceptor protein’s shape, ultimately producing a response in the cell. Hence, a given cell responds to the signal mole- cules that fit the particular set of receptor proteins it pos- sesses, and ignores those for which it lacks receptors. The Hunt for Receptor Proteins The characterization of receptor proteins has presented a very difficult technical problem, because of their relative scarcity in the cell. Because these proteins may constitute less than 0.01% of the total mass of protein in a cell, puri- fying them is analogous to searching for a particular grain of sand in a sand dune! However, two recent techniques have enabled cell biologists to make rapid progress in this area. 7.1 Cells signal one another with chemicals. Cytoplasm Signal molecules Extracellular surface FIGURE 7.2 Cell surface receptors recognize only specific molecules. Signal molecules will bind only to those cells displaying receptor proteins with a shape into which they can fit snugly. Types of Cell Signaling Cells communicate through any of four basic mechanisms, depending primarily on the distance between the signaling and responding cells (figure 7.3). In ad- dition to using these four basic mecha- nisms, some cells actually send signals to themselves, secreting signals that bind to specific receptors on their own plasma membranes. This process, called autocrine signaling, is thought to play an important role in reinforcing developmental changes. Direct Contact As we saw in chapter 6, the surface of a eukaryotic cell is a thicket of proteins, carbohydrates, and lipids attached to and extending outward from the plasma membrane. When cells are very close to one another, some of the mol- ecules on the cells’ plasma membranes may bind together in specific ways. Many of the important interactions between cells in early development occur by means of direct contact be- tween cell surfaces (figure 7.3a). We’ll examine contact-dependent interac- tions more closely later in this chapter. Paracrine Signaling Signal molecules released by cells can diffuse through the extracellular fluid to other cells. If those molecules are taken up by neighboring cells, destroyed by extracellular enzymes, or quickly removed from the extracellular fluid in some other way, their influence is restricted to cells in the immediate vicinity of the releasing cell. Signals with such short-lived, local effects are called paracrine signals (figure 7.3b). Like direct contact, paracrine signaling plays an im- portant role in early development, coordinating the activi- ties of clusters of neighboring cells. Endocrine Signaling If a released signal molecule remains in the extracellular fluid, it may enter the organism’s circulatory system and travel widely throughout the body. These longer lived signal mole- cules, which may affect cells very distant from the releasing cell, are called hormones, and this type of intercellular com- munication is known as endocrine signaling (figure 7.3c). Chapter 58 discusses endocrine signaling in detail. Both ani- mals and plants use this signaling mechanism extensively. Synaptic Signaling In animals, the cells of the nervous system provide rapid communication with distant cells. Their signal molecules, neurotransmitters, do not travel to the distant cells through the circulatory system like hormones do. Rather, the long, fiberlike extensions of nerve cells release neuro- transmitters from their tips very close to the target cells (figure 7.3d). The narrow gap between the two cells is called a chemical synapse. While paracrine signals move through the fluid between cells, neurotransmitters cross the synapse and persist only briefly. We will examine synaptic signaling more fully in chapter 54. Adjacent cells can signal others by direct contact, while nearby cells that are not touching can communicate through paracrine signals. Two other systems mediate communication over longer distances: in endocrine signaling the blood carries hormones to distant cells, and in synaptic signaling nerve cells secrete neurotransmitters from long cellular extensions close to the responding cells. Chapter 7 Cell-Cell Interactions 125 (d) Synaptic signaling Nerve cell Neurotransmitter Synaptic gap Target cell (c) Endocrine signaling Hormone secretion into blood by endocrine gland Blood vessel Distant target cells Gap junction (b) Paracrine signaling Adjacent target cells Secretory cell (a) Direct contact FIGURE 7.3 Four kinds of cell signaling. Cells communicate in several ways. (a) Two cells in direct contact with each other may send signals across gap junctions. (b) In paracrine signaling, secretions from one cell have an effect only on cells in the immediate area. (c) In endocrine signaling, hormones are released into the circulatory system, which carries them to the target cells. (d) Chemical synaptic signaling involves transmission of signal molecules, called neurotransmitters, from a neuron over a small synaptic gap to the target cell. Intracellular Receptors All cell signaling pathways share certain common elements, including a chemical signal that passes from one cell to an- other and a receptor that receives the signal in or on the target cell. We’ve looked at the sorts of signals that pass from one cell to another. Now let’s consider the nature of the receptors that receive the signals. Table 7.1 summarizes the types of receptors we will discuss in this chapter. Many cell signals are lipid-soluble or very small mole- cules that can readily pass across the plasma membrane of the target cell and into the cell, where they interact with a receptor. Some bind to protein receptors located in the cy- toplasm; others pass across the nuclear membrane as well and bind to receptors within the nucleus. These intracel- lular receptors (figure 7.4) may trigger a variety of re- sponses in the cell, depending on the receptor. 126 Part II Biology of the Cell 7.2 Proteins in the cell and on its surface receive signals from other cells. Signal molecule- binding siteInhibitor protein DNA binding domain Transcription-activating domain Signal molecule Signal molecule- binding domain FIGURE 7.4 Basic structure of a gene-regulating intracellular receptor. These receptors are located within the cell and function in the reception of signals such as steroid hormones, vitamin D, and thyroid hormone. Table 7.1 Cell Communicating Mechanisms Mechanism Structure Function Example INTRACELLULAR RECEPTORS No extracellular signal-binding Receives signals from lipid-soluble Receptors for NO, steroid site or noncharged, nonpolar small hormone, vitamin D, and molecules thyroid hormone CELL SURFACE RECEPTORS Chemically gated Multipass transmembrane Molecular “gates” triggered Neurons ion channels protein forming a central pore chemically to open or close Enzymic receptors Single-pass transmembrane Binds signal extracellularly, Phosphorylation of protein protein catalyzes response intracellularly kinases G-protein-linked Seven-pass transmembrane Binding of signal to receptor causes Peptide hormones, rod receptors protein with cytoplasmic GTP to bind a G protein; G protein, cells in the eyes binding site for G protein with attached GTP, detaches to deliver the signal inside the cell PHYSICAL CONTACT WITH OTHER CELLS Surface markers Variable; integral proteins or Identify the cell MHC complexes, blood glycolipids in cell membrane groups, antibodies Tight junctions Tightly bound, leakproof, Organizing junction: holds cells Junctions between fibrous protein “belt” that together such that material passes epithelial cells in the gut surrounds cell through but not between the cells Desmosomes Intermediate filaments of Anchoring junction: “buttons” Epithelium cytoskeleton linked to adjoining cells together cells through cadherins Adherens junctions Transmembrane fibrous Anchoring junction: “roots” Tissues with high proteins extracellular matrix to cytoskeleton mechanical stress, such as the skin Gap junctions Six transmembrane connexon Communicating junction: allows Excitable tissue such as proteins creating a “pipe” passage of small molecules from heart muscle that connects cells cell to cell in a tissue Plasmodesmata Cytoplasmic connections Communicating junction between Plant tissues between gaps in adjoining plant cells plant cell walls Receptors That Act as Gene Regulators Some intracellular receptors act as regulators of gene transcription. Among them are the receptors for steroid hormones, such as cortisol, estrogen, and progesterone, as well as the receptors for a number of other small, lipid- soluble signal molecules, such as vitamin D and thyroid hormone. All of these receptors have similar structures; the genes that code for them may well be the evolutionary descendants of a single ancestral gene. Because of their structural similarities, they are all part of the intracellular receptor superfamily. Each of these receptors has a binding site for DNA. In its inactive state, the receptor typically cannot bind DNA because an inhibitor protein occupies the binding site. When the signal molecule binds to another site on the re- ceptor, the inhibitor is released and the DNA binding site is exposed (figure 7.5). The receptor then binds to a spe- cific nucleotide sequence on the DNA, which activates (or, in a few instances, suppresses) a particular gene, usually lo- cated adjacent to the regulatory site. The lipid-soluble signal molecules that intracellular re- ceptors recognize tend to persist in the blood far longer than water-soluble signals. Most water-soluble hormones break down within minutes, and neurotransmitters within seconds or even milliseconds. A steroid hormone like corti- sol or estrogen, on the other hand, persists for hours. The target cell’s response to a lipid-soluble cell signal can vary enormously, depending on the nature of the cell. This is true even when different target cells have the same intracellular receptor, for two reasons: First, the binding site for the receptor on the target DNA differs from one cell type to another, so that different genes are affected when the signal-receptor complex binds to the DNA, and second, most eukaryotic genes have complex controls. We will discuss them in detail in chapter 16, but for now it is sufficient to note that several different regulatory proteins are usually involved in reading a eu- karyotic gene. Thus the intracellular receptor interacts with different signals in different tissues. Depending on the cell-specific controls operating in different tissues, the effect the intracellular receptor produces when it binds with DNA will vary. Receptors That Act as Enzymes Other intracellular receptors act as enzymes. A very inter- esting example is the receptor for the signal molecule, ni- tric oxide (NO). A small gas molecule, NO diffuses readily out of the cells where it is produced and passes directly into neighboring cells, where it binds to the enzyme guanylyl cyclase. Binding of NO activates the enzyme, enabling it to catalyze the synthesis of cyclic guanosine monophosphate (GMP), an intracellular messenger molecule that produces cell-specific responses such as the relaxation of smooth muscle cells. NO has only recently been recognized as a signal mole- cule in vertebrates. Already, however, a wide variety of roles have been documented. For example, when the brain sends a nerve signal relaxing the smooth muscle cells lining the walls of vertebrate blood vessels, the signal molecule acetylcholine released by the nerve near the muscle does not interact with the muscle cell directly. Instead, it causes nearby epithelial cells to produce NO, which then causes the smooth muscle to relax, allowing the vessel to expand and thereby increase blood flow. Many target cells possess intracellular receptors, which are activated by substances that pass through the plasma membrane. Chapter 7 Cell-Cell Interactions 127 DNA binding site blocked Transcription activating domain DNA binding site exposed Cortisol Inhibitor Signal molecule- binding domain FIGURE 7.5 How intracellular receptors regulate gene transcription. In this model, the binding of the steroid hormone cortisol to a DNA regulatory protein causes it to alter its shape. The inhibitor is released, exposing the DNA binding site of the regulatory protein. The DNA binds to the site, positioning a specific nucleotide sequence over the transcription activating domain of the receptor and initiating transcription. Cell Surface Receptors Most signal molecules are water-soluble, including neuro- transmitters, peptide hormones, and the many proteins that multicellular organisms employ as “growth factors” during development. Water-soluble signals cannot diffuse through cell membranes. Therefore, to trigger responses in cells, they must bind to receptor proteins on the sur- face of the cell. These cell surface receptors (figure 7.6) convert the extracellular signal to an intracellular one, re- sponding to the binding of the signal molecule by produc- ing a change within the cell’s cytoplasm. Most of a cell’s receptors are cell surface receptors, and almost all of them belong to one of three receptor superfamilies: chemically gated ion channels, enzymic receptors, and G-protein- linked receptors. Chemically Gated Ion Channels Chemically gated ion channels are receptor proteins that ions pass through. The receptor proteins that bind many neurotransmitters have the same basic structure (figure 7.6a). Each is a “multipass” transmembrane protein, mean- ing that the chain of amino acids threads back and forth across the plasma membrane several times. In the center of the protein is a pore that connects the extracellular fluid with the cytoplasm. The pore is big enough for ions to pass through, so the protein functions as an ion channel. The channel is said to be chemically gated because it opens when a chemical (the neurotransmitter) binds to it. The type of ion (sodium, potassium, calcium, chloride, for ex- ample) that flows across the membrane when a chemically gated ion channel opens depends on the specific three- dimensional structure of the channel. Enzymic Receptors Many cell surface receptors either act as enzymes or are di- rectly linked to enzymes (figure 7.6b). When a signal mole- cule binds to the receptor, it activates the enzyme. In al- most all cases, these enzymes are protein kinases, enzymes that add phosphate groups to proteins. Most enzymic re- ceptors have the same general structure. Each is a single- pass transmembrane protein (the amino acid chain passes through the plasma membrane only once); the portion that binds the signal molecule lies outside the cell, and the por- tion that carries out the enzyme activity is exposed to the cytoplasm. 128 Part II Biology of the Cell (a) Chemically gated ion channel Signal G protein Activated G protein Enzyme or ion channel Activated enzyme or ion channel Ions (b) Enzymic receptor (c) G-protein-linked receptor Signal Inactive catalytic domain Active catalytic domain FIGURE 7.6 Cell surface receptors. (a) Chemically gated ion channels are multipass transmembrane proteins that form a pore in the cell membrane. This pore is opened or closed by chemical signals. (b) Enzymic receptors are single-pass transmembrane proteins that bind the signal on the extracellular surface. A catalytic region on their cytoplasmic portion then initiates enzymatic activity inside the cell. (c) G-protein- linked receptors bind to the signal outside the cell and to G proteins inside the cell. The G protein then activates an enzyme or ion channel, mediating the passage of a signal from the cell’s surface to its interior. G-Protein-Linked Receptors A third class of cell surface receptors acts indirectly on enzymes or ion channels in the plasma membrane with the aid of an assisting protein, called a guanosine triphos- phate (GTP)-binding protein, or G protein (figure 7.6c). Receptors in this category use G proteins to mediate pas- sage of the signal from the membrane surface into the cell interior. How G-Protein-Linked Receptors Work. G pro- teins are mediators that initiate a diffusible signal in the cytoplasm. They form a transient link between the recep- tor on the cell surface and the signal pathway within the cytoplasm. Importantly, this signal has a relatively short life span whose active age is determined by GTP. When a signal arrives, it finds the G protein nestled into the G- protein-linked receptor on the cytoplasmic side of the plasma membrane. Once the signal molecule binds to the receptor, the G-protein-linked receptor changes shape. This change in receptor shape twists the G protein, caus- ing it to bind GTP. The G protein can now diffuse away from the receptor. The “activated” complex of a G pro- tein with attached GTP is then free to initiate a number of events. However, this activation is short-lived, because GTP has a relatively short life span (seconds to minutes). This elegant arrangement allows the G proteins to acti- vate numerous pathways, but only in a transient manner. In order for a pathway to “stay on,” there must be a con- tinuous source of incoming extracellular signals. When the rate of external signal drops off, the pathway shuts down. The Largest Family of Cell Surface Receptors. Sci- entists have identified more than 100 different G- protein-linked receptors, more than any other kind of cell surface receptor. They mediate an incredible range of cell signals, including peptide hormones, neurotrans- mitters, fatty acids, and amino acids. Despite this great variation in specificity, however, all G-protein-linked re- ceptors whose amino acid sequences are known have a similar structure. They are almost certainly closely re- lated in an evolutionary sense, arising from a single an- cestral sequence. Each of these G-protein-linked recep- tors is a seven-pass transmembrane protein (figure 7.7)—a single polypeptide chain that threads back and forth across the lipid bilayer seven times, creating a chan- nel through the membrane. Evolutionary Origin of G-Protein-Linked Receptors. As research revealed the structure of G-protein-linked re- ceptors, an interesting pattern emerged: the same seven- pass structural motif is seen again and again, in sensory re- ceptors such as the light-activated rhodopsin protein in the vertebrate eye, in the light-activated bacteriorhodopsin proton pump that plays a key role in bacterial photosynthe- sis, in the receptor that recognizes the yeast mating factor protein discussed earlier, and in many other sensory recep- tors. Vertebrate rhodopsin is in fact a G-protein-linked re- ceptor and utilizes a G protein. Bacteriorhodopsin is not. The occurrence of the seven-pass structural motif in both, and in so many other G-protein-linked receptors, suggests that this motif is a very ancient one, and that G-protein- linked receptors may have evolved from sensory receptors of single-celled ancestors. Discovery of G Proteins. Martin Rodbell of the Na- tional Institute of Environmental Health Sciences and Alfred Gilman of the University of Texas Southwestern Medical Center received the 1994 Nobel Prize for Medi- cine or Physiology for their work on G proteins. Rodbell and Gilman’s work has proven to have significant ramifi- cations. G proteins are involved in the mechanism em- ployed by over half of all medicines in use today. Study- ing G proteins will vastly expand our understanding of how these medicines work. Furthermore, the investiga- tion of G proteins should help elucidate how cells com- municate in general and how they contribute to the over- all physiology of organisms. As Gilman says, G proteins are “involved in everything from sex in yeast to cognition in humans.” Most receptors are located on the surface of the plasma membrane. Chemically gated ion channels open or close when signal molecules bind to the channel, allowing specific ions to diffuse through. Enzyme receptors typically activate intracellular proteins by phosphorylation. G-protein-linked receptors activate an intermediary protein, which then effects the intracellular change. Chapter 7 Cell-Cell Interactions 129 Protein signal-binding site G-protein-binding sites COOH NH 2 FIGURE 7.7 The G-protein-linked receptor is a seven-pass transmembrane protein. Initiating the Intracellular Signal Some enzymic receptors and most G- protein-linked receptors carry the sig- nal molecule’s message into the target cell by utilizing other substances to relay the message within the cyto- plasm. These other substances, small molecules or ions commonly called second messengers or intracellular mediators, alter the behavior of partic- ular proteins by binding to them and changing their shape. The two most widely used second messengers are cyclic adenosine monophosphate (cAMP) and calcium. cAMP All animal cells studied thus far use cAMP as a second messenger (chap- ter 56 discusses cAMP in detail). To see how cAMP typically works as a messenger, let’s examine what hap- pens when the hormone epinephrine binds to a particular type of G- protein-linked receptor called the β- adrenergic receptor (figure 7.8). When epinephrine binds with this re- ceptor, it activates a G protein, which then stimulates the enzyme adenylyl cyclase to produce large amounts of cAMP within the cell (figure 7.9a). The cAMP then binds to and activates the enzyme α-kinase, which adds phosphates to specific proteins in the cell. The effect this phosphorylation has on cell function depends on the identity of the cell and the proteins that are phosphorylated. In muscle cells, for example, the α-kinase phosphorylates and thereby acti- vates enzymes that stimulate the breakdown of glycogen into glucose and inhibit the synthesis of glycogen from glucose. Glucose is then more available to the muscle cells for metabolism. Calcium Calcium (Ca ++ ) ions serve even more widely than cAMP as second messengers. Ca ++ levels inside the cytoplasm of a cell are normally very low (less than 10 H110027 M), while outside the cell and in the endoplasmic reticulum Ca ++ levels are quite high (about 10 H110023 M). Chemically gated calcium chan- nels in the endoplasmic reticulum membrane act as switches; when they open, Ca ++ rushes into the cytoplasm and triggers proteins sensitive to Ca ++ to initiate a variety of activities. For example, the efflux of Ca ++ from the endo- plasmic reticulum causes skeletal muscle cells to contract and some endocrine cells to secrete hormones. The gated Ca ++ channels are opened by a G-protein- linked receptor. In response to signals from other cells, the receptor activates its G protein, which in turn acti- vates the enzyme, phospholipase C. This enzyme catalyzes the production of inositol trisphosphate (IP 3 ) from phospho- lipids in the plasma membrane. The IP 3 molecules diffuse through the cytoplasm to the endoplasmic reticulum and bind to the Ca ++ channels. This opens the channels and allows Ca ++ to flow from the endoplasmic reticulum into the cytoplasm (figure 7.9b). Ca ++ initiates some cellular responses by binding to calmodulin, a 148-amino acid cytoplasmic protein that con- tains four binding sites for Ca ++ (figure 7.10). When four Ca ++ ions are bound to calmodulin, the calmodulin/Ca ++ complex binds to other proteins, and activates them. Cyclic AMP and Ca ++ often behave as second messengers, intracellular substances that relay messages from receptors to target proteins. 130 Part II Biology of the Cell 7.3 Follow the journey of information into the cell. Extracellular Intracellular NH 3 + COO - Oligosaccharide unit FIGURE 7.8 Structure of the β-adrenergic receptor. The receptor is a G-protein-linked molecule which, when it binds to an extracellular signal molecule, stimulates voluminous production of cAMP inside the cell, which then effects the cellular change. Chapter 7 Cell-Cell Interactions 131 Signal molecule Signal molecule Cell surface receptor cAMP pathway Ca ++ pathway Adenylyl cyclase G protein cAMP Target protein Nucleus Cell membrane Cytoplasm Nucleus Cell membrane Cytoplasm Cell surface receptor Phospholipase C G protein Ca ++ Target protein Inositol trisphosphate intermediaryEndoplasmic reticulum ATP (a) (b) FIGURE 7.9 How second messengers work. (a) The cyclic AMP (cAMP) pathway. An extracellular receptor binds to a signal molecule and, through a G protein, activates the membrane-bound enzyme, adenylyl cyclase. This enzyme catalyzes the synthesis of cAMP, which binds to the target protein to initiate the cellular change. (b) The calcium (Ca ++ ) pathway. An extracellular receptor binds to another signal molecule and, through another G protein, activates the enzyme phospholipase C. This enzyme stimulates the production of inositol trisphosphate, which binds to and opens calcium channels in the membrane of the endoplasmic reticulum. Ca ++ is released into the cytoplasm, effecting a change in the cell. Ca ++ Ca ++ Ca ++ Ca ++ Ca ++ Inactive protein Active protein Calmodulin Calmodulin (a) (b) FIGURE 7.10 Calmodulin. (a) Calmodulin is a protein containing 148 amino acid residues that mediates Ca ++ function. (b) When four Ca ++ are bound to the calmodulin molecule, it undergoes a conformational change that allows it to bind to other cytoplasmic proteins and effect cellular responses. Amplifying the Signal: Protein Kinase Cascades Both enzyme-linked and G-protein-linked receptors re- ceive signals at the surface of the cell, but as we’ve seen, the target cell’s response rarely takes place there. In most cases the signals are relayed to the cytoplasm or the nucleus by second messengers, which influence the activity of one or more enzymes or genes and so alter the behavior of the cell. But most signaling molecules are found in such low concentrations that their diffusion across the cytoplasm would take a great deal of time unless the signal is ampli- fied. Therefore, most enzyme-linked and G-protein-linked receptors use a chain of other protein messengers to am- plify the signal as it is being relayed to the nucleus. How is the signal amplified? Imagine a relay race where, at the end of each stage, the finishing runner tags five new runners to start the next stage. The number of runners would increase dramatically as the race progresses: 1, then 5, 25, 125, and so on. The same sort of process takes place as a signal is passed from the cell surface to the cytoplasm or nucleus. First the receptor activates a stage-one protein, almost always by phosphorylating it. The receptor either adds a phosphate group directly, or, it activates a G protein that goes on to activate a second protein that does the phosphorylation. Once activated, each of these stage-one proteins in turn activates a large number of stage-two pro- 132 Part II Biology of the Cell Signal molecule Receptor protein Activated adenylyl cyclase Amplification Amplification Amplification Amplification GTP G protein 2 1 3 4 5 6 7 Enzymatic product Enzyme Protein kinase cAMP Not yet activated FIGURE 7.11 Signal amplification. Amplification at many steps of the cell-signaling process can ultimately produce a large response by the cell. One cell surface receptor (1), for example, may activate many G protein molecules (2), each of which activates a molecule of adenylyl cyclase (3), yielding an enormous production of cAMP (4). Each cAMP molecule in turn will activate a protein kinase (5), which can phosphorylate and thereby activate several copies of a specific enzyme (6). Each of those enzymes can then catalyze many chemical reactions (7). Starting with 10 H1100210 M of signaling molecule, one cell surface receptor can trigger the production of 10 H110026 M of one of the products, an amplification of four orders of magnitude. teins; then each of them activates a large number of stage- three proteins, and so on (figure 7.11). A single cell surface receptor can thus stimulate a cascade of protein kinases to amplify the signal. The Vision Amplification Cascade Let’s trace a protein amplification cascade to see exactly how one works. In vision, a single light-activated rhodopsin (a G-protein-linked receptor) activates hundreds of mole- cules of the G protein transducin in the first stage of the relay. In the second stage, each transducin causes an en- zyme to modify thousands of molecules of a special inside- the-cell messenger called cyclic GMP (figure 7.12). (We will discuss cyclic GMP in more detail later.) In about 1 second, a single rhodopsin signal passing through this two- step cascade splits more than 10 5 (100,000) cyclic GMP molecules (figure 7.13)! The rod cells of humans are suffi- ciently sensitive to detect brief flashes of 5 photons. The Cell Division Amplification Cascade The amplification of signals traveling from the plasma membrane to the nucleus can be even more complex than the process we’ve just described. Cell division, for example, is controlled by a receptor that acts as a protein kinase. The receptor responds to growth-promoting signals by phos- phorylating an intracellular protein called ras, which then activates a series of interacting phosphorylation cascades, some with five or more stages. If the ras protein becomes hyperactive for any reason, the cell acts as if it is being con- stantly stimulated to divide. Ras proteins were first discov- ered in cancer cells. A mutation of the gene that encodes ras had caused it to become hyperactive, resulting in unre- strained cell proliferation. Almost one-third of human can- cers have such a mutation in a ras gene. A small number of surface receptors can generate a vast intracellular response, as each stage of the pathway amplifies the next. Chapter 7 Cell-Cell Interactions 133 Sugar Guanine Phosphate CH 2 O – O OH O O O NH 2N N N N O P Na + Na + One rhodopsin molecule absorbs one photon, which activates 500 transducin molecules, which activate 500 phosphodiesterase molecules, which hydrolyze 10 5 cyclic GMP molecules, which Na + close 250 Na + channels, preventing 10 6 –10 7 Na + per second from entering the cell for a period of 1 second, which hyperpolarizes the rod cell membrane by 1 mV, sending a visual signal to the brain. FIGURE 7.13 The role of signal amplification in vision. In this vertebrate rod cell (the cells of the eye responsible for interpreting light and dark), one single rhodopsin pigment molecule, when excited by a photon, ultimately yields 100,000 split cGMP molecules, which will then effect a change in the membrane of the rod cell, which will be interpreted by the organism as a visual event. FIGURE 7.12 Cyclic GMP. Cyclic GMP is a guanosine monophosphate nucleotide molecule with the single phosphate group attached to a sugar residue in two places (this cyclic part is shown in yellow). Cyclic GMP is an important second messenger linking G proteins to signal transduction pathways within the cytoplasm. The Expression of Cell Identity With the exception of a few primitive types of organisms, the hallmark of multicellular life is the development of highly specialized groups of cells called tissues, such as blood and muscle. Remarkably, each cell within a tissue performs the functions of that tissue and no other, even though all cells of the body are derived from a single fertil- ized cell and contain the same genetic information. How do cells sense where they are, and how do they “know” which type of tissue they belong to? Tissue-Specific Identity Markers As it develops, each animal cell type acquires a unique set of cell surface molecules. These molecules serve as markers proclaiming the cells’ tissue-specific identity. Other cells that make direct physical contact with them “read” the markers. Glycolipids. Most tissue-specific cell surface markers are glycolipids, lipids with carbohydrate heads. The glycolipids on the surface of red blood cells are also responsible for the differences among A, B, and O blood types. As the cells in a tissue divide and differentiate, the population of cell surface glycolipids changes dramatically. MHC Proteins. The immune system uses other cell sur- face markers to distinguish between “self” and “nonself” cells. All of the cells of a given individual, for example, have the same “self” markers, called major histocompatibility com- plex (MHC) proteins. Because practically every individual makes a different set of MHC proteins, they serve as dis- tinctive identity tags for each individual. The MHC pro- teins and other self-identifying markers are single-pass pro- teins anchored in the plasma membrane, and many of them are members of a large superfamily of receptors, the im- munoglobulins (figure 7.14). Cells of the immune system continually inspect the other cells they encounter in the body, triggering the destruction of cells that display foreign or “nonself” identity markers. The immune systems of vertebrates, described in detail in chapter 57, shows an exceptional ability to distinguish self from nonself. However, other vertebrates and even some simple animals like sponges are able to make this distinc- tion to some degree, even though they lack a complex im- mune system. Every cell contains a specific array of marker proteins on its surface. These markers identify each type of cell in a very precise way. 134 Part II Biology of the Cell 7.4 Cell surface proteins mediate cell-cell interactions. Constant region Variable region Disulfide bond s s s s s s s s s s H9251 chain H9252 chain T Receptor ss ss s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s B Receptor Light chain Light chain Heavy chains Cell membrane s s s s s s MHC-II H9251 chain H9252 chain s s s s s s MHC-I H9251 chain H9252-2 microglobulin SS s FIGURE 7.14 Structure of the immunoglobulin family of cell surface marker proteins. T and B cell receptors help mediate the immune response in organisms by recognizing and binding to foreign cell markers. MHC antigens label cells as “self,” so that the immune system attacks only invading entities, such as bacteria, viruses, and usually even the cells of transplanted organs! Intercellular Adhesion Not all physical contacts between cells in a multicellular organism are fleeting touches. In fact, most cells are in physical contact with other cells at all times, usually as members of organized tissues such as those in the lungs, heart, or gut. These cells and the mass of other cells clus- tered around them form long-lasting or permanent connec- tions with each other called cell junctions (figure 7.15). The nature of the physical connections between the cells of a tissue in large measure determines what the tissue is like. Indeed, a tissue’s proper functioning often depends criti- cally upon how the individual cells are arranged within it. Just as a house cannot maintain its structure without nails and cement, so a tissue cannot maintain its characteristic architecture without the appropriate cell junctions. Cells attach themselves to one another with long- lasting bonds called cell junctions. Chapter 7 Cell-Cell Interactions 135 Microvilli Tight junction Adherens junction (anchoring junction) Intermediate filament Desmosome (anchoring junction) Gap junction (communicating junction) Hemidesmosome (anchoring junction) Basal lamina FIGURE 7.15 A summary of cell junction types. Gut epithelial cells are used here to illustrate the comparative structures and locations of common cell junctions. Tight Junctions Cell junctions are divided into three categories, based upon the functions they serve (figure 7.16): tight junc- tions, anchoring junctions, and com- municating junctions. Sometimes called occluding junc- tions, tight junctions connect the plasma membranes of adjacent cells in a sheet, preventing small molecules from leaking between the cells and through the sheet (figure 7.17). This allows the sheet of cells to act as a wall within the organ, keeping mole- cules on one side or the other. Creating Sheets of Cells The cells that line an animal’s diges- tive tract are organized in a sheet only one cell thick. One surface of the sheet faces the inside of the tract and the other faces the extracellular space where blood vessels are lo- cated. Tight junctions encircle each cell in the sheet, like a belt cinched around a pair of pants. The junc- tions between neighboring cells are so securely attached that there is no space between them for leakage. Hence, nutrients absorbed from the food in the digestive tract must pass directly through the cells in the sheet to enter the blood. Partitioning the Sheet The tight junctions between the cells lining the digestive tract also partition the plasma membranes of these cells into separate compartments. Transport proteins in the membrane facing the inside of the tract carry nutrients from that side to the cytoplasm of the cells. Other proteins, located in the membrane on the opposite side of the cells, transport those nutrients from the cytoplasm to the extra- cellular fluid, where they can enter the blood. For the sheet to absorb nutrients properly, these proteins must remain in the correct locations within the fluid membrane. Tight junctions effectively segregate the proteins on opposite sides of the sheet, preventing them from drifting within the membrane from one side of the sheet to the other. When tight junctions are experimentally disrupted, just this sort of migration occurs. Tight junctions connect the plasma membranes of adjacent cells into sheets. 136 Part II Biology of the Cell ER Tight junction Cell 1 Cell 2 Cell 3 Lumen (a) Tight junction Cell 1 2 Cytoskeletal filament Inter- cellular space Extracellular matrix Intracellular attachment proteins Plasma membranes Transmembrane linking proteins (b) Anchoring junction Central tubule Smooth Cell 1 Cell 2 Primary cell wall Middle lamella Plasma membrane (c) Communicating junction Cell FIGURE 7.16 The three types of cell junctions. These three models represent current thinking on how the structures of the three major types of cell junctions facilitate their function: (a) tight junction; (b) anchoring junction; (c) communicating junction. Cell 1 Cell 2 Cell 3 Blood Glucose Apical surface Lumen of gut Tight junction Plasma membranes of adjacent cells Intercellular space Extracellular fluid FIGURE 7.17 Tight junctions. Encircling the cell like a tight belt, these intercellular contacts ensure that materials move through the cells rather than between them. Anchoring Junctions Anchoring junctions mechanically attach the cytoskele- ton of a cell to the cytoskeletons of other cells or to the extracellular matrix. They are commonest in tissues sub- ject to mechanical stress, such as muscle and skin epithelium. Cadherin and Intermediate Filaments: Desmosomes Anchoring junctions called desmosomes connect the cy- toskeletons of adjacent cells (figure 7.18), while hemidesmosomes anchor epithelial cells to a basement membrane. Proteins called cadherins, most of which are single-pass transmembrane glycoproteins, create the criti- cal link. A variety of attachment proteins link the short cy- toplasmic end of a cadherin to the intermediate filaments in the cytoskeleton. The other end of the cadherin molecule projects outward from the plasma membrane, joining di- rectly with a cadherin protruding from an adjacent cell in a firm handshake binding the cells together. Connections between proteins tethered to the interme- diate filaments are much more secure than connections be- tween free-floating membrane proteins. Proteins are sus- pended within the membrane by relatively weak interactions between the nonpolar portions of the protein and the membrane lipids. It would not take much force to pull an untethered protein completely out of the mem- brane, as if pulling an unanchored raft out of the water. Chapter 7 Cell-Cell Interactions 137 Adjacent plasma membranes Cytoplasmic protein plaque Cadherin Intercellular space Cytoskeletal filaments anchored to cytoplasmic plaque 0.1 μm(a) FIGURE 7.18 Desmosomes. (a) Desmosomes anchor adjacent cells to each other. (b) Cadherin proteins create the adhering link between adjoining cells. (b) Cadherin and Actin Filaments Cadherins can also connect the actin frame- works of cells in cadherin-mediated junc- tions (figure 7.19). When they do, they form less stable links between cells than when they connect intermediate filaments. Many kinds of actin-linking cadherins occur in dif- ferent tissues, as well as in the same tissue at different times. During vertebrate develop- ment, the migration of neurons in the em- bryo is associated with changes in the type of cadherin expressed on their plasma mem- branes. This suggests that gene-controlled changes in cadherin expression may provide the migrating cells with a “roadmap” to their destination. Integrin-Mediated Links Anchoring junctions called adherens junc- tions are another type of junction that con- nects the actin filaments of one cell with those of neighboring cells or with the extra- cellular matrix (figure 7.20). The linking proteins in these junctions are members of a large superfamily of cell surface receptors called integrins. Each integrin is a trans- membrane protein composed of two differ- ent glycoprotein subunits that extend out- ward from the plasma membrane. Together, these subunits bind a protein component of the extracellular matrix, like two hands clasping a pole. There appear to be many different kinds of integrin (cell biologists have identified 20), each with a slightly dif- ferent shaped “hand.” The exact component of the matrix that a given cell binds to de- pends on which combination of integrins that cell has in its plasma membrane. Anchoring junctions attach the cytoskeleton of a cell to the matrix surrounding the cell, or to the cytoskeleton of another cell. 138 Part II Biology of the Cell β β α γ x Extracellular domains of cadherin protein Adjoining cell membrane Plasma membrane Cytoplasm Cytoplasm Cadherin of adjoining cell Actin 10 nm COOH Intracellular attachment proteins NH 2 FIGURE 7.19 A cadherin-mediated junction. The cadherin molecule is anchored to actin in the cytoskeleton and passes through the membrane to interact with the cadherin of an adjoining cell. Extracellular matrix protein Plasma membrane Cytoplasm Integrin subunitIntegrin subunit Actin 10 nm COOHHOOC S S FIGURE 7.20 An integrin-mediated junction. These adherens junctions link the actin filaments inside cells to their neighbors and to the extracellular matrix. Communicating Junctions Many cells communicate with adjacent cells through direct connections, called communicating junctions. In these junctions, a chemical signal passes directly from one cell to an adjacent one. Communicating junctions establish direct physical connections that link the cytoplasms of two cells together, permitting small molecules or ions to pass from one to the other. In animals, these direct communication channels between cells are called gap junctions. In plants, they are called plasmodesmata. Gap Junctions in Animals Communicating junctions called gap junctions are com- posed of structures called connexons, complexes of six identical transmembrane proteins (figure 7.21). The pro- teins in a connexon are arranged in a circle to create a channel through the plasma membrane that protrudes sev- eral nanometers from the cell surface. A gap junction forms when the connexons of two cells align perfectly, creating an open channel spanning the plasma membranes of both cells. Gap junctions provide passageways large enough to permit small substances, such as simple sugars and amino acids, to pass from the cytoplasm of one cell to that of the next, yet small enough to prevent the passage of larger molecules such as proteins. The connexons hold the plasma membranes of the paired cells about 4 nanometers apart, in marked contrast to the more-or-less direct contact between the lipid bilayers in a tight junction. Gap junction channels are dynamic structures that can open or close in response to a variety of factors, including Ca ++ and H + ions. This gating serves at least one important function. When a cell is damaged, its plasma membrane often becomes leaky. Ions in high concentrations outside the cell, such as Ca ++ , flow into the damaged cell and shut its gap junction channels. This isolates the cell and so pre- vents the damage from spreading to other cells. Plasmodesmata in Plants In plants, cell walls separate every cell from all others. Cell- cell junctions occur only at holes or gaps in the walls, where the plasma membranes of adjacent cells can come into contact with each other. Cytoplasmic connections that form across the touching plasma membranes are called plasmodesmata (figure 7.22). The majority of living cells within a higher plant are connected with their neighbors by these junctions. Plasmodesmata function much like gap junctions in animal cells, although their structure is more complex. Unlike gap junctions, plasmodesmata are lined with plasma membrane and contain a central tubule that connects the endoplasmic reticulum of the two cells. Communicating junctions permit the controlled passage of small molecules or ions between cells. Chapter 7 Cell-Cell Interactions 139 Two adjacent connexons forming an open channel between cells Adjacent plasma membranes Connexon Channel (diameter 1.5 nm) Intercellular space "Gap" of 2-4 nm FIGURE 7.21 Gap junctions. Connexons in gap junctions create passageways that connect the cytoplasms of adjoining cells. Gap junctions readily allow the passage of small molecules and ions required for rapid communication (such as in heart tissue), but do not allow the passage of larger molecules like proteins. Vacuoles Cytoplasm Primary cell wall Middle lamella Plasmodesmata Nuclei FIGURE 7.22 Plasmodesmata. Plant cells can communicate through specialized openings in their cell walls, called plasmodesmata, where the cytoplasms of adjoining cells are connected. 140 Part II Biology of the Cell Chapter 7 Summary Questions Media Resources 7.1 Cells signal one another with chemicals. ? Cell signaling is accomplished through the recognition of signal molecules by target cells. 1. What determines which signal molecules in the extracellular environment a cell will respond to? 2. How do paracrine, endocrine, and synaptic signaling differ? ? The binding of a signal molecule to an intracellular receptor usually initiates transcription of specific regions of DNA, ultimately resulting in the production of specific proteins. ? Cell surface receptors bind to specific molecules in the extracellular fluid. In some cases, this binding causes the receptor to enzymatically alter other (usually internal) proteins, typically through phosphorylation. ? G proteins behave as intracellular shuttles, moving from an activated receptor to other areas in the cell. 3. Describe two of the ways in which intracellular receptors control cell activities. 4. What structural features are characteristic of chemically gated ion channels, and how are these features related to the function of the channels? 5. What are G proteins? How do they participate in cellular responses mediated by G- protein-linked receptors? 7.2 Proteins in the cell and on its surface receive signals from other cells. ? There are usually several amplifying steps between the binding of a signal molecule to a cell surface receptor and the response of the cell. These steps often involve phosphorylation by protein kinases. 6. How does the binding of a single signal molecule to a cell surface receptor result in an amplified response within the target cell? 7.3 Follow the journey of information into the cell. ? Tight junctions and desmosomes enable cells to adhere in tight, leakproof sheets, holding the cells together such that materials cannot pass between them. ? Gap junctions (in animals) and plasmodesmata (in plants) permit small substances to pass directly from cell to cell through special passageways. 7. What are the functions of tight junctions? What are the functions of desmosomes and adherens junctions, and what proteins are involved in these junctions? 8. What are the molecular components that make up gap junctions? What sorts of substances can pass through gap junctions? 9. Where are plasmodesmata found? What cellular constituents are found in plasmodesmata? 7.4 Cell surface proteins mediate cell-cell interactions. http://www.mhhe.com/raven6e http://www.biocourse.com ? Cell Interactions ? Student Research: Retrograde Messengers between Nerve Cells ? Student Research: Vertebrate Limb formation ? Exploration: Cell-Cell Interactions ? Scientists on Science: G Proteins 141 How Do Proteins Help Chlorophyll Carry Out Photosynthesis? Much public attention in recent years has been focused on high-profile science—headline-creating advances in the Human Genome Project, genetic engineering, and the battle against AIDS and cancer. Meanwhile, great advances have been made more quietly in other areas of biology. Among the greatest of these achievements has been the unmasking in the last decade of the underlying mechanism of photosynthesis. In photosynthesis, photons of light are absorbed by chlorophyll molecules, causing them to donate a high- energy electron that is put to work making NADPH and pumping protons to produce ATP. When researchers looked at the light-absorbing chloro- phylls that carry out photosynthesis more closely, they found the chlorophylls to be arranged in clusters called photosystems, supported by proteins and accessory pig- ments. Within a photosystem, hundreds of chlorophyll molecules act like antennae, absorbing light and passing the energy they capture inward to a single chlorophyll mole- cule that acts as the reaction center. This chlorophyll acts as the primary electron donor of photosynthesis. Once it releases a light-energized electron, the complex series of chemical events we call photosynthesis begins, and, like a falling row of dominos, is difficult to stop. Plants possess two kinds of photosystems that work together to harvest light energy. One of them, called photo- system I, is similar to a simpler photosystem found in pho- tosynthetic bacteria, and is thought to have evolved from it. Photosystem I has been the subject of intense research. In its reaction center, a pair of chlorophyll molecules act as the trap for photon energy, passing an excited electron on to an acceptor molecule outside the reaction center. This moves the photon energy away from the chlorophylls, and is the key conversion of light energy to chemical energy, the very heart of photosynthesis. Because the pair of chlorophyll molecules in the reaction center of photosystem I absorb light at a wavelength of 700 nm, they are together given the name P 700 . The P 700 dimer is positioned within the photosystem by two related proteins that act as scaffolds. These proteins, discovered less than 10 years ago, turn out to play a pivotal role in the pho- tosynthetic process. Passing back and forth across the inter- nal chloroplast membranes 11 times, they form a molecular frame that positions P 700 to accept energy from other chlorophyll molecules of the photosystem, and to donate a photo-excited electron to an acceptor molecule outside the photosystem. Recent research suggests that the role of these scaffold proteins, called PsaA and PsaB, is far more active than the passive support provided by a scaffold. Analysis of highly purified photosystems carried out in 1995 revealed that the distribution of electric charge over the two halves of the P 700 dimer is highly asymmetric—one chlorophyll molecule exhibits a far greater charge density than the other. Because the two chlorophyll molecules of P 700 are themselves identical, this suggests that the PsaA and PsaB proteins are actively modulating the physicochemical properties of the chlorophyll. How can a protein pull off this physical-chemical sleight-of-hand? Just what are these proteins doing to the chlorophyll molecules? To look more closely at what is going on, you have to first figure out what part of the pro- tein to look at. One way to get a handle on this problem is to compare the amino acid sequences of PsaA and PsaB with that of the bacterial photosystem from which they are thought to have evolved. It is likely that such an important part of the sequence would have been conserved and will be found in all three. Several sequences are indeed conserved, but most of them prove not to interact directly with chlorophyll. One, however, is a promising candidate. A single amino acid in the helix X domain (that is, the tenth pass of the PsaB protein across the membrane), dubbed His-656, is con- served in all sequences, and is positioned right where the PsaB protein touches the P 700 chlorophyll (see above). This amino acid, a histidine, has become the focus of recent efforts to clarify how proteins help chlorophyll carry out photosynthesis. Part Stroma Thylakoid space XI 700 Psa B protein 650 736 600 450 500 IX VIII VII 398 X 550 H P 700 III Energetics The proposed antenna complex of the PsaB protein. Position 656 is a histidine (H) in the tenth pass (helix X) of the PsaB protein across the thylakoid membrane within chloroplasts. This histidine is where the PsaB protein makes contact with a P 700 chlorophyll molecule. Real People Doing Real Science The Experiment To determine the importance of His-656, and more gener- ally of the helix X domain of the PsaB protein, Professor Andrew Webber of Arizona State University, working with his research team and the group of Professor Wolfgang Lubitz at Technische Universitat Berlin, has created site- directed mutations of His-656 in the photosynthetic protist Chlamydomonas reinhardtii. C. reinhardtii is widely used to study photosynthesis because of the ease with which lab ex- periments can be done. Webber and his collaborators set out to change the amino acid located at position 656 of PsaB, and then to look and see what effect the change had on photosynthesis. If His-656 indeed plays a critical role in modifying the P 700 chlorophylls, then a change at that position to a different amino acid should have profound effects. Creating PsaB Proteins Mutant at Position 656. The first and key step in Webber’s experimental approach was to genetically alter the chloroplast of C. reinhardtii, intro- ducing a mutation of the PsaB gene at the His-656 posi- tion. To do this, the team employed site-specific mutagen- esis to construct mutant plasmids pHN(B656) and pHS(B656), inserting a gene carrying either the Ser or Asn amino acids in place of His. The two mutation-carrying plasmids were then cloned into C. reinhardtii, cells carrying the mutant plasmids isolated, and presence of the mutated gene directly confirmed by sequencing the DNA. Characterizing the Effects of 656 Mutations. Once re- searchers confirmed that the C. reinhardtii chloroplast DNA now contained the mutant forms of the PsaB gene, they proceeded to test the function of the mutated PsaB protein in coordinating P 700 , examining interior thylakoid membranes isolated from the chloroplasts. To do this, the researchers measured the oxidation midpoint potentials of the P 700 complexes, an indication of how tightly the chloro- phyll molecules are holding onto their electrons. The researchers further characterized the P 700 com- plexes by measuring the changes in absorbance of the mu- tants versus the wild type to see if the mutations altered the spectral properties of the P 700 chlorophylls. The Results The results of the examination of the oxidation midpoint potentials revealed that the influence of the PsaB protein on P 700 had been profoundly altered by the mutations. The midpoint potential of P 700 in the wild type was deter- mined to be 447+6 mV, while the midpoint potential had increased to 487+6 mV in both the PsaB mutant I, HN(B656), and the PsaB mutant II, HS(B656) (see graph a). This increase in the oxidation midpoint potential by ap- proximately 40 mV indicates that the mutations to the His residue significantly altered the redox property of P 700 and, therefore, that His-656 is closely interacting with one of the chlorophyll molecules of the P 700 dimer. These results and this conclusion are further supported by changes observed in the spectral properties of the mutants and wild type (see graph b). There is a reduction and a slight shift in the 696 nm bleaching band (dip in absorbance) in PsaB mutant I toward the blue end of the spectrum and a new bleaching band appearing at 667 nm, both changes in the spectral properties of chlorophyll induced by the muta- tional changes in the PsaB protein. Ultimately, the researchers conclude that the His-656 of PsaB directly coordinates the central magnesium atom of one of the two chlorophyll molecules of P 700 . Their results are consistent with a model of photosystem I in which the first six spans of PsaB constitute an antenna domain for receiving energy from other chlorophylls and the last five membrane spans interact with the P 700 reaction complex. Relative absorbance dif ference at 826 nm Relative absorbance change 0 H110021 (b)(a) Wild type Mutant I Wild type Mutant I Mutant II 1.0 0.8 0.6 0.4 0.2 0.0 H110020.2 0.25 0.30 0.35 0.40 0.45 Potential (volts) Wavelength (nm) 0.50 0.55 0.60 0.65 500 550 600 650 700 Effect of altering position 656. (a) When P 700 interacts with normal and mutant forms of PsaB, the midpoint potentials are 447+6 mV in the wild type and 487+6 in the mutants, the mutant value being about 40 mV higher. (b) The bleaching band (dip in the absorbance) of P 700 is shifted to the blue (left) and exhibits a new bleaching band at 667 nm when interacting with mutant forms of PsaB. To explore this experiment further, go to the Virtual Lab at www.mhhe.com/raven6/vlab3.mhtml 143 8 Energy and Metabolism Concept Outline 8.1 The laws of thermodynamics describe how energy changes. The Flow of Energy in Living Things. Potential energy is present in the valance electrons of atoms, and so can be transferred from one molecule to another. The Laws of Thermodynamics. Energy is never lost but as it is transferred, more and more of it dissipates as heat, a disordered form of energy. Free Energy. In a chemical reaction, the energy released or supplied is the difference in bond energies between reactants and products, corrected for disorder. Activation Energy. To start a chemical reaction, an input of energy is required to destabilize existing chemical bonds. 8.2 Enzymes are biological catalysts. Enzymes. Globular proteins called enzymes catalyze chemical reactions within cells. How Enzymes Work. Enzymes have sites on their surface shaped to fit their substrates snugly, forcing chemically reactive groups close enough to facilitate a reaction. Enzymes Take Many Forms. Some enzymes are associated in complex groups; others are not even proteins. Factors Affecting Enzyme Activity. Each enzyme works most efficiently at its optimal temperature and pH. Metal ions or other substances often help enzymes carry out their catalysis. 8.3 ATP is the energy currency of life. What Is ATP? Cells store and release energy from the phosphate bonds of ATP, the energy currency of the cell. 8.4 Metabolism is the chemical life of a cell. Biochemical Pathways: The Organizational Units of Metabolism. Biochemical pathways are the organizational units of metabolism. The Evolution of Metabolism. The major metabolic processes evolved over a long period, building on what came before. L ife can be viewed as a constant flow of energy, channeled by organisms to do the work of living. Each of the signif- icant properties by which we define life—order, growth, re- production, responsiveness, and internal regulation— requires a constant supply of energy (figure 8.1). Deprived of a source of energy, life stops. Therefore, a comprehensive study of life would be impossible without discussing bioener- getics, the analysis of how energy powers the activities of liv- ing systems. In this chapter, we will focus on energy—on what it is and how organisms capture, store, and use it. FIGURE 8.1 Lion at lunch. Energy that this lion extracts from its meal of giraffe will be used to power its roar, fuel its running, and build a bigger lion. Oxidation-Reduction Energy flows into the biological world from the sun, which shines a constant beam of light on the earth. It is estimated that the sun provides the earth with more than 13 × 10 23 calories per year, or 40 million billion calories per second! Plants, algae, and certain kinds of bacteria capture a frac- tion of this energy through photosynthesis. In photosyn- thesis, energy garnered from sunlight is used to combine small molecules (water and carbon dioxide) into more com- plex molecules (sugars). The energy is stored as potential energy in the covalent bonds between atoms in the sugar molecules. Recall from chapter 2 that an atom consists of a central nucleus surrounded by one or more orbiting elec- trons, and a covalent bond forms when two atomic nuclei share valence electrons. Breaking such a bond requires en- ergy to pull the nuclei apart. Indeed, the strength of a cova- lent bond is measured by the amount of energy required to break it. For example, it takes 98.8 kcal to break one mole (6.023 × 10 23 ) of carbon-hydrogen (C—H) bonds. During a chemical reaction, the energy stored in chemical bonds may transfer to new bonds. In some of these reactions, electrons actually pass from one atom or molecule to another. When an atom or molecule loses an electron, it is said to be oxidized, and the process by which this occurs is called oxidation. The name reflects the fact that in biological systems oxygen, which attracts electrons strongly, is the most common electron accep- 144 Part III Energetics The Flow of Energy in Living Things Energy is defined as the capacity to do work. It can be con- sidered to exist in two states. Kinetic energy is the energy of motion (figure 8.2). Moving objects perform work by causing other matter to move. Potential energy is stored energy. Objects that are not actively moving but have the capacity to do so possess potential energy. A boulder perched on a hilltop has potential energy; as it begins to roll downhill, some of its potential energy is converted into kinetic energy. Much of the work that living organisms carry out involves transforming potential energy to kinetic energy. Energy can take many forms: mechanical energy, heat, sound, electric current, light, or radioactive radiation. Be- cause it can exist in so many forms, there are many ways to measure energy. The most convenient is in terms of heat, because all other forms of energy can be converted into heat. In fact, the study of energy is called thermodynam- ics, meaning heat changes. The unit of heat most com- monly employed in biology is the kilocalorie (kcal). One kilocalorie is equal to 1000 calories (cal), and one calorie is the heat required to raise the temperature of one gram of water one degree Celsius (°C). (It is important not to con- fuse calories with a term related to diets and nutrition, the Calorie with a capital C, which is actually another term for kilocalorie.) Another energy unit, often used in physics, is the joule; one joule equals 0.239 cal. 8.1 The laws of thermodynamics describe how energy changes. (a) Potential energy (b) Kinetic energy FIGURE 8.2 Potential and kinetic energy. (a) Objects that have the capacity to move but are not moving have potential energy. The energy required to move the ball up the hill is stored as potential energy. (b) Objects that are in motion have kinetic energy. The stored energy is released as kinetic energy as the ball rolls down the hill. tor. Conversely, when an atom or molecule gains an elec- tron, it is said to be reduced, and the process is called re- duction. Oxidation and reduction always take place to- gether, because every electron that is lost by an atom through oxidation is gained by some other atom through reduction. Therefore, chemical reactions of this sort are called oxidation-reduction (redox) reactions (figure 8.3). Energy is transferred from one molecule to another via redox reactions. The reduced form of a molecule thus has a higher level of energy than the oxidized form (figure 8.4). Oxidation-reduction reactions play a key role in the flow of energy through biological systems because the electrons that pass from one atom to another carry energy with them. The amount of energy an electron possesses depends on how far it is from the nucleus and how strongly the nu- cleus attracts it. Light (and other forms of energy) can add energy to an electron and boost it to a higher energy level. When this electron departs from one atom (oxidation) and moves to another (reduction), the electron’s added energy is transferred with it, and the electron orbits the second atom’s nucleus at the higher energy level. The added en- ergy is stored as potential chemical energy that the atom can later release when the electron returns to its original energy level. Energy is the capacity to do work, either actively (kinetic energy) or stored for later use (potential energy). Energy is transferred with electrons. Oxidation is the loss of an electron; reduction is the gain of one. Chapter 8 Energy and Metabolism 145 Product NAD + H H H H NAD + NAD + NAD NAD H Energy-rich molecule 1. Enzymes that harvest hydrogen atoms have a binding site for NAD + located near the substrate binding site. NAD + and an energy-rich molecule bind to the enzyme. 3. NADH then diffuses away and is available to other molecules. 2. In an oxidation-reduction reaction, a hydrogen atom is transferred to NAD + , forming NADH. FIGURE 8.3 An oxidation-reduction reaction. Cells use a chemical called NAD + to carry out oxidation-reduction reactions. Energetic electrons are often paired with a proton as a hydrogen atom. Molecules that gain energetic electrons are said to be reduced, while ones that lose energetic electrons are said to be oxidized. NAD + oxidizes energy-rich molecules by acquiring their hydrogens (in the figure, this proceeds 1→2→3) and then reduces other molecules by giving the hydrogens to them (in the figure, this proceeds 3→2→1). FIGURE 8.4 Redox reactions. Oxidation is the loss of an electron; reduction is the gain of an electron. In this example, the charges of molecules A and B are shown in small circles to the upper right of each molecule. Molecule A loses energy as it loses an electron, while molecule B gains energy as it gains an electron. Gain of electron (reduction) Low energy e – AB High energy Loss of electron (oxidation) A+ oo B + + – A* B* The Laws of Thermodynamics Running, thinking, singing, reading these words—all activities of living organisms involve changes in energy. A set of univer- sal laws we call the Laws of Thermody- namics govern all energy changes in the universe, from nuclear reactions to the buzzing of a bee. The First Law of Thermodynamics The first of these universal laws, the First Law of Thermodynamics, con- cerns the amount of energy in the uni- verse. It states that energy cannot be cre- ated or destroyed; it can only change from one form to another (from potential to kinetic, for example). The total amount of energy in the universe remains constant. The lion eating a giraffe in figure 8.1 is in the process of acquiring energy. Rather than creating new energy or cap- turing the energy in sunlight, the lion is merely transferring some of the potential energy stored in the giraffe’s tissues to its own body (just as the giraffe obtained the potential energy stored in the plants it ate while it was alive). Within any living organism, this chemical potential energy can be shifted to other molecules and stored in different chemical bonds, or it can convert into other forms, such as kinetic energy, light, or electricity. During each conversion, some of the energy dissipates into the environment as heat, a measure of the random motions of molecules (and, hence, a mea- sure of one form of kinetic energy). Energy continuously flows through the biological world in one direction, with new energy from the sun constantly entering the system to replace the energy dissipated as heat. Heat can be harnessed to do work only when there is a heat gradient, that is, a temperature difference between two areas (this is how a steam engine functions). Cells are too small to maintain significant internal temperature differ- ences, so heat energy is incapable of doing the work of cells. Thus, although the total amount of energy in the uni- verse remains constant, the energy available to do work de- creases, as progressively more of it dissipates as heat. The Second Law of Thermodynamics The Second Law of Thermodynamics concerns this trans- formation of potential energy into heat, or random molecular motion. It states that the disorder (more formally called en- tropy) in the universe is continuously increasing. Put simply, disorder is more likely than order. For example, it is much more likely that a column of bricks will tumble over than that a pile of bricks will arrange themselves spontaneously to form a column. In general, energy transformations proceed sponta- neously to convert matter from a more ordered, less stable form, to a less ordered, more stable form (figure 8.5). Entropy Entropy is a measure of the disorder of a system, so the Second Law of Thermodynamics can also be stated simply as “entropy increases.” When the universe formed, it held all the potential energy it will ever have. It has become pro- gressively more disordered ever since, with every energy exchange increasing the amount of entropy. The First Law of Thermodynamics states that energy cannot be created or destroyed; it can only undergo conversion from one form to another. The Second Law of Thermodynamics states that disorder (entropy) in the universe is increasing. As energy is used, more and more of it is converted to heat, the energy of random molecular motion. 146 Part III Energetics Disorder happens “spontaneously” Organization requires energy FIGURE 8.5 Entropy in action. As time elapses, a child’s room becomes more disorganized. It takes effort to clean it up. Free Energy It takes energy to break the chemical bonds that hold the atoms in a molecule together. Heat energy, because it in- creases atomic motion, makes it easier for the atoms to pull apart. Both chemical bonding and heat have a significant influence on a molecule, the former reducing disorder and the latter increasing it. The net effect, the amount of en- ergy actually available to break and subsequently form other chemical bonds, is called the free energy of that molecule. In a more general sense, free energy is defined as the energy available to do work in any system. In a mole- cule within a cell, where pressure and volume usually do not change, the free energy is denoted by the symbol G (for “Gibbs’ free energy,” which limits the system being considered to the cell). G is equal to the energy contained in a molecule’s chemical bonds (called enthalpy and desig- nated H) minus the energy unavailable because of disorder (called entropy and given the symbol S) times the absolute temperature, T, in degrees Kelvin (K = °C + 273): G = H – TS Chemical reactions break some bonds in the reactants and form new bonds in the products. Consequently, reac- tions can produce changes in free energy. When a chemical reaction occurs under conditions of constant temperature, pressure, and volume—as do most biological reactions— the change in free energy (?G) is simply: ?G = ?H – T ?S The change in free energy, or ?G, is a fundamental property of chemical reactions. In some reactions, the ?G is positive. This means that the products of the reaction contain more free energy than the reactants; the bond energy (H) is higher or the disorder (S) in the system is lower. Such reactions do not proceed spontaneously because they require an input of energy. Any reaction that requires an input of energy is said to be en- dergonic (“inward energy”). In other reactions, the ?G is negative. The products of the reaction contain less free energy than the reactants; ei- ther the bond energy is lower or the disorder is higher, or both. Such reactions tend to proceed spontaneously. Any chemical reaction will tend to proceed spontaneously if the difference in disorder (T ?S) is greater than the difference in bond energies between reactants and products (?H). Note that spontaneous does not mean the same thing as in- stantaneous. A spontaneous reaction may proceed very slowly. These reactions release the excess free energy as heat and are thus said to be exergonic (“outward energy”). Figure 8.6 sums up these reactions. Free energy is the energy available to do work. Within cells, the change in free energy (?G) is the difference in bond energies between reactants and products (?H), minus any change in the degree of disorder of the system (T ?S). Any reaction whose products contain less free energy than the reactants (?G is negative) will tend to proceed spontaneously. Chapter 8 Energy and Metabolism 147 Reactant Reactant Product Product ExergonicEndergonic Energy released Energy supplied Energy is released. Energy must be supplied. FIGURE 8.6 Energy in chemical reactions. (a) In an endergonic reaction, the products of the reaction contain more energy than the reactants, and the extra energy must be supplied for the reaction to proceed. (b) In an exergonic reaction, the products contain less energy than the reactants, and the excess energy is released. (a) (b) Activation Energy If all chemical reactions that release free energy tend to occur spontaneously, why haven’t all such reactions already occurred? One reason they haven’t is that most reactions require an input of energy to get started. Before it is possi- ble to form new chemical bonds, even bonds that contain less energy, it is first necessary to break the existing bonds, and that takes energy. The extra energy required to desta- bilize existing chemical bonds and initiate a chemical reac- tion is called activation energy (figure 8.7a). The rate of an exergonic reaction depends on the acti- vation energy required for the reaction to begin. Reac- tions with larger activation energies tend to proceed more slowly because fewer molecules succeed in over- coming the initial energy hurdle. Activation energies are not constant, however. Stressing particular chemical bonds can make them easier to break. The process of in- fluencing chemical bonds in a way that lowers the activa- tion energy needed to initiate a reaction is called cataly- sis, and substances that accomplish this are known as catalysts (figure 8.7b). Catalysts cannot violate the basic laws of thermody- namics; they cannot, for example, make an endergonic re- action proceed spontaneously. By reducing the activation energy, a catalyst accelerates both the forward and the re- verse reactions by exactly the same amount. Hence, it does not alter the proportion of reactant ultimately con- verted into product. 148 Part III Energetics To grasp this, imagine a bowling ball resting in a shal- low depression on the side of a hill. Only a narrow rim of dirt below the ball prevents it from rolling down the hill. Now imagine digging away that rim of dirt. If you remove enough dirt from below the ball, it will start to roll down the hill—but removing dirt from below the ball will never cause the ball to roll UP the hill! Removing the lip of dirt simply allows the ball to move freely; gravity determines the direction it then travels. Lowering the resistance to the ball’s movement will promote the movement dictated by its position on the hill. Similarly, the direction in which a chemical reaction proceeds is determined solely by the difference in free en- ergy. Like digging away the soil below the bowling ball on the hill, catalysts reduce the energy barrier preventing the reaction from proceeding. Catalysts don’t favor endergonic reactions any more than digging makes the hypothetical bowling ball roll uphill. Only exergonic reactions can pro- ceed spontaneously, and catalysts cannot change that. What catalysts can do is make a reaction proceed much faster. The rate of a reaction depends on the activation energy necessary to initiate it. Catalysts reduce the activation energy and so increase the rates of reactions, although they do not change the final proportions of reactants and products. Activation energy Reactant Product Activation energy Catalyzed Uncatalyzed Product Energy released Energy supplied Reactant FIGURE 8.7 Activation energy and catalysis. (a) Exergonic reactions do not necessarily proceed rapidly because energy must be supplied to destabilize existing chemical bonds. This extra energy is the activation energy for the reaction. (b) Catalysts accelerate particular reactions by lowering the amount of activation energy required to initiate the reaction. (a) (b) Enzymes The chemical reactions within living organisms are regu- lated by controlling the points at which catalysis takes place. Life itself is, therefore, regulated by catalysts. The agents that carry out most of the catalysis in living organ- isms are proteins called enzymes. (There is increasing evi- dence that some types of biological catalysis are carried out by RNA molecules.) The unique three-dimensional shape of an enzyme enables it to stabilize a temporary association between substrates, the molecules that will undergo the reaction. By bringing two substrates together in the correct orientation, or by stressing particular chemical bonds of a substrate, an enzyme lowers the activation energy required for new bonds to form. The reaction thus proceeds much more quickly than it would without the enzyme. Because the enzyme itself is not changed or consumed in the reac- tion, only a small amount of an enzyme is needed, and it can be used over and over. As an example of how an enzyme works, let’s consider the reaction of carbon dioxide and water to form carbonic acid. This important enzyme-catalyzed reaction occurs in vertebrate red blood cells: CO 2 + H 2 O ?→ H 2 CO 3 carbon water carbonic dioxide acid This reaction may proceed in either direction, but because it has a large activation energy, the reaction is very slow in the absence of an enzyme: perhaps 200 molecules of car- bonic acid form in an hour in a cell. Reactions that proceed this slowly are of little use to a cell. Cells overcome this problem by employing an enzyme within their cytoplasm called carbonic anhydrase (enzyme names usually end in “–ase”). Under the same conditions, but in the presence of carbonic anhydrase, an estimated 600,000 molecules of car- bonic acid form every second! Thus, the enzyme increases the reaction rate more than 10 million times. Thousands of different kinds of enzymes are known, each catalyzing one or a few specific chemical reactions. By facilitating particular chemical reactions, the enzymes in a cell determine the course of metabolism—the collec- tion of all chemical reactions—in that cell. Different types of cells contain different sets of enzymes, and this differ- ence contributes to structural and functional variations among cell types. The chemical reactions taking place within a red blood cell differ from those that occur within a nerve cell, in part because the cytoplasm and membranes of red blood cells and nerve cells contain different arrays of enzymes. Cells use proteins called enzymes as catalysts to lower activation energies. Chapter 8 Energy and Metabolism 149 8.2 Enzymes are biological catalysts. Catalysis: A Closer Look at Carbonic Anhydrase of the enzyme is a deep cleft traversing the enzyme, as if it had been cut with the blade of an ax. Deep within the cleft, some 1.5 nm from the surface, are located three his- tidines, their imidazole (nitrogen ring) groups all pointed at the same place in the center of the cleft. Together they hold a zinc ion firmly in position. This zinc ion will be the cutting blade of the catalytic process. Here is how the zinc catalyzes the reac- tion. Immediately adjacent to the position of the zinc atom in the cleft are a group of amino acids that recognize and bind carbon dioxide. The zinc atom interacts with this carbon dioxide molecule, orienting it in the plane of the cleft. Meanwhile, water bound to the zinc is rapidly converted to hydroxide ion. This hydroxide ion is now precisely po- sitioned to attack the carbon dioxide. When it does so, HCO 3 – is formed—and the en- zyme is unchanged (figure 8.A). Carbonic anhydrase is an effective cata- lyst because it brings its two substrates into close proximity and optimizes their orien- tation for reaction. Other enzymes use other mechanisms. Many, for example, use charged amino acids to polarize substrates or electronegative amino acids to stress particular bonds. Whatever the details of the reaction, however, the precise posi- tioning of substrates achieved by the par- ticular shape of the enzyme always plays a key role. One of the most rapidly acting enzymes in the human body is carbonic anhydrase, which plays a key role in blood by convert- ing dissolved CO 2 into carbonic acid, which dissociates into bicarbonate and hydrogen ions: CO 2 + H 2 O → H 2 CO 3 → HCO 3 – + H + Fully 70% of the CO 2 transported by the blood is transported as bicarbonate ion. This reaction is exergonic, but its energy of activation is significant, so that little con- version to bicarbonate occurs sponta- neously. In the presence of the enzyme car- bonic anhydrase, however, the rate of the reaction accelerates by a factor of more than 10 million! How does carbonic anhydrase catalyze this reaction so effectively? The active site Zn ++ Zn ++ HIS HIS HIS HIS HIS HIS H O – C O O H O C O – O FIGURE 8.A How Enzymes Work Most enzymes are globular proteins with one or more pockets or clefts on their surface called active sites (figure 8.8). Substrates bind to the enzyme at these active sites, forming an enzyme-substrate complex. For catalysis to occur within the complex, a substrate molecule must fit precisely into an active site. When that happens, amino acid side groups of the enzyme end up in close proximity to certain bonds of the substrate. These side groups interact chemically with the substrate, usually stressing or distorting a particular bond and consequently lowering the activation energy needed to break the bond. The substrate, now a product, then dissociates from the enzyme. Proteins are not rigid. The binding of a substrate in- duces the enzyme to adjust its shape slightly, leading to a better induced fit between enzyme and substrate (figure 8.9). This interaction may also facilitate the binding of other substrates; in such cases, the substrate itself “activates” the enzyme to receive other substrates. Enzymes typically catalyze only one or a few similar chemical reactions because they are specific in their choice of substrates. This specificity is due to the active site of the enzyme, which is shaped so that only a certain substrate molecule will fit into it. 150 Part III Energetics Active site (a) Substrate (b) FIGURE 8.8 How the enzyme lysozyme works. (a) A groove runs through lysozyme that fits the shape of the polysaccharide (a chain of sugars) that makes up bacterial cell walls. (b) When such a chain of sugars, indicated in yellow, slides into the groove, its entry induces the protein to alter its shape slightly and embrace the substrate more intimately. This induced fit positions a glutamic acid residue in the protein next to the bond between two adjacent sugars, and the glutamic acid “steals” an electron from the bond, causing it to break. The substrate, sucrose, consists of glucose and fructose bonded together. 1 The substrate binds to the enzyme, forming an enzyme-substrate complex. 2 The binding of the substrate and enzyme places stress on the glucose-fructose bond, and the bond breaks. 3 Products are released, and the enzyme is free to bind other substrates. 4 Bond Enzyme Active site H 2 O Glucose Fructose FIGURE 8.9 The catalytic cycle of an enzyme. Enzymes increase the speed with which chemical reactions occur, but they are not altered themselves as they do so. In the reaction illustrated here, the enzyme sucrase is splitting the sugar sucrose (present in most candy) into two simpler sugars: glucose and fructose. (1) First, the sucrose substrate binds to the active site of the enzyme, fitting into a depression in the enzyme surface. (2) The binding of sucrose to the active site forms an enzyme-substrate complex and induces the sucrase molecule to alter its shape, fitting more tightly around the sucrose. (3) Amino acid residues in the active site, now in close proximity to the bond between the glucose and fructose components of sucrose, break the bond. (4) The enzyme releases the resulting glucose and fructose fragments, the products of the reaction, and is then ready to bind another molecule of sucrose and run through the catalytic cycle once again. This cycle is often summarized by the equation: E + S ? [ ES ] ? E + P, where E = enzyme, S = substrate, ES = enzyme-substrate complex, and P = products. Enzymes Take Many Forms While many enzymes are suspended in the cytoplasm of cells, free to move about and not attached to any structure, other enzymes function as integral parts of cell structures and organelles. Multienzyme Complexes Often in cells the several enzymes catalyzing the different steps of a sequence of reactions are loosely associated with one another in non- covalently bonded assemblies called multienzyme complexes. The bacter- ial pyruvate dehydrogenase mul- tienzyme complex seen in figure 8.10 contains enzymes that carry out three sequential reactions in ox- idative metabolism. Each complex has multiple copies of each of the three enzymes—60 protein subunits in all. The many subunits work in concert, like a tiny factory. Multienzyme complexes offer significant advantages in catalytic efficiency: 1. The rate of any enzyme reaction is limited by the fre- quency with which the enzyme collides with its sub- strate. If a series of sequential reactions occurs within a multienzyme complex, the product of one reaction can be delivered to the next enzyme without releasing it to diffuse away. 2. Because the reacting substrate never leaves the com- plex during its passage through the series of reactions, the possibility of unwanted side reactions is eliminated. 3. All of the reactions that take place within the mul- tienzyme complex can be controlled as a unit. In addition to pyruvate dehydrogenase, which controls entry to the Krebs cycle, several other key processes in the cell are catalyzed by multienzyme complexes. One well-studied system is the fatty acid synthetase complex that catalyzes the synthesis of fatty acids from two-carbon precursors. There are seven different enzymes in this multienzyme complex, and the reaction intermediates re- main associated with the complex for the entire series of reactions. Not All Biological Catalysts Are Proteins Until a few years ago, most biology textbooks contained statements such as “Enzymes are the catalysts of biological systems.” We can no longer make that statement without qualification. As discussed in chapter 4, Tom Cech and his colleagues at the University of Colorado reported in 1981 that certain reactions involving RNA molecules appear to be catalyzed in cells by RNA itself, rather than by enzymes. This initial observation has been corroborated by addi- tional examples of RNA catalysis in the last few years. Like enzymes, these RNA catalysts, which are loosely called “ri- bozymes,” greatly accelerate the rate of particular biochem- ical reactions and show extraordinary specificity with re- spect to the substrates on which they act. There appear to be at least two sorts of ribozymes. Those that carry out intramolecular catalysis have folded structures and catalyze reactions on themselves. Those that carry out intermolecular catalysis act on other mole- cules without themselves being changed in the process. Many important cellular reactions involve small RNA molecules, including reactions that chip out unnecessary sections from RNA copies of genes, that prepare ribo- somes for protein synthesis, and that facilitate the replica- tion of DNA within mitochondria. In all of these cases, the possibility of RNA catalysis is being actively investi- gated. It seems likely, particularly in the complex process of photosynthesis, that both enzymes and RNA play im- portant catalytic roles. The ability of RNA, an informational molecule, to act as a catalyst has stirred great excitement among biologists, as it appears to provide a potential answer to the “chicken- and-egg” riddle posed by the spontaneous origin of life hy- pothesis discussed in chapter 3. Which came first, the pro- tein or the nucleic acid? It now seems at least possible that RNA may have evolved first and catalyzed the formation of the first proteins. Not all biological catalysts float free in the cytoplasm. Some are part of other structures, and some are not even proteins. Chapter 8 Energy and Metabolism 151 (a) FIGURE 8.10 The enzyme pyruvate dehydrogenase. The enzyme (model, a) that carries out the oxidation of pyruvate is one of the most complex enzymes known—it has 60 subunits, many of which can be seen in the electron micrograph (b) (200,000×). Factors Affecting Enzyme Activity The rate of an enzyme-catalyzed reaction is affected by the concentration of substrate, and of the enzyme that works on it. In addition, any chemical or physical factor that alters the enzyme’s three-dimensional shape—such as tempera- ture, pH, salt concentration, and the binding of specific regulatory molecules—can affect the enzyme’s ability to catalyze the reaction. Temperature Increasing the temperature of an uncatalyzed reaction will increase its rate because the additional heat represents an increase in random molecular movement. The rate of an enzyme-catalyzed reaction also increases with temperature, but only up to a point called the temperature optimum (fig- ure 8.11a). Below this temperature, the hydrogen bonds and hydrophobic interactions that determine the enzyme’s shape are not flexible enough to permit the induced fit that is optimum for catalysis. Above the temperature optimum, these forces are too weak to maintain the enzyme’s shape against the increased random movement of the atoms in the enzyme. At these higher temperatures, the enzyme de- natures, as we described in chapter 3. Most human enzymes have temperature optima between 35°C and 40°C, a range that includes normal body temperature. Bacteria that live in hot springs have more stable enzymes (that is, enzymes held together more strongly), so the temperature optima for those enzymes can be 70°C or higher. pH Ionic interactions between oppositely charged amino acid residues, such as glutamic acid (–) and lysine (+), also hold enzymes together. These interactions are sensitive to the hydrogen ion concentration of the fluid the enzyme is dis- solved in, because changing that concentration shifts the balance between positively and negatively charged amino acid residues. For this reason, most enzymes have a pH op- timum that usually ranges from pH 6 to 8. Those enzymes able to function in very acid environments are proteins that maintain their three-dimensional shape even in the pres- ence of high levels of hydrogen ion. The enzyme pepsin, for example, digests proteins in the stomach at pH 2, a very acidic level (figure 8.11b). Inhibitors and Activators Enzyme activity is sensitive to the presence of specific sub- stances that bind to the enzyme and cause changes in its shape. Through these substances, a cell is able to regulate which enzymes are active and which are inactive at a partic- ular time. This allows the cell to increase its efficiency and to control changes in its characteristics during develop- ment. A substance that binds to an enzyme and decreases its activity is called an inhibitor. Very often, the end product of a biochemical pathway acts as an inhibitor of an early re- action in the pathway, a process called feedback inhibition (to be discussed later). Enzyme inhibition occurs in two ways: competitive inhibitors compete with the substrate for the same bind- ing site, displacing a percentage of substrate molecules from the enzymes; noncompetitive inhibitors bind to the enzyme in a location other than the active site, changing the shape of the enzyme and making it unable to bind to the substrate (figure 8.12). Most noncompeti- tive inhibitors bind to a specific portion of the enzyme called an allosteric site (Greek allos, “other” + steros, “form”). These sites serve as chemical on/off switches; the binding of a substance to the site can switch the en- zyme between its active and inactive configurations. A substance that binds to an allosteric site and reduces en- zyme activity is called an allosteric inhibitor (figure 8.12b). Alternatively, activators bind to allosteric sites and keep the enzymes in their active configurations, thereby increasing enzyme activity. 152 Part III Energetics 30 Optimum temperature for human enzyme Optimal temperature for enzyme from hotsprings bacterium Optimum pH for pepsin Rate of reaction Temperature of reaction (°C) pH of reaction Rate of reaction Optimum pH for trypsin 40 50 60 70 80 1 2 3 4 5 6 7 8 9 (a) (b) FIGURE 8.11 Enzymes are sensitive to their environment. The activity of an enzyme is influenced by both (a) temperature and (b) pH. Most human enzymes, such as the protein-degrading enzyme trypsin, work best at temperatures of about 40°C and within a pH range of 6 to 8. Enzyme Cofactors Enzyme function is often assisted by additional chemical components known as cofactors. For example, the active sites of many enzymes contain metal ions that help draw electrons away from substrate molecules. The enzyme carboxypeptidase digests proteins by employing a zinc ion (Zn ++ ) in its active site to remove electrons from the bonds joining amino acids. Other elements, such as molybdenum and manganese, are also used as cofactors. Like zinc, these substances are required in the diet in small amounts. When the cofactor is a nonprotein organic molecule, it is called a coenzyme. Many vitamins are parts of coenzymes. In numerous oxidation-reduction reactions that are cat- alyzed by enzymes, the electrons pass in pairs from the ac- tive site of the enzyme to a coenzyme that serves as the electron acceptor. The coenzyme then transfers the elec- trons to a different enzyme, which releases them (and the energy they bear) to the substrates in another reaction. Often, the electrons pair with protons (H + ) as hydrogen atoms. In this way, coenzymes shuttle energy in the form of hydrogen atoms from one enzyme to another in a cell. One of the most important coenzymes is the hydrogen acceptor nicotinamide adenine dinucleotide (NAD + ) (figure 8.13). The NAD + molecule is composed of two nucleotides bound together. As you may recall from chapter 3, a nucleotide is a five-carbon sugar with one or more phosphate groups attached to one end and an or- ganic base attached to the other end. The two nu- cleotides that make up NAD + , nicotinamide monophos- phate (NMP) and adenine monophosphate (AMP), are joined head-to-head by their phosphate groups. The two nucleotides serve different functions in the NAD + mole- cule: AMP acts as the core, providing a shape recognized by many enzymes; NMP is the active part of the mole- cule, contributing a site that is readily reduced (that is, easily accepts electrons). When NAD + acquires an electron and a hydrogen atom (actually, two electrons and a proton) from the active site of an enzyme, it is reduced to NADH. The NADH molecule now carries the two energetic electrons and the proton. The oxidation of energy-containing molecules, which pro- vides energy to cells, involves stripping electrons from those molecules and donating them to NAD + . As we’ll see, much of the energy of NADH is transferred to another molecule. Enzymes have an optimum temperature and pH, at which the enzyme functions most effectively. Inhibitors decrease enzyme activity, while activators increase it. The activity of enzymes is often facilitated by cofactors, which can be metal ions or other substances. Cofactors that are nonprotein organic molecules are called coenzymes. Chapter 8 Energy and Metabolism 153 (a) Competitive inhibition (b) Noncompetitive inhibition Competitive inhibitor inteferes with active site of enzyme so substrate cannot bind Allosteric inhibitor changes shape of enzyme so it cannot bind to substrate Enzyme Substrate Enzyme Substrate FIGURE 8.12 How enzymes can be inhibited. (a) In competitive inhibition, the inhibitor interferes with the active site of the enzyme. (b) In noncompetitive inhibition, the inhibitor binds to the enzyme at a place away from the active site, effecting a conformational change in the enzyme so that it can no longer bind to its substrate. N + OCH 2 HH P O – O O OPO O – OH OH OH OH HH O CH 2 HH HH O C O NH 2 N N N N NH 2 NMP reactive group AMP group FIGURE 8.13 The chemical structure of nicotinamide adenine dinucleotide (NAD + ). This key cofactor is composed of two nucleotides, NMP and AMP, attached head-to-head. What Is ATP? The chief energy currency all cells use is a molecule called adenosine triphosphate (ATP). Cells use their supply of ATP to power almost every energy-requiring process they carry out, from making sugars, to supplying activation en- ergy for chemical reactions, to actively transporting sub- stances across membranes, to moving through their envi- ronment and growing. Structure of the ATP Molecule Each ATP molecule is a nucleotide composed of three smaller components (figure 8.14). The first component is a five-carbon sugar, ribose, which serves as the backbone to which the other two subunits are attached. The second component is adenine, an organic molecule composed of two carbon-nitrogen rings. Each of the nitrogen atoms in the ring has an unshared pair of electrons and weakly at- tracts hydrogen ions. Adenine, therefore, acts chemically as a base and is usually referred to as a nitrogenous base (it is one of the four nitrogenous bases found in DNA and RNA). The third component of ATP is a triphosphate group (a chain of three phosphates). How ATP Stores Energy The key to how ATP stores energy lies in its triphosphate group. Phosphate groups are highly negatively charged, so they repel one another strongly. Because of the electrosta- tic repulsion between the charged phosphate groups, the two covalent bonds joining the phosphates are unstable. The ATP molecule is often referred to as a “coiled spring,” the phosphates straining away from one another. The unstable bonds holding the phosphates together in the ATP molecule have a low activation energy and are easily broken. When they break, they can transfer a consid- erable amount of energy. In most reactions involving ATP, only the outermost high-energy phosphate bond is hy- drolyzed, cleaving off the phosphate group on the end. When this happens, ATP becomes adenosine diphos- phate (ADP), and energy equal to 7.3 kcal/mole is released under standard conditions. The liberated phosphate group usually attaches temporarily to some intermediate mole- cule. When that molecule is dephosphorylated, the phos- phate group is released as inorganic phosphate (P i ). How ATP Powers Energy-Requiring Reactions Cells use ATP to drive endergonic reactions. Such reac- tions do not proceed spontaneously, because their products possess more free energy than their reactants. However, if the cleavage of ATP’s terminal high-energy bond releases more energy than the other reaction consumes, the overall energy change of the two coupled reactions will be exer- gonic (energy releasing) and they will both proceed. Be- cause almost all endergonic reactions require less energy than is released by the cleavage of ATP, ATP can provide most of the energy a cell needs. The same feature that makes ATP an effective energy donor—the instability of its phosphate bonds—precludes it from being a good long-term energy storage molecule. Fats and carbohydrates serve that function better. Most cells do not maintain large stockpiles of ATP. Instead, they typically have only a few seconds’ supply of ATP at any given time, and they continually produce more from ADP and P i . The instability of its phosphate bonds makes ATP an excellent energy donor. 154 Part III Energetics 8.3 ATP is the energy currency of life. Triphosphate group High-energy bonds O == P — O – O – — O O == P — O – O O == P — O O – — AMP core CH 2 HH OH OH HH O N N N N NH 2 Adenine Ribose (a) (b) FIGURE 8.14 The ATP molecule. (a) The model and (b) structural diagram both show that like NAD + , ATP has a core of AMP. In ATP the reactive group added to the end of the AMP phosphate group is not another nucleotide but rather a chain of two additional phosphate groups. The bonds connecting these two phosphate groups to each other and to AMP are energy-storing bonds. Biochemical Pathways: The Organizational Units of Metabolism This living chemistry, the total of all chemical reactions carried out by an organism, is called metabolism (Greek metabole, “change”). Those reactions that expend energy to make or transform chemical bonds are called anabolic reac- tions, or anabolism. Reactions that harvest energy when chemical bonds are broken are called catabolic reactions, or catabolism. Organisms contain thousands of different kinds of en- zymes that catalyze a bewildering variety of reactions. Many of these reactions in a cell occur in sequences called biochemical pathways. In such pathways, the product of one reaction becomes the substrate for the next (figure 8.15). Biochemical pathways are the organizational units of metabolism, the elements an organism controls to achieve coherent metabolic activity. Most sequential enzyme steps in biochemical pathways take place in specific compart- ments of the cell; the steps of the citric acid cycle (chapter 9), for example, occur inside mitochondria. By determining where many of the enzymes that catalyze these steps are lo- cated, we can “map out” a model of metabolic processes in the cell. How Biochemical Pathways Evolved In the earliest cells, the first biochemical processes proba- bly involved energy-rich molecules scavenged from the en- vironment. Most of the molecules necessary for these processes are thought to have existed in the “organic soup” of the early oceans. The first catalyzed reactions are thought to have been simple, one-step reactions that brought these molecules together in various combinations. Eventually, the energy-rich molecules became depleted in the external environment, and only organisms that had evolved some means of making those molecules from other substances in the environment could survive. Thus, a hypo- thetical reaction, F + ?→ H G where two energy-rich molecules (F and G) react to pro- duce compound H and release energy, became more com- plex when the supply of F in the environment ran out. A new reaction was added in which the depleted molecule, F, is made from another molecule, E, which was also present in the environment: E ?→ F + ?→ H G When the supply of E in turn became depleted, organisms that were able to make it from some other available precur- sor, D, survived. When D became depleted, those organ- isms in turn were replaced by ones able to synthesize D from another molecule, C: C ?→ D ?→ E ?→ F + ?→ H G This hypothetical biochemical pathway would have evolved slowly through time, with the final reactions in the pathway evolving first and earlier reactions evolving later. Looking at the pathway now, we would say that the organ- ism, starting with compound C, is able to synthesize H by means of a series of steps. This is how the biochemical pathways within organisms are thought to have evolved— not all at once, but one step at a time, backward. Chapter 8 Energy and Metabolism 155 8.4 Metabolism is the chemical life of a cell. Product Enzyme 1 Enzyme 3 Enzyme 4 Substrate Enzyme 2 FIGURE 8.15 A biochemical pathway. The original substrate is acted on by enzyme 1, changing the substrate to a new form recognized by enzyme 2. Each enzyme in the pathway acts on the product of the previous stage. How Biochemical Pathways Are Regulated For a biochemical pathway to operate effi- ciently, its activity must be coordinated and regulated by the cell. Not only is it unnecessary to synthesize a compound when plenty is already present, doing so would waste energy and raw materials that could be put to use elsewhere. It is, there- fore, advantageous for a cell to temporar- ily shut down biochemical pathways when their products are not needed. The regulation of simple biochemical pathways often depends on an elegant feedback mechanism: the end product of the pathway binds to an allosteric site on the enzyme that catalyzes the first reaction in the pathway. In the hypothetical path- way we just described, the enzyme catalyz- ing the reaction C ?→ D would possess an allosteric site for H, the end product of the pathway. As the pathway churned out its product and the amount of H in the cell increased, it would become increas- ingly likely that one of the H molecules would encounter the allosteric site on the C ?→ D enzyme. If the product H func- tioned as an allosteric inhibitor of the en- zyme, its binding to the enzyme would es- sentially shut down the reaction C ?→ D. Shutting down this reaction, the first reaction in the pathway, effectively shuts down the whole pathway. Hence, as the cell produces in- creasing quantities of the product H, it automatically in- hibits its ability to produce more. This mode of regulation is called feedback inhibition (figure 8.16). A biochemical pathway is an organized series of reactions, often regulated as a unit. 156 Part III Energetics endergonic reaction A chemical reaction to which energy from an outside source must be added before the reaction proceeds; the opposite of an exergonic reaction. entropy A measure of the randomness or disorder of a system. In cells, it is a measure of how much energy has become so dis- persed (usually as evenly distributed heat) that it is no longer available to do work. exergonic reaction. An energy-yielding chemical reaction. Exergonic reactions tend to proceed spontaneously, although activa- tion energy is required to initiate them. free energy Energy available to do work. kilocalorie 1000 calories. A calorie is the heat required to raise the temperature of 1 gram of water by 1°C. metabolism The sum of all chemical processes occurring within a living cell or organism. oxidation The loss of an electron by an atom or molecule. It occurs simultaneously with reduction of some other atom or mole- cule because an electron that is lost by one is gained by another. reduction The gain of an electron by an atom or molecule. Oxidation-reduction re- actions are an important means of energy transfer within living systems. substrate A molecule on which an en- zyme acts; the initial reactant in an enzyme- catalyzed reaction. activation energy The energy required to destabilize chemical bonds and to initiate a chemical reaction. catalysis Acceleration of the rate of a chemical reaction by lowering the activa- tion energy. coenzyme A nonprotein organic mole- cule that plays an accessory role in enzyme- catalyzed reactions, often by acting as a donor or acceptor of electrons. NAD + is a coenzyme. A Vocabulary of Metabolism End product Initial substrate Intermediate A Intermediate B End product End-product inhibition + (b) Initial substrate Intermediate A Intermediate B End product Enzyme 1 Enzyme 2 Enzyme 3 Enzyme 2 Enzyme 3 No end-product inhibition (a) Enzyme 1 FIGURE 8.16 Feedback inhibition. (a) A biochemical pathway with no feedback inhibition. (b) A biochemical pathway in which the final end product becomes the allosteric effector for the first enzyme in the pathway. In other words, the formation of the pathway’s final end product stops the pathway. The Evolution of Metabolism Metabolism has changed a great deal as life on earth has evolved. This has been particularly true of the reactions or- ganisms use to capture energy from the sun to build or- ganic molecules (anabolism), and then break down organic molecules to obtain energy (catabolism). These processes, the subject of the next two chapters, evolved in concert with each other. Degradation The most primitive forms of life are thought to have ob- tained chemical energy by degrading, or breaking down, organic molecules that were abiotically produced. The first major event in the evolution of metabolism was the origin of the ability to harness chemical bond energy. At an early stage, organisms began to store this energy in the bonds of ATP, an energy carrier used by all organisms today. Glycolysis The second major event in the evolution of metabolism was glycolysis, the initial breakdown of glucose. As proteins evolved diverse catalytic functions, it became possible to capture a larger fraction of the chemical bond energy in or- ganic molecules by breaking chemical bonds in a series of steps. For example, the progressive breakdown of the six- carbon sugar glucose into three-carbon molecules is per- formed in a series of 10 steps that results in the net produc- tion of two ATP molecules. The energy for the synthesis of ATP is obtained by breaking chemical bonds and forming new ones with less bond energy, the energy difference being channeled into ATP production. This biochemical pathway is called glycolysis. Glycolysis undoubtedly evolved early in the history of life on earth, since this biochemical pathway has been retained by all living organisms. It is a chemical process that does not appear to have changed for well over 3 billion years. Anaerobic Photosynthesis The third major event in the evolution of metabolism was anaerobic photosynthesis. Early in the history of life, some organisms evolved a different way of generating ATP, called photosynthesis. Instead of obtaining energy for ATP synthesis by reshuffling chemical bonds, as in glycolysis, these organisms developed the ability to use light to pump protons out of their cells, and to use the resulting proton gradient to power the production of ATP, a process called chemiosmosis. Photosynthesis evolved in the absence of oxygen and works well without it. Dissolved H 2 S, present in the oceans beneath an atmosphere free of oxygen gas, served as a ready source of hydrogen atoms for building organic molecules. Free sulfur was produced as a by-product of this reaction. Nitrogen Fixation Nitrogen fixation was the fourth major step in the evolu- tion of metabolism. Proteins and nucleic acids cannot be synthesized from the products of photosynthesis because both of these biologically critical molecules contain ni- trogen. Obtaining nitrogen atoms from N 2 gas, a process called nitrogen fixation, requires the breaking of an N≡N triple bond. This important reaction evolved in the hydrogen-rich atmosphere of the early earth, an atmos- phere in which no oxygen was present. Oxygen acts as a poison to nitrogen fixation, which today occurs only in oxygen-free environments, or in oxygen-free compart- ments within certain bacteria. Oxygen-Forming Photosynthesis The substitution of H 2 O for H 2 S in photosynthesis was the fifth major event in the history of metabolism. Oxygen- forming photosynthesis employs H 2 O rather than H 2 S as a source of hydrogen atoms and their associated electrons. Because it garners its hydrogen atoms from reduced oxygen rather than from reduced sulfur, it generates oxygen gas rather than free sulfur. More than 2 billion years ago, small cells capable of car- rying out this oxygen-forming photosynthesis, such as cyanobacteria, became the dominant forms of life on earth. Oxygen gas began to accumulate in the atmosphere. This was the beginning of a great transition that changed condi- tions on earth permanently. Our atmosphere is now 20.9% oxygen, every molecule of which is derived from an oxygen-forming photosynthetic reaction. Aerobic Respiration Aerobic respiration is the sixth and final event in the his- tory of metabolism. This cellular process harvests energy by stripping energetic electrons from organic molecules. Aerobic respiration employs the same kind of proton pumps as photosynthesis, and is thought to have evolved as a modification of the basic photosynthetic machinery. However, the hydrogens and their associated electrons are not obtained from H 2 S or H 2 O, as in photosynthesis, but rather from the breakdown of organic molecules. Biologists think that the ability to carry out photosyn- thesis without H 2 S first evolved among purple nonsulfur bacteria, which obtain their hydrogens from organic com- pounds instead. It was perhaps inevitable that among the descendants of these respiring photosynthetic bacteria, some would eventually do without photosynthesis en- tirely, subsisting only on the energy and hydrogens de- rived from the breakdown of organic molecules. The mi- tochondria within all eukaryotic cells are thought to be their descendants. Six major innovations highlight the evolution of metabolism as we know it today. Chapter 8 Energy and Metabolism 157 ? Energy Conversion ? Catalysis ? Thermodynamics ? Coupled Reactions 158 Part III Energetics Chapter 8 Summary Questions Media Resources 8.1 The laws of thermodynamics describe how energy changes. ? Energy is the capacity to bring about change, to provide motion against a force, or to do work. ? Kinetic energy is actively engaged in doing work, while potential energy has the capacity to do so. ? An oxidation-reduction (redox) reaction is one in which an electron is taken from one atom or molecule (oxidation) and donated to another (reduction). ? The First Law of Thermodynamics states that the amount of energy in the universe is constant; energy is neither lost nor created. ? The Second Law of Thermodynamics states that disorder in the universe (entropy) tends to increase. ? Any chemical reaction whose products contain less free energy than the original reactants can proceed spontaneously. However, the difference in free energy does not determine the rate of the reaction. ? The rate of a reaction depends on the amount of activation energy required to break existing bonds. ? Catalysis is the process of lowering activation energies by stressing chemical bonds. 1. What is the difference between anabolism and catabolism? 2. Define oxidation and reduction. Why must these two reactions always occur in concert? 3. State the First and Second Laws of Thermodynamics. 4. What is heat? What is entropy? What is free energy? 5. What is the difference between an exergonic and an endergonic reaction? Which type of reaction tends to proceed spontaneously? 6. Define activation energy. How does a catalyst affect the final proportion of reactant converted into product? ? Enzymes are the major catalysts of cells; they affect the rate of a reaction but not the ultimate balance between reactants and products. ? Cells contain many different enzymes, each of which catalyzes a specific reaction. ? The specificity of an enzyme is due to its active site, which fits only one or a few types of substrate molecules. 7. How are the rates of enzyme-catalyzed reactions affected by temperature? What is the molecular basis for the effect on reaction rate? 8. What is the difference between the active site and an allosteric site on an enzyme? 8.2 Enzymes are biological catalysts. ? Cells obtain energy from photosynthesis and the oxidation of organic molecules and use it to manufacture ATP from ADP and phosphate. ? The energy stored in ATP is then used to drive endergonic reactions. 9. What part of the ATP molecule contains the bond that is employed to provide energy for most of the endergonic reactions in cells? 8.3 ATP is the energy currency of life. ? Generally, the final reactions of a biochemical pathway evolved first; preceding reactions in the pathway were added later, one step at a time. 10. What is a biochemical pathway? How does feedback inhibition regulate the activity of a biochemical pathway? 8.4 Metabolism is the chemical life of a cell. ? Exploration: Thermodynamics ? Exploration: Kinetics ? Enzymes ? ATP ? Feedback Inhibition http://www.mhhe.com/raven6e http://www.biocourse.com 159 9 How Cells Harvest Energy Concept Outline 9.1 Cells harvest the energy in chemical bonds. Using Chemical Energy to Drive Metabolism. The energy in C—H, C—O, and other chemical bonds can be captured and used to fuel the synthesis of ATP. 9.2 Cellular respiration oxidizes food molecules. An Overview of Glucose Catabolism. The chemical energy in sugar is harvested by both substrate-level phosphorylation and by aerobic respiration. Stage One: Glycolysis. The 10 reactions of glycolysis capture energy from glucose by reshuffling the bonds. Stage Two: The Oxidation of Pyruvate. Pyruvate, the product of glycolysis, is oxidized to acetyl-CoA. Stage Three: The Krebs Cycle. In a series of reactions, electrons are stripped from acetyl-CoA. Harvesting Energy by Extracting Electrons. The respiration of glucose is a series of oxidation-reduction reactions which involve stripping electrons from glucose and using the energy of these electrons to power the synthesis of ATP. Stage Four: The Electron Transport Chain. The electrons harvested from glucose pass through a chain of membrane proteins that use the energy to pump protons, driving the synthesis of ATP. Summarizing Aerobic Respiration. The oxidation of glucose by aerobic respiration in eukaryotes produces up to three dozen ATP molecules, over half the energy in the chemical bonds of glucose. Regulating Aerobic Respiration. High levels of ATP tend to shut down cellular respiration by feedback-inhibiting key reactions. 9.3 Catabolism of proteins and fats can yield considerable energy. Glucose Is Not the Only Source of Energy. Proteins and fats are dismantled and the products fed into cellular respiration. 9.4 Cells can metabolize food without oxygen. Fermentation. Fermentation allows continued metabolism in the absence of oxygen by donating the electrons harvested in glycolysis to organic molecules. L ife is driven by energy. All the activities organisms carry out—the swimming of bacteria, the purring of a cat, your reading of these words—use energy. In this chap- ter, we will discuss the processes all cells use to derive chemical energy from organic molecules and to convert that energy to ATP. We will consider photosynthesis, which uses light energy rather than chemical energy, in de- tail in chapter 10. We examine the conversion of chemical energy to ATP first because all organisms, both photosyn- thesizers and the organisms that feed on them (like the field mice in figure 9.1), are capable of harvesting energy from chemical bonds. As you will see, though, this process and photosynthesis have much in common. FIGURE 9.1 Harvesting chemical energy. Organisms such as these harvest mice depend on the energy stored in the chemical bonds of the food they eat to power their life processes. fireplace. In both instances, the reactants are carbohydrates and oxygen, and the products are carbon dioxide, water, and energy: C 6 H 12 O 6 + 6 O 2 ?→ 6 CO 2 + 6 H 2 O + energy (heat or ATP) The change in free energy in this reaction is –720 kilo- calories (–3012 kilojoules) per mole of glucose under the conditions found within a cell (the traditional value of –686 kilocalories, or –2870 kJ, per mole refers to stan- dard conditions—room temperature, one atmosphere of pressure, etc.). This change in free energy results largely from the breaking of the six C—H bonds in the glucose molecule. The negative sign indicates that the products possess less free energy than the reactants. The same amount of energy is released whether glucose is catabo- lized or burned, but when it is burned most of the energy is released as heat. This heat cannot be used to perform work in cells. The key to a cell’s ability to harvest useful energy from the catabolism of food molecules such as glucose is its conversion of a portion of the energy into a more useful form. Cells do this by using some of the en- ergy to drive the production of ATP, a molecule that can power cellular activities. 160 Part III Energetics Using Chemical Energy to Drive Metabolism Plants, algae, and some bacteria harvest the en- ergy of sunlight through photosynthesis, convert- ing radiant energy into chemical energy. These organisms, along with a few others that use chemical energy in a similar way, are called au- totrophs (“self-feeders”). All other organisms live on the energy autotrophs produce and are called heterotrophs (“fed by others”). At least 95% of the kinds of organisms on earth—all ani- mals and fungi, and most protists and bacteria— are heterotrophs. Where is the chemical energy in food, and how do heterotrophs harvest it to carry out the many tasks of living (figure 9.2)? Most foods contain a variety of carbohydrates, proteins, and fats, all rich in energy-laden chemical bonds. Carbohydrates and fats, for example, possess many carbon-hydrogen (C—H), as well as carbon-oxygen (C—O) bonds. The job of ex- tracting energy from this complex organic mix- ture is tackled in stages. First, enzymes break the large molecules down into smaller ones, a process called digestion. Then, other enzymes dismantle these fragments a little at a time, har- vesting energy from C—H and other chemical bonds at each stage. This process is called ca- tabolism. While you obtain energy from many of the constituents of food, it is traditional to focus first on the catabolism of carbohydrates. We will follow the six-carbon sugar, glucose (C 6 H 12 O 6 ), as its chemical bonds are progressively har- vested for energy. Later, we will come back and examine the catabolism of proteins and fats. Cellular Respiration The energy in a chemical bond can be visualized as poten- tial energy borne by the electrons that make up the cova- lent bond. Cells harvest this energy by putting the elec- trons to work, often to produce ATP, the energy currency of the cell. Afterward, the energy-depleted electron (associ- ated with a proton as a hydrogen atom) is donated to some other molecule. When oxygen gas (O 2 ) accepts the hydro- gen atom, water forms, and the process is called aerobic respiration. When an inorganic molecule other than oxy- gen accepts the hydrogen, the process is called anaerobic respiration. When an organic molecule accepts the hydro- gen atom, the process is called fermentation. Chemically, there is little difference between the catabo- lism of carbohydrates in a cell and the burning of wood in a 9.1 Cells harvest the energy in chemical bonds. FIGURE 9.2 Start every day with a good breakfast. The carbohydrates, proteins, and fats in this fish contain energy that the bear’s cells can use to power their daily activities. The ATP Molecule Adenosine triphosphate (ATP) is the energy currency of the cell, the molecule that transfers the energy captured during respiration to the many sites that use energy in the cell. How is ATP able to transfer energy so readily? Recall from chapter 8 that ATP is composed of a sugar (ribose) bound to an organic base (adenine) and a chain of three phosphate groups. As shown in figure 9.3, each phosphate group is negatively charged. Because like charges repel each other, the linked phosphate groups push against the bond that holds them together. Like a cocked mousetrap, the linked phosphates store the energy of their electrostatic repulsion. Transferring a phosphate group to another mol- ecule relaxes the electrostatic spring of ATP, at the same time cocking the spring of the molecule that is phosphory- lated. This molecule can then use the energy to undergo some change that requires work. How Cells Use ATP Cells use ATP to do most of those activities that require work. One of the most obvious is movement. Some bacte- ria swim about, propelling themselves through the water by rapidly spinning a long, tail-like flagellum, much as a ship moves by spinning a propeller. During your development as an embryo, many of your cells moved about, crawling over one another to reach new positions. Movement also occurs within cells. Tiny fibers within muscle cells pull against one another when muscles contract. Mitochondria pass a meter or more along the narrow nerve cells that con- nect your feet with your spine. Chromosomes are pulled by microtubules during cell division. All of these movements by cells require the expenditure of ATP energy. A second major way cells use ATP is to drive endergonic reactions. Many of the synthetic activities of the cell are en- dergonic, because building molecules takes energy. The chemical bonds of the products of these reactions contain more energy, or are more organized, than the reactants. The reaction can’t proceed until that extra energy is sup- plied to the reaction. It is ATP that provides this needed energy. How ATP Drives Endergonic Reactions How does ATP drive an endergonic reaction? The en- zyme that catalyzes the endergonic reaction has two bind- ing sites on its surface, one for the reactant and another for ATP. The ATP site splits the ATP molecule, liberat- ing over 7 kcal (30 kJ) of chemical energy. This energy pushes the reactant at the second site “uphill,” driving the endergonic reaction. (In a similar way, you can make water in a swimming pool leap straight up in the air, de- spite the fact that gravity prevents water from rising spon- taneously—just jump in the pool! The energy you add going in more than compensates for the force of gravity holding the water back.) When the splitting of ATP molecules drives an energy- requiring reaction in a cell, the two parts of the reaction— ATP-splitting and endergonic—take place in concert. In some cases, the two parts both occur on the surface of the same enzyme; they are physically linked, or “coupled,” like two legs walking. In other cases, a high-energy phosphate from ATP attaches to the protein catalyzing the ender- gonic process, activating it (figure 9.4). Coupling energy- requiring reactions to the splitting of ATP in this way is one of the key tools cells use to manage energy. The catabolism of glucose into carbon dioxide and water in living organisms releases about 720 kcal (3012 kJ) of energy per mole of glucose. This energy is captured in ATP, which stores the energy by linking charged phosphate groups near one another. When the phosphate bonds in ATP are hydrolyzed, energy is released and available to do work. Chapter 9 How Cells Harvest Energy 161 Triphosphate group Sugar Adenine NH 2 OP CH 2 O O O – P O O O – P O – O O – OH OH O N N N N FIGURE 9.3 Structure of the ATP molecule. ATP is composed of an organic base and a chain of phosphates attached to opposite ends of a five- carbon sugar. Notice that the charged regions of the phosphate chain are close to one another. These like charges tend to repel one another, giving the bonds that hold them together a particularly high energy transfer potential. ADP Inactive Active ATP P FIGURE 9.4 How ATP drives an endergonic reaction. In many cases, a phosphate group split from ATP activates a protein, catalyzing an endergonic process. An Overview of Glucose Catabolism Cells are able to make ATP from the catabolism of organic molecules in two different ways. 1. Substrate-level phosphoryla- tion. In the first, called substrate- level phosphoryla- tion, ATP is formed by transfer- ring a phosphate group directly to ADP from a phosphate-bear- ing intermediate (figure 9.5). During glycolysis, discussed below, the chemical bonds of glu- cose are shifted around in reac- tions that provide the energy re- quired to form ATP. 2. Aerobic respiration. In the second, called aerobic respira- tion, ATP forms as electrons are harvested, transferred along the electron transport chain, and eventually donated to oxygen gas. Eukary- otes produce the majority of their ATP from glucose in this way. In most organisms, these two processes are combined. To harvest energy to make ATP from the sugar glucose in the presence of oxygen, the cell carries out a complex se- ries of enzyme-catalyzed reactions that occur in four stages: the first stage captures energy by substrate-level phosphorylation through glycolysis, the following three stages carry out aerobic respiration by oxidizing the end product of glycolysis. Glycolysis Stage One: Glycolysis. The first stage of extracting en- ergy from glucose is a 10-reaction biochemical pathway called glycolysis that produces ATP by substrate-level phosphorylation. The enzymes that catalyze the glycolytic reactions are in the cytoplasm of the cell, not bound to any membrane or organelle. Two ATP molecules are used up early in the pathway, and four ATP molecules are formed by substrate-level phosphorylation. This yields a net of two ATP molecules for each molecule of glucose catabo- lized. In addition, four electrons are harvested as NADH that can be used to form ATP by aerobic respiration. Still, the total yield of ATP is small. When the glycolytic process is completed, the two molecules of pyruvate that are formed still contain most of the energy the original glucose molecule held. Aerobic Respiration Stage Two: Pyruvate Oxidation. In the second stage, pyruvate, the end product from glycolysis, is converted into carbon dioxide and a two-carbon molecule called acetyl- CoA. For each molecule of pyruvate converted, one mole- cule of NAD + is reduced to NADH. Stage Three: The Krebs Cycle. The third stage intro- duces this acetyl-CoA into a cycle of nine reactions called the Krebs cycle, named after the British biochemist, Sir Hans Krebs, who discovered it. (The Krebs cycle is also called the citric acid cycle, for the citric acid, or citrate, formed in its first step, and less commonly, the tricar- boxylic acid cycle, because citrate has three carboxyl groups.) In the Krebs cycle, two more ATP molecules are extracted by substrate-level phosphorylation, and a large number of electrons are removed by the reduction of NAD + to NADH. Stage Four: Electron Transport Chain. In the fourth stage, the energetic electrons carried by NADH are em- ployed to drive the synthesis of a large amount of ATP by the electron transport chain. Pyruvate oxidation, the reactions of the Krebs cycle, and ATP production by electron transport chains occur within many forms of bacteria and inside the mitochondria of all eukaryotes. Recall from chapter 5 that mitochondria are thought to have evolved from bacteria. Although plants and algae can produce ATP by photosynthesis, they also pro- duce ATP by aerobic respiration, just as animals and other nonphotosynthetic eukaryotes do. Figure 9.6 provides an overview of aerobic respiration. 162 Part III Energetics 9.2 Cellular respiration oxidizes food molecules. P PEP Enzyme ADP Adenosine P P Pyruvate ATP Adenosine P P P FIGURE 9.5 Substrate-level phosphorylation. Some molecules, such as phosphoenolpyruvate (PEP), possess a high-energy phosphate bond similar to the bonds in ATP. When PEP’s phosphate group is transferred enzymatically to ADP, the energy in the bond is conserved and ATP is created. Anaerobic Respiration In the presence of oxygen, cells can respire aerobically, using oxygen to accept the electrons harvested from food molecules. In the absence of oxygen to accept the electrons, some organisms can still respire anaerobically, using inor- ganic molecules to accept the electrons. For example, many bacteria use sulfur, nitrate, or other inorganic com- pounds as the electron acceptor in place of oxygen. Methanogens. Among the heterotrophs that practice anaerobic respiration are primitive archaebacteria such as the thermophiles discussed in chapter 4. Some of these, called methanogens, use CO 2 as the electron acceptor, re- ducing CO 2 to CH 4 (methane) with the hydrogens derived from organic molecules produced by other organisms. Sulfur Bacteria. Evidence of a second anaerobic respi- ratory process among primitive bacteria is seen in a group of rocks about 2.7 billion years old, known as the Woman River iron formation. Organic material in these rocks is enriched for the light isotope of sulfur, 32 S, rela- tive to the heavier isotope 34 S. No known geochemical process produces such enrichment, but biological sulfur reduction does, in a process still carried out today by cer- tain primitive bacteria. In this sulfate respiration, the bacteria derive energy from the reduction of inorganic sulfates (SO 4 ) to H 2 S. The hydrogen atoms are obtained from organic molecules other organisms produce. These bacteria thus do the same thing methanogens do, but they use SO 4 as the oxidizing (that is, electron-accepting) agent in place of CO 2 . The sulfate reducers set the stage for the evolution of photosynthesis, creating an environment rich in H 2 S. As discussed in chapter 8, the first form of photosynthesis ob- tained hydrogens from H 2 S using the energy of sunlight. In aerobic respiration, the cell harvests energy from glucose molecules in a sequence of four major pathways: glycolysis, pyruvate oxidation, the Krebs cycle, and the electron transport chain. Oxygen is the final electron acceptor. Anaerobic respiration donates the harvested electrons to other inorganic compounds. Chapter 9 How Cells Harvest Energy 163 NADH NADH H 2 O CO 2 Extracellular fluid Lactate Mitochondrion Plasma membrane ATP ATP ATP E le ct ro n tr a n s p o rt s y s te m O 2 NADH Krebs cycle Acetyl-CoA Pyruvate Cytoplasm GlycolysisGlucose FIGURE 9.6 An overview of aerobic respiration. Stage One: Glycolysis The metabolism of primitive organisms focused on glu- cose. Glucose molecules can be dismantled in many ways, but primitive organisms evolved a glucose-catabolizing process that releases enough free energy to drive the syn- thesis of ATP in coupled reactions. This process, called glycolysis, occurs in the cytoplasm and involves a se- quence of 10 reactions that convert glucose into 2 three- carbon molecules of pyruvate (figure 9.7). For each mole- cule of glucose that passes through this transformation, the cell nets two ATP molecules by substrate-level phos- phorylation. Priming The first half of glycolysis consists of five sequential reac- tions that convert one molecule of glucose into two mole- cules of the three-carbon compound, glyceraldehyde 3- phosphate (G3P). These reactions demand the expenditure of ATP, so they are an energy-requiring process. Step A: Glucose priming. Three reactions “prime” glucose by changing it into a compound that can be cleaved readily into 2 three-carbon phosphorylated mole- cules. Two of these reactions require the cleavage of ATP, so this step requires the cell to use two ATP molecules. Step B: Cleavage and rearrangement. In the first of the remaining pair of reactions, the six-carbon product of step A is split into 2 three-carbon molecules. One is G3P, and the other is then converted to G3P by the sec- ond reaction (figure 9.8). 164 Part III Energetics OVERVIEW OF GLYCOLYSIS 12 3 (Starting material) 6-carbon sugar diphosphate 6-carbon glucose 2 P P 6-carbon sugar diphosphate P P 3-carbon sugar phosphate P 3-carbon sugar phosphate P 3-carbon sugar phosphate P 3-carbon sugar phosphate P Priming reactions. Glycolysis begins with the addition of energy. Two high- energy phosphates from two molecules of ATP are added to the six-carbon molecule glucose, producing a six-carbon molecule with two phosphates. 3-carbon pyruvate 2 NADH Cleavage reactions. Then, the six-carbon molecule with two phosphates is split in two, forming two three-carbon sugar phosphates. Energy-harvesting reactions. Finally, in a series of reactions, each of the two three- carbon sugar phosphates is converted to pyruvate. In the process, an energy-rich hydrogen is harvested as NADH, and two ATP molecules are formed. 3-carbon pyruvate ATP ATP 2 NADH ATP FIGURE 9.7 How glycolysis works. Chapter 9 How Cells Harvest Energy 165 1. Phosphorylation of glucose by ATP. Glucose Hexokinase Phosphoglucoisomerase Phosphofructokinase Glyceraldehyde 3- phosphate (G3P) P i P i Dihydroxyacetone phosphate Glucose 6-phosphate Fructose 6-phosphate Fructose 1,6-bisphosphate Isomerase Triose phosphate dehydrogenase Aldolase 1,3-Bisphosphoglycerate (BPG) 1,3-Bisphosphoglycerate (BPG) 3-Phosphoglycerate (3PG) 3-Phosphoglycerate (3PG) 2-Phosphoglycerate (2PG) 2-Phosphoglycerate (2PG) Phosphoenolpyruvate (PEP) Phosphoenolpyruvate (PEP) Pyruvate Pyruvate 2–3. Rearrangement, followed by a second ATP phosphorylation. 4–5. The six-carbon molecule is split into two three-carbon molecules—one G3P, another that is converted into G3P in another reaction. 6. Oxidation followed by phosphorylation produces two NADH molecules and two molecules of BPG, each with one high-energy phosphate bond. 7. Removal of high-energy phosphate by two ADP molecules produces two ATP molecules and leaves two 3PG molecules. 8–9. Removal of water yields two PEP molecules, each with a high-energy phosphate bond. 10. Removal of high-energy phosphate by two ADP molecules produces two ATP molecules and two pyruvate molecules. 1 2 3 6 Phosphoglycerokinase 7 Phosphoglyceromutase 8 EnolaseH 2 OH 2 O 9 Pyruvate kinase 10 4,5 ADP ATP ADP ATP ADP ATP ADP ATP ADP ATP ADP ATP NADH NAD + NADH NAD + O CO O CH 2 OH CH 2 P CO C O – O CH 3 PCO C O – O CH 2 PCOH C O – O CH 2 OH CHOH PO C O – O CH 2 CHOH PO C H O CH 2 POCH 2 P O CH 2 O PO CH 2 OH CH 2 OH CH 2 POCH 2 CHOH P P O O CO CH 2 O O FIGURE 9.8 The glycolytic pathway. The first five reactions convert a molecule of glucose into two molecules of G3P. The second five reactions convert G3P into pyruvate. Substrate-Level Phosphorylation In the second half of glycolysis, five more reactions convert G3P into pyruvate in an energy-yielding process that gen- erates ATP. Overall, then, glycolysis is a series of 10 en- zyme-catalyzed reactions in which some ATP is invested in order to produce more. Step C: Oxidation. Two electrons and one proton are transferred from G3P to NAD + , forming NADH. Note that NAD + is an ion, and that both electrons in the new covalent bond come from G3P. Step D: ATP generation. Four reactions convert G3P into another three-carbon molecule, pyruvate. This process generates two ATP molecules (see figure 9.5). Because each glucose molecule is split into two G3P molecules, the overall reaction sequence yields two mol- ecules of ATP, as well as two molecules of NADH and two of pyruvate: 4 ATP (2 ATP for each of the 2 G3P molecules in step D) – 2 ATP (used in the two reactions in step A ) 2 ATP Under the nonstandard conditions within a cell, each ATP molecule produced represents the capture of about 12 kcal (50 kJ) of energy per mole of glucose, rather than the 7.3 traditionally quoted for standard conditions. This means glycolysis harvests about 24 kcal/mole (100 kJ/mole). This is not a great deal of energy. The total en- ergy content of the chemical bonds of glucose is 686 kcal (2870 kJ) per mole, so glycolysis harvests only 3.5% of the chemical energy of glucose. Although far from ideal in terms of the amount of en- ergy it releases, glycolysis does generate ATP. For more than a billion years during the anaerobic first stages of life on earth, it was the primary way heterotrophic organ- isms generated ATP from organic molecules. Like many biochemical pathways, glycolysis is believed to have evolved backward, with the last steps in the process being the most ancient. Thus, the second half of glycolysis, the ATP-yielding breakdown of G3P, may have been the original process early heterotrophs used to generate ATP. The synthesis of G3P from glucose would have ap- peared later, perhaps when alternative sources of G3P were depleted. All Cells Use Glycolysis The glycolytic reaction sequence is thought to have been among the earliest of all biochemical processes to evolve. It uses no molecular oxygen and occurs readily in an anaero- bic environment. All of its reactions occur free in the cyto- plasm; none is associated with any organelle or membrane structure. Every living creature is capable of carrying out glycolysis. Most present-day organisms, however, can ex- tract considerably more energy from glucose through aero- bic respiration. Why does glycolysis take place even now, since its en- ergy yield in the absence of oxygen is comparatively so paltry? The answer is that evolution is an incremental process: change occurs by improving on past successes. In catabolic metabolism, glycolysis satisfied the one essential evolutionary criterion: it was an improvement. Cells that could not carry out glycolysis were at a competitive disad- vantage, and only cells capable of glycolysis survived the early competition of life. Later improvements in catabolic metabolism built on this success. Glycolysis was not dis- carded during the course of evolution; rather, it served as the starting point for the further extraction of chemical energy. Metabolism evolved as one layer of reactions added to another, just as successive layers of paint cover the walls of an old building. Nearly every present-day or- ganism carries out glycolysis as a metabolic memory of its evolutionary past. Closing the Metabolic Circle: The Regeneration of NAD + Inspect for a moment the net reaction of the glycolytic se- quence: Glucose + 2 ADP + 2 P i + 2 NAD + ?→ 2 Pyruvate + 2 ATP + 2 NADH + 2 H + + 2 H 2 O You can see that three changes occur in glycolysis: (1) glu- cose is converted into two molecules of pyruvate; (2) two molecules of ADP are converted into ATP via substrate level phosphorylation; and (3) two molecules of NAD + are reduced to NADH. The Need to Recycle NADH As long as food molecules that can be converted into glu- cose are available, a cell can continually churn out ATP to drive its activities. In doing so, however, it accumulates NADH and depletes the pool of NAD + molecules. A cell does not contain a large amount of NAD + , and for glycoly- sis to continue, NADH must be recycled into NAD + . Some other molecule than NAD + must ultimately accept the hy- drogen atom taken from G3P and be reduced. Two mole- cules can carry out this key task (figure 9.9): 1. Aerobic respiration. Oxygen is an excellent elec- tron acceptor. Through a series of electron transfers, the hydrogen atom taken from G3P can be donated to oxygen, forming water. This is what happens in the cells of eukaryotes in the presence of oxygen. Because air is rich in oxygen, this process is also referred to as aerobic metabolism. 166 Part III Energetics 2. Fermentation. When oxygen is unavailable, an or- ganic molecule can accept the hydrogen atom instead. Such fermentation plays an important role in the me- tabolism of most organisms (figure 9.10), even those capable of aerobic respiration. The fate of the pyruvate that is produced by glycolysis depends upon which of these two processes takes place. The aerobic respiration path starts with the oxidation of pyruvate to a molecule called acetyl-CoA, which is then further oxidized in a series of reactions called the Krebs cycle. The fermentation path, by contrast, involves the re- duction of all or part of pyruvate. We will start by examin- ing aerobic respiration, then look briefly at fermentation. Glycolysis generates a small amount of ATP by reshuffling the bonds of glucose molecules. In glycolysis, two molecules of NAD + are reduced to NADH. NAD + must be regenerated for glycolysis to continue unabated. Chapter 9 How Cells Harvest Energy 167 NADH Pyruvate With oxygen Without oxygen Acetyl-CoA Lactate Ethanol NAD + O 2 NAD + NADH NAD + NADH CO 2 Acetaldehyde H 2 O Krebs cycle FIGURE 9.9 What happens to pyruvate, the product of glycolysis? In the presence of oxygen, pyruvate is oxidized to acetyl-CoA, which enters the Krebs cycle. In the absence of oxygen, pyruvate is instead reduced, accepting the electrons extracted during glycolysis and carried by NADH. When pyruvate is reduced directly, as in muscle cells, the product is lactate. When CO 2 is first removed from pyruvate and the product, acetaldehyde, is then reduced, as in yeast cells, the product is ethanol. FIGURE 9.10 Fermentation. The conversion of pyruvate to ethanol takes place naturally in grapes left to ferment on vines, as well as in fermentation vats of crushed grapes. Yeasts carry out the process, but when their conversion increases the ethanol concentration to about 12%, the toxic effects of the alcohol kill the yeast cells. What is left is wine. Stage Two: The Oxidation of Pyruvate In the presence of oxygen, the oxidation of glucose that be- gins in glycolysis continues where glycolysis leaves off— with pyruvate. In eukaryotic organisms, the extraction of additional energy from pyruvate takes place exclusively in- side mitochondria. The cell harvests pyruvate’s consider- able energy in two steps: first, by oxidizing pyruvate to form acetyl-CoA, and then by oxidizing acetyl-CoA in the Krebs cycle. Producing Acetyl-CoA Pyruvate is oxidized in a single “decarboxylation” reaction that cleaves off one of pyruvate’s three carbons. This car- bon then departs as CO 2 (figure 9.11, top). This reaction produces a two-carbon fragment called an acetyl group, as well as a pair of electrons and their associated hydrogen, which reduce NAD + to NADH. The reaction is complex, involving three intermediate stages, and is catalyzed within mitochondria by a multienzyme complex. As chapter 8 noted, such a complex organizes a series of enzymatic steps so that the chemical intermediates do not diffuse away or undergo other reactions. Within the complex, component polypeptides pass the substrates from one en- zyme to the next, without releasing them. Pyruvate dehy- drogenase, the complex of enzymes that removes CO 2 from pyruvate, is one of the largest enzymes known: it contains 60 subunits! In the course of the reaction, the acetyl group removed from pyruvate combines with a cofactor called coenzyme A (CoA), forming a compound known as acetyl-CoA: Pyruvate + NAD + + CoA ?→ Acetyl-CoA + NADH + CO 2 This reaction produces a molecule of NADH, which is later used to produce ATP. Of far greater significance than the reduction of NAD + to NADH, however, is the produc- tion of acetyl-CoA (figure 9.11, bottom). Acetyl-CoA is im- portant because so many different metabolic processes gen- erate it. Not only does the oxidation of pyruvate, an intermediate in carbohydrate catabolism, produce it, but the metabolic breakdown of proteins, fats, and other lipids also generate acetyl-CoA. Indeed, almost all molecules ca- tabolized for energy are converted into acetyl-CoA. Acetyl- CoA is then channeled into fat synthesis or into ATP pro- duction, depending on the organism’s energy requirements. Acetyl-CoA is a key point of focus for the many catabolic processes of the eukaryotic cell. Using Acetyl-CoA Although the cell forms acetyl-CoA in many ways, only a limited number of processes use acetyl-CoA. Most of it is either directed toward energy storage (lipid synthesis, for example) or oxidized in the Krebs cycle to produce ATP. Which of these two options is taken depends on the level of ATP in the cell. When ATP levels are high, the oxida- tive pathway is inhibited, and acetyl-CoA is channeled into fatty acid synthesis. This explains why many animals (humans included) develop fat reserves when they con- sume more food than their bodies require. Alternatively, when ATP levels are low, the oxidative pathway is stimu- lated, and acetyl-CoA flows into energy-producing oxida- tive metabolism. In the second energy-harvesting stage of glucose catabolism, pyruvate is decarboxylated, yielding acetyl- CoA, NADH, and CO 2 . This process occurs within the mitochondrion. 168 Part III Energetics Coenzyme A Acetyl group Acetyl coenzyme A ATPFat CO 2 Protein Lipid NADH NAD + Pyruvate Glycolysis FIGURE 9.11 The oxidation of pyruvate. This complex reaction involves the reduction of NAD + to NADH and is thus a significant source of metabolic energy. Its product, acetyl-CoA, is the starting material for the Krebs cycle. Almost all molecules that are catabolized for energy are converted into acetyl-CoA, which is then channeled into fat synthesis or into ATP production. Stage Three: The Krebs Cycle After glycolysis catabolizes glucose to produce pyruvate, and pyruvate is oxidized to form acetyl-CoA, the third stage of extracting energy from glucose begins. In this third stage, acetyl-CoA is oxidized in a series of nine reactions called the Krebs cycle. These reactions occur in the matrix of mitochondria. In this cycle, the two-carbon acetyl group of acetyl-CoA combines with a four-carbon molecule called oxaloacetate (figure 9.12). The resulting six-carbon mole- cule then goes through a sequence of electron-yielding oxi- dation reactions, during which two CO 2 molecules split off, restoring oxaloacetate. The oxaloacetate is then recycled to bind to another acetyl group. In each turn of the cycle, a new acetyl group replaces the two CO 2 molecules lost, and more electrons are extracted to drive proton pumps that generate ATP. Overview of the Krebs Cycle The nine reactions of the Krebs cycle occur in two steps: Step A: Priming. Three reactions prepare the six- carbon molecule for energy extraction. First, acetyl- CoA joins the cycle, and then chemical groups are rearranged. Step B: Energy extraction. Four of the six reactions in this step are oxidations in which electrons are re- moved, and one generates an ATP equivalent directly by substrate-level phosphorylation. Chapter 9 How Cells Harvest Energy 169 OVERVIEW OF THE KREBS CYCLE 123 Finally, the resulting four-carbon molecule is further oxidized (hydrogens removed to form FADH 2 and NADH). This regenerates the four-carbon starting material, completing the cycle. Then, the resulting six-carbon mole- cule is oxidized (a hydrogen removed to form NADH) and decarboxylated (a carbon removed to form CO 2 ). Next, the five-carbon molecule is oxidized and decarboxylated again, and a coupled reaction generates ATP. The Krebs cycle begins when a two- carbon fragment is transferred from acetyl-CoA to a four-carbon molecule (the starting material). 5-carbon molecule4-carbon molecule (Acetyl-CoA) 6-carbon molecule 4-carbon molecule (Starting material) CoA– CoA NADH CO 2 NADH CO 2 ATP NADH FADH 2 6-carbon molecule 4-carbon molecule 4-carbon molecule (Starting material) FIGURE 9.12 How the Krebs cycle works. The Reactions of the Krebs Cycle The Krebs cycle consists of nine sequential reactions that cells use to extract energetic electrons and drive the synthe- sis of ATP (figure 9.13). A two-carbon group from acetyl- CoA enters the cycle at the beginning, and two CO 2 mole- cules and several electrons are given off during the cycle. Reaction 1: Condensation. The two-carbon group from acetyl-CoA joins with a four-carbon molecule, oxaloac- etate, to form a six-carbon molecule, citrate. This conden- sation reaction is irreversible, committing the two-carbon acetyl group to the Krebs cycle. The reaction is inhibited when the cell’s ATP concentration is high and stimulated when it is low. Hence, when the cell possesses ample amounts of ATP, the Krebs cycle shuts down and acetyl- CoA is channeled into fat synthesis. Reactions 2 and 3: Isomerization. Before the oxidation reactions can begin, the hydroxyl (—OH) group of citrate must be repositioned. This is done in two steps: first, a water molecule is removed from one carbon; then, water is added to a different carbon. As a result, an —H group and an —OH group change positions. The product is an iso- mer of citrate called isocitrate. Reaction 4: The First Oxidation. In the first energy- yielding step of the cycle, isocitrate undergoes an oxidative decarboxylation reaction. First, isocitrate is oxidized, yield- ing a pair of electrons that reduce a molecule of NAD + to NADH. Then the oxidized intermediate is decarboxylated; the central carbon atom splits off to form CO 2 , yielding a five-carbon molecule called α-ketoglutarate. Reaction 5: The Second Oxidation. Next, α-ketoglutarate is decarboxylated by a multienzyme complex similar to pyruvate dehydrogenase. The succinyl group left after the removal of CO 2 joins to coenzyme A, forming succinyl- CoA. In the process, two electrons are extracted, and they reduce another molecule of NAD + to NADH. Reaction 6: Substrate-Level Phosphorylation. The linkage between the four-carbon succinyl group and CoA is a high-energy bond. In a coupled reaction similar to those that take place in glycolysis, this bond is cleaved, and the energy released drives the phosphorylation of guanosine diphosphate (GDP), forming guanosine triphosphate (GTP). GTP is readily converted into ATP, and the four- carbon fragment that remains is called succinate. Reaction 7: The Third Oxidation. Next, succinate is oxidized to fumarate. The free energy change in this re- action is not large enough to reduce NAD + . Instead, flavin adenine dinucleotide (FAD) is the electron accep- tor. Unlike NAD + , FAD is not free to diffuse within the mitochondrion; it is an integral part of the inner mito- chondrial membrane. Its reduced form, FADH 2 , con- tributes electrons to the electron transport chain in the membrane. Reactions 8 and 9: Regeneration of Oxaloacetate. In the final two reactions of the cycle, a water molecule is added to fumarate, forming malate. Malate is then oxi- dized, yielding a four-carbon molecule of oxaloacetate and two electrons that reduce a molecule of NAD + to NADH. Oxaloacetate, the molecule that began the cycle, is now free to combine with another two-carbon acetyl group from acetyl-CoA and reinitiate the cycle. The Products of the Krebs Cycle In the process of aerobic respiration, glucose is entirely consumed. The six-carbon glucose molecule is first cleaved into a pair of three-carbon pyruvate molecules during gly- colysis. One of the carbons of each pyruvate is then lost as CO 2 in the conversion of pyruvate to acetyl-CoA; two other carbons are lost as CO 2 during the oxidations of the Krebs cycle. All that is left to mark the passing of the glu- cose molecule into six CO 2 molecules is its energy, some of which is preserved in four ATP molecules and in the re- duced state of 12 electron carriers. Ten of these carriers are NADH molecules; the other two are FADH 2 . The Krebs cycle generates two ATP molecules per molecule of glucose, the same number generated by glycolysis. More importantly, the Krebs cycle and the oxidation of pyruvate harvest many energized electrons, which can be directed to the electron transport chain to drive the synthesis of much more ATP. 170 Part III Energetics Chapter 9 How Cells Harvest Energy 171 Oxaloacetate (4C) Krebs cycle Mitochondrial membrane Oxidation of pyruvate Pyruvate Acetyl-CoA (2C) CoA-SH CoA-SH Malate (4C) Fumarate (4C) Succinate (4C) α-Ketoglutarate (5C) Isocitrate (6C) Citrate (6C) Citrate synthetase Succinyl-CoA (4C) The oxidation of succinate produces FADH 2 . The dehydrogenation of malate produces a third NADH, and the cycle returns to its starting point. A second oxidative decarboxylation produces a second NADH with the release of a second CO 2 . Oxidative decarboxylation produces NADH with the release of CO 2 . The cycle begins when a 2C unit from acetyl-CoA reacts with a 4C molecule (oxaloacetate) to produce citrate (6C). H 2 O NADH NAD + NAD + NADH NAD + NADH NAD + NADH FAD FADH 2 CO COO – COO – CH 2 C S CoA O CH 3 COO – COO – CH 2 CH 2 CHHO COO – COO – CH 2 CH COO – COO – HC COO – CO S CoA CH 2 CH 2 COO – COO – CO CH 2 CH 2 COO – HC CHHO COO – COO – CH 2 COO – CHO COO – COO – CH 2 CH 2 CO 2 CO 2 CO 2 1 Isocitrate dehydrogenase 4 Malate dehydrogenase 9 Succinate dehydrogenase 7 Fumarase8 α-Ketoglutarate dehydrogenase Succinyl-CoA synthetase 5 6 Aconitase 2 3 CoA-SH GTP GDP + P i ATP ADP FIGURE 9.13 The Krebs cycle. This series of reactions takes place within the matrix of the mitochondrion. For the complete breakdown of a molecule of glucose, the two molecules of acetyl-CoA produced by glycolysis and pyruvate oxidation will each have to make a trip around the Krebs cycle. Follow the different carbons through the cycle, and notice the changes that occur in the carbon skeletons of the molecules as they proceed through the cycle. Harvesting Energy by Extracting Electrons To understand how cells direct some of the energy released during glucose catabolism into ATP production, we need to take a closer look at the electrons in the C—H bonds of the glucose molecule. We stated in chapter 8 that when an electron is removed from one atom and donated to an- other, the electron’s potential energy of position is also transferred. In this process, the atom that receives the elec- tron is reduced. We spoke of reduction in an all-or-none fashion, as if it involved the complete transfer of an elec- tron from one atom to another. Often this is just what hap- pens. However, sometimes a reduction simply changes the degree of sharing within a covalent bond. Let us now revisit that discussion and consider what happens when the trans- fer of electrons is incomplete. A Closer Look at Oxidation-Reduction The catabolism of glucose is an oxidation-reduction reac- tion. The covalent electrons in the C—H bonds of glucose are shared approximately equally between the C and H atoms because carbon and hydrogen nuclei have about the same affinity for valence electrons (that is, they exhibit sim- ilar electronegativity). However, when the carbon atoms of glucose react with oxygen to form carbon dioxide, the elec- trons in the new covalent bonds take a different position. Instead of being shared equally, the electrons that were as- sociated with the carbon atoms in glucose shift far toward the oxygen atom in CO 2 because oxygen is very electroneg- ative. Since these electrons are pulled farther from the car- bon atoms, the carbon atoms of glucose have been oxidized (loss of electrons) and the oxygen atoms reduced (gain of electrons). Similarly, when the hydrogen atoms of glucose combine with oxygen atoms to form water, the oxygen atoms draw the shared electrons strongly toward them; again, oxygen is reduced and glucose is oxidized. In this re- action, oxygen is an oxidizing (electron-attracting) agent because it oxidizes the atoms of glucose. Releasing Energy The key to understanding the oxidation of glucose is to focus on the energy of the shared electrons. In a covalent bond, energy must be added to remove an electron from an atom, just as energy must be used to roll a boulder up a hill. The more electronegative the atom, the steeper the energy hill that must be climbed to pull an electron away from it. However, energy is released when an electron is shifted away from a less electronegative atom and closer to a more electronegative atom, just as energy is released when a boulder is allowed to roll down a hill. In the catabolism of glucose, energy is released when glucose is oxidized, as electrons relocate closer to oxygen (figure 9.14). Glucose is an energy-rich food because it has an abun- dance of C—H bonds. Viewed in terms of oxidation- reduction, glucose possesses a wealth of electrons held far from their atoms, all with the potential to move closer to- ward oxygen. In oxidative respiration, energy is released not simply because the hydrogen atoms of the C—H bonds are transferred from glucose to oxygen, but be- cause the positions of the valence electrons shift. This shift releases energy that can be used to make ATP. Harvesting the Energy in Stages It is generally true that the larger the release of energy in any single step, the more of that energy is released as heat (random molecular motion) and the less there is available to be channeled into more useful paths. In the combustion of gasoline, the same amount of energy is released whether all of the gasoline in a car’s gas tank explodes at once, or whether the gasoline burns in a series of very small explo- sions inside the cylinders. By releasing the energy in gaso- line a little at a time, the harvesting efficiency is greater and 172 Part III Energetics Electron transport chain Low energy High energy Energy for synthesis of Electrons from food Formation of water ATP e – e – FIGURE 9.14 How electron transport works. This diagram shows how ATP is generated when electrons transfer from one energy level to another. Rather than releasing a single explosive burst of energy, electrons “fall” to lower and lower energy levels in steps, releasing stored energy with each fall as they tumble to the lowest (most electronegative) electron acceptor. more of the energy can be used to push the pistons and move the car. The same principle applies to the oxidation of glucose inside a cell. If all of the hydrogens were transferred to oxy- gen in one explosive step, releasing all of the free energy at once, the cell would recover very little of that energy in a useful form. Instead, cells burn their fuel much as a car does, a little at a time. The six hydrogens in the C—H bonds of glucose are stripped off in stages in the series of enzyme-catalyzed reactions collectively referred to as gly- colysis and the Krebs cycle. We have had a great deal to say about these reactions already in this chapter. Recall that the hydrogens are removed by transferring them to a coenzyme carrier, NAD + (figure 9.15). Discussed in chapter 8, NAD + is a very versatile electron acceptor, shuttling energy-bearing electrons throughout the cell. In harvesting the energy of glucose, NAD + acts as the primary electron acceptor. Following the Electrons As you examine these reactions, try not to become confused by the changes in electrical charge. Always follow the elec- trons. Enzymes extract two hydrogens—that is, two elec- trons and two protons—from glucose and transfer both electrons and one of the protons to NAD + . The other pro- ton is released as a hydrogen ion, H + , into the surrounding solution. This transfer converts NAD + into NADH; that is, two negative electrons and one positive proton are added to one positively charged NAD + to form NADH, which is electrically neutral. Energy captured by NADH is not harvested all at once. Instead of being transferred directly to oxygen, the two electrons carried by NADH are passed along the electron transport chain if oxygen is present. This chain consists of a series of molecules, mostly proteins, embedded within the inner membranes of mitochondria. NADH delivers elec- trons to the top of the electron transport chain and oxygen captures them at the bottom. The oxygen then joins with hydrogen ions to form water. At each step in the chain, the electrons move to a slightly more electronegative carrier, and their positions shift slightly. Thus, the electrons move down an energy gradient. The entire process releases a total of 53 kcal/mole (222 kJ/mole) under standard conditions. The transfer of electrons along this chain allows the energy to be extracted gradually. In the next section, we will dis- cuss how this energy is put to work to drive the production of ATP. The catabolism of glucose involves a series of oxidation-reduction reactions that release energy by repositioning electrons closer to oxygen atoms. Energy is thus harvested from glucose molecules in gradual steps, using NAD + as an electron carrier. Chapter 9 How Cells Harvest Energy 173 N + OCH 2 H P O O O H O OP H - O - O O P - O O HO OH O CH 2 H HO OH HO OH O CNH 2 + 2H Reduction Oxidation NAD + : oxidized form of nicotinamide Adenine N N N N H NH 2 N OCH 2 H P O - O O O HHO O H HO OH O CH 2 H O CNH 2 + H + NADH: reduced form of nicotinamide Adenine N N N N H NH 2 FIGURE 9.15 NAD + and NADH. This dinucleotide serves as an “electron shuttle” during cellular respiration. NAD + accepts electrons from catabolized macromolecules and is reduced to NADH. Stage Four: The Electron Transport Chain The NADH and FADH 2 molecules formed during the first three stages of aerobic respiration each contain a pair of electrons that were gained when NAD + and FAD were re- duced. The NADH molecules carry their electrons to the inner mitochondrial membrane, where they transfer the electrons to a series of membrane-associated proteins col- lectively called the electron transport chain. Moving Electrons through the Electron Transport Chain The first of the proteins to receive the electrons is a com- plex, membrane-embedded enzyme called NADH dehy- drogenase. A carrier called ubiquinone then passes the electrons to a protein-cytochrome complex called the bc 1 complex. This complex, along with others in the chain, oper- ates as a proton pump, driving a proton out across the mem- brane. Cytochromes are respiratory proteins that contain heme groups, complex carbon rings with many alternating single and double bonds and an iron atom in the center. The electron is then carried by another carrier, cy- tochrome c, to the cytochrome oxidase complex. This com- plex uses four such electrons to reduce a molecule of oxy- gen, each oxygen then combines with two hydrogen ions to form water: O 2 + 4 H + + 4 e - ?→ 2 H 2 O This series of membrane-associated electron carriers is collectively called the electron transport chain (figure 9.16). NADH contributes its electrons to the first protein of the electron transport chain, NADH dehydrogenase. FADH 2 , which is always attached to the inner mitochon- drial membrane, feeds its electrons into the electron trans- port chain later, to ubiquinone. It is the availability of a plentiful electron acceptor (often oxygen) that makes oxidative respiration possible. As we’ll see in chapter 10, the electron transport chain used in aerobic respiration is similar to, and may well have evolved from, the chain employed in aerobic photosynthesis. The electron transport chain is a series of five membrane-associated proteins. Electrons delivered by NADH and FADH 2 are passed from protein to protein along the chain, like a baton in a relay race. 174 Part III Energetics Intermembrane space Mitochondrial matrix Inner mitochondrial membrane NAD + Q C NADH H 2 O 2H + + O 2 + H + H + H + H + NADH dehydrogenase bc 1 complex Cytochrome oxidase complex FADH 2 H11002 1 2 FIGURE 9.16 The electron transport chain. High-energy electrons harvested from catabolized molecules are transported (red arrows) by mobile electron carriers (ubiquinone, marked Q, and cytochrome c, marked C) along a chain of membrane proteins. Three proteins use portions of the electrons’ energy to pump protons (blue arrows) out of the matrix and into the intermembrane space. The electrons are finally donated to oxygen to form water. Building an Electrochemical Gradient In eukaryotes, aerobic metabolism takes place within the mitochondria present in virtually all cells. The internal compartment, or matrix, of a mitochondrion contains the enzymes that carry out the reactions of the Krebs cycle. As the electrons harvested by oxidative respiration are passed along the electron transport chain, the energy they release transports protons out of the matrix and into the outer compartment, sometimes called the intermembrane space. Three transmembrane proteins in the inner mito- chondrial membrane (see figure 9.16) actually accomplish the transport. The flow of excited electrons induces a change in the shape of these pump proteins, which causes them to transport protons across the membrane. The electrons contributed by NADH activate all three of these proton pumps, while those contributed by FADH 2 acti- vate only two. Producing ATP: Chemiosmosis As the proton concentration in the intermembrane space rises above that in the matrix, the matrix becomes slightly negative in charge. This internal negativity attracts the positively charged protons and induces them to reenter the matrix. The higher outer concentration tends to drive pro- tons back in by diffusion; since membranes are relatively impermeable to ions, most of the protons that reenter the matrix pass through special proton channels in the inner mitochondrial membrane. When the protons pass through, these channels synthesize ATP from ADP + P i within the ma- trix. The ATP is then transported by facilitated diffusion out of the mito- chondrion and into the cell’s cyto- plasm. Because the chemical forma- tion of ATP is driven by a diffusion force similar to osmosis, this process is referred to as chemiosmosis (fig- ure 9.17). Thus, the electron transport chain uses electrons harvested in aerobic respiration to pump a large number of protons across the inner mitochon- drial membrane. Their subsequent reentry into the mitochondrial matrix drives the synthesis of ATP by chemiosmosis. Figure 9.18 summa- rizes the overall process. The electrons harvested from glucose are pumped out of the mitochondrial matrix by the electron transport chain. The return of the protons into the matrix generates ATP. Chapter 9 How Cells Harvest Energy 175 Intermembrane space Mitochondrial matrix Na-K pump ATP ADP + P i NADH H + H + H + H + H + H + NAD + Inner mitochondrial membrane Proton pump ATP synthase FIGURE 9.17 Chemiosmosis. NADH transports high-energy electrons harvested from the catabolism of macromolecules to “proton pumps” that use the energy to pump protons out of the mitochondrial matrix. As a result, the concentration of protons in the intermembrane space rises, inducing protons to diffuse back into the matrix. Many of the protons pass through special channels that couple the reentry of protons to the production of ATP. H + H + H + H + O 2 O 2 + Q C ATP32 ATP2 Pyruvate from cytoplasm Electron transport system Channel protein H 2 O CO 2 Acetyl-CoA Krebs cycle FADH 2 NADH NADH Intermembrane space Mitochondrial matrix Inner mitochondrial membrane 1. Electrons are harvested and carried to the transport system. 2. Electrons provide energy to pump protons across the membrane. 3. Oxygen joins with protons to form water. 4. Protons diffuse back in, driving the synthesis of ATP. 2H + H11002 1 2 FIGURE 9.18 ATP generation during the Krebs cycle and electron transport chain. This process begins with pyruvate, the product of glycolysis, and ends with the synthesis of ATP. Summarizing Aerobic Respiration How much metabolic energy does a cell actually gain from the electrons har- vested from a molecule of glucose, using the electron transport chain to produce ATP by chemiosmosis? Theoretical Yield The chemiosmotic model suggests that one ATP molecule is generated for each proton pump activated by the electron transport chain. Since the elec- trons from NADH activate three pumps and those from FADH 2 activate two, we would expect each molecule of NADH and FADH 2 to generate three and two ATP molecules, respectively. However, because eukaryotic cells carry out glycolysis in their cytoplasm and the Krebs cycle within their mitochon- dria, they must transport the two mole- cules of NADH produced during gly- colysis across the mitochondrial membranes, which requires one ATP per molecule of NADH. Thus, the net ATP production is decreased by two. Therefore, the overall ATP production resulting from aerobic respiration theo- retically should be 4 (from substrate- level phosphorylation during glycolysis) + 30 (3 from each of 10 molecules of NADH) + 4 (2 from each of 2 mole- cules of FADH 2 ) – 2 (for transport of glycolytic NADH) = 36 molecules of ATP (figure 9.19). Actual Yield The amount of ATP actually produced in a eukaryotic cell during aerobic respiration is somewhat lower than 36, for two reasons. First, the inner mitochondrial membrane is somewhat “leaky” to protons, allowing some of them to reenter the matrix without passing through ATP-generating channels. Second, mitochondria often use the proton gra- dient generated by chemiosmosis for purposes other than ATP synthesis (such as transporting pyruvate into the ma- trix). Consequently, the actual measured values of ATP generated by NADH and FADH 2 are closer to 2.5 for each NADH and 1.5 for each FADH 2 . With these correc- tions, the overall harvest of ATP from a molecule of glu- cose in a eukaryotic cell is closer to 4 (from substrate-level phosphorylation) + 25 (2.5 from each of 10 molecules of NADH) + 3 (1.5 from each of 2 molecules of FADH 2 ) – 2 (transport of glycolytic NADH) = 30 molecules of ATP. The catabolism of glucose by aerobic respiration, in contrast to glycolysis, is quite efficient. Aerobic respiration in a eukaryotic cell harvests about 7.3 × 30 ÷ 686 = 32% of the energy available in glucose. (By comparison, a typical car converts only about 25% of the energy in gasoline into useful energy.) The efficiency of oxidative respiration at harvesting energy establishes a natural limit on the maxi- mum length of food chains. The high efficiency of aerobic respiration was one of the key factors that fostered the evolution of heterotrophs. With this mechanism for producing ATP, it became feasible for nonphotosynthetic organisms to derive metabolic energy ex- clusively from the oxidative breakdown of other organisms. As long as some organisms captured energy by photosynthe- sis, others could exist solely by feeding on them. Oxidative respiration produces approximately 30 molecules of ATP from each molecule of glucose in eukaryotic cells. This represents more than half of the energy in the chemical bonds of glucose. 176 Part III Energetics Glycolysis2 26ATP Pyruvate Glucose Acetyl-CoA NADH 24ATPNADH 2 ATP 2 ATP 618ATPNADH 24ATP Total net ATP yield = 36 ATP FADH 2 Krebs cycle ATP FIGURE 9.19 Theoretical ATP yield. The theoretical yield of ATP harvested from glucose by aerobic respiration totals 36 molecules. Regulating Aerobic Respiration When cells possess plentiful amounts of ATP, the key reac- tions of glycolysis, the Krebs cycle, and fatty acid break- down are inhibited, slowing ATP production. The regula- tion of these biochemical pathways by the level of ATP is an example of feedback inhibition. Conversely, when ATP levels in the cell are low, ADP levels are high; and ADP ac- tivates enzymes in the pathways of carbohydrate catabolism to stimulate the production of more ATP. Control of glucose catabolism occurs at two key points of the catabolic pathway (figure 9.20). The control point in glycolysis is the enzyme phosphofructokinase, which catalyzes reaction 3, the conversion of fructose phosphate to fructose bisphosphate. This is the first reaction of gly- colysis that is not readily reversible, committing the sub- strate to the glycolytic sequence. High levels of ADP rela- tive to ATP (implying a need to convert more ADP to ATP) stimulate phosphofructokinase, committing more sugar to the catabolic pathway; so do low levels of citrate (implying the Krebs cycle is not running at full tilt and needs more input). The main control point in the oxida- tion of pyruvate occurs at the committing step in the Krebs cycle with the enzyme pyruvate decarboxylase. It is inhibited by high levels of NADH (implying no more is needed). Another control point in the Krebs cycle is the enzyme citrate synthetase, which catalyzes the first reaction, the conversion of oxaloacetate and acetyl-CoA into citrate. High levels of ATP inhibit citrate synthetase (as well as pyruvate decarboxylase and two other Krebs cycle en- zymes), shutting down the catabolic pathway. Relative levels of ADP and ATP regulate the catabolism of glucose at key committing reactions. Chapter 9 How Cells Harvest Energy 177 A Vocabulary of ATP Generation which the final electron acceptor is or- ganic; it includes aerobic and anaerobic respiration. chemiosmosis The passage of high- energy electrons along the electron trans- port chain, which is coupled to the pump- ing of protons across a membrane and the return of protons to the original side of the membrane through ATP-generating channels. fermentation Alternative ATP-producing pathway performed by some cells in the ab- sence of oxygen, in which the final electron acceptor is an organic molecule. maximum efficiency The maximum number of ATP molecules generated by oxidizing a substance, relative to the free energy of that substance; in organisms, the actual efficiency is usually less than the maximum. oxidation The loss of an electron. In cel- lular respiration, high-energy electrons are stripped from food molecules, oxidizing them. photosynthesis The chemiosmotic gen- eration of ATP and complex organic mole- cules powered by the energy derived from light. substrate-level phosphorylation The generation of ATP by the direct transfer of a phosphate group to ADP from another phosphorylated molecule. aerobic respiration The portion of cellu- lar respiration that requires oxygen as an electron acceptor; it includes pyruvate oxi- dation, the Krebs cycle, and the electron transport chain. anaerobic respiration Cellular respiration in which inorganic electron acceptors other than oxygen are used; it includes glycolysis. cellular respiration The oxidation of organic molecules to produce ATP in ATP Krebs cycle Electron transport chain and chemiosmosis NADH Citrate Glucose ADP Fructose 6-phosphate Fructose 1,6-bisphosphate Pyruvate Pyruvate decarboxylase Acetyl-CoA Phosphofructokinase Inhibits Activates Activates Inhibits FIGURE 9.20 Control of glucose catabolism. The relative levels of ADP and ATP control the catabolic pathway at two key points: the committing reactions of glycolysis and the Krebs cycle. Glucose Is Not the Only Source of Energy Thus far we have discussed oxidative respiration of glucose, which organisms obtain from the digestion of carbohy- drates or from photosynthesis. Other organic molecules than glucose, partic- ularly proteins and fats, are also impor- tant sources of energy (figure 9.21). Cellular Respiration of Protein Proteins are first broken down into their individual amino acids. The nitrogen- containing side group (the amino group) is then removed from each amino acid in a process called deamina- tion. A series of reactions convert the carbon chain that remains into a mole- cule that takes part in glycolysis or the Krebs cycle. For example, alanine is converted into pyruvate, glutamate into α-ketoglutarate (figure 9.22), and aspar- tate into oxaloacetate. The reactions of glycolysis and the Krebs cycle then ex- tract the high-energy electrons from these molecules and put them to work making ATP. 178 Part III Energetics 9.3 Catabolism of proteins and fats can yield considerable energy. Macromolecule degradation Cell building blocks Nucleic acids Proteins Lipids and fats Polysaccharides Nucleotides Amino acids Fatty acidsSugars NH 3 H 2 OCO 2 Deamination H9252-oxidationGlycolysis Oxidative respiration Ultimate metabolic products Pyruvate Acetyl-CoA Krebs cycle FIGURE 9.21 How cells extract chemical energy. All eukaryotes and many prokaryotes extract energy from organic molecules by oxidizing them. The first stage of this process, breaking down macromolecules into their constituent parts, yields little energy. The second stage, oxidative or aerobic respiration, extracts energy, primarily in the form of high-energy electrons, and produces water and carbon dioxide. H9251-Ketoglutarate C — — — O O H — C — — —— H — C — HO — — — C — — — HO — C — H H H Glutamate H 2 N C — — — O O O H — C — — — —— H — C — — — — C — — — HO HO C H H NH 2 Urea FIGURE 9.22 Deamination. After proteins are broken down into their amino acid constituents, the amino groups are removed from the amino acids to form molecules that participate in glycolysis and the Krebs cycle. For example, the amino acid glutamate becomes α-ketoglutarate, a Krebs cycle molecule, when it loses its amino group. Cellular Respiration of Fat Fats are broken down into fatty acids plus glycerol. The tails of fatty acids typically have 16 or more —CH 2 links, and the many hydrogen atoms in these long tails provide a rich harvest of energy. Fatty acids are oxidized in the ma- trix of the mitochondrion. Enzymes there remove the two- carbon acetyl groups from the end of each fatty acid tail until the entire fatty acid is converted into acetyl groups (figure 9.23). Each acetyl group then combines with coen- zyme A to form acetyl-CoA. This process is known as H9252- oxidation. How much ATP does the catabolism of fatty acids pro- duce? Let’s compare a hypothetical six-carbon fatty acid with the six-carbon glucose molecule, which we’ve said yields about 30 molecules of ATP in a eukaryotic cell. Two rounds of β-oxidation would convert the fatty acid into three molecules of acetyl-CoA. Each round requires one molecule of ATP to prime the process, but it also produces one molecule of NADH and one of FADH 2 . These mole- cules together yield four molecules of ATP (assuming 2.5 ATPs per NADH and 1.5 ATPs per FADH 2 ). The oxida- tion of each acetyl-CoA in the Krebs cycle ultimately pro- duces an additional 10 molecules of ATP. Overall, then, the ATP yield of a six-carbon fatty acid would be approxi- mately 8 (from two rounds of β-oxidation) – 2 (for priming those two rounds) + 30 (from oxidizing the three acetyl- CoAs) = 36 molecules of ATP. Therefore, the respiration of a six-carbon fatty acid yields 20% more ATP than the respiration of glucose. Moreover, a fatty acid of that size would weigh less than two-thirds as much as glucose, so a gram of fatty acid contains more than twice as many kilo- calories as a gram of glucose. That is why fat is a storage molecule for excess energy in many types of animals. If ex- cess energy were stored instead as carbohydrate, as it is in plants, animal bodies would be much bulkier. Proteins, fats, and other organic molecules are also metabolized for energy. The amino acids of proteins are first deaminated, while fats undergo a process called β-oxidation. Chapter 9 How Cells Harvest Energy 179 OH O Fatty acid C H H C H H C Fatty acid C H H C H H C O FAD FADH 2 ATP AMP + PP i CoA CoAFatty acid C H C H C O CoA Fatty acid C HO H C H H C O CoA Fatty acid C C H H C OO CoA NAD + NADH H 2 O Acetyl-CoA Krebs cycle CoA FIGURE 9.23 β-oxidation. Through a series of reactions known as β-oxidation, the last two carbons in a fatty acid tail combine with coenzyme A to form acetyl-CoA, which enters the Krebs cycle. The fatty acid, now two carbons shorter, enters the pathway again and keeps reentering until all its carbons have been used to form acetyl-CoA molecules. Each round of β-oxidation uses one molecule of ATP and generates one molecule each of FADH 2 and NADH, not including the molecules generated from the Krebs cycle. 180 Part III Energetics Even with oxidative metabolism, approx- imately two-thirds of the available energy is lost at each trophic level, and that puts a limit on how long a food chain can be. Most food chains, like the one illustrated in figure 9.A, involve only three or rarely four trophic levels. Too much energy is lost at each transfer to allow chains to be much longer than that. For example, it would be impossi- ble for a large human population to subsist by eating lions captured from the Serengeti Plain of Africa; the amount of grass available there would not support enough zebras and other herbivores to maintain the number of lions needed to feed the human population. Thus, the ecological complexity of our world is fixed in a fundamental way by the chemistry of oxidative respiration. When organisms became able to extract energy from organic molecules by oxidative metabolism, this constraint became far less severe, because the efficiency of oxidative respiration is estimated to be about 52 to 63%. This increased efficiency results in the transmission of much more energy from one trophic level to another than does glycolysis. (A trophic level is a step in the movement of energy through an ecosys- tem.) The efficiency of oxidative metabo- lism has made possible the evolution of food chains, in which autotrophs are con- sumed by heterotrophs, which are con- sumed by other heterotrophs, and so on. You will read more about food chains in chapter 28. It has been estimated that a heterotroph limited to glycolysis captures only 3.5% of the energy in the food it consumes. Hence, if such a heterotroph preserves 3.5% of the energy in the autotrophs it consumes, then any other heterotrophs that consume the first hetertroph will capture through glycol- ysis 3.5% of the energy in it, or 0.12% of the energy available in the original au- totrophs. A very large base of autotrophs would thus be needed to support a small number of heterotrophs. Metabolic Efficiency and the Length of Food Chains FIGURE 9.A A food chain in the savannas, or open grasslands, of East Africa. Stage 1: Photosynthesizer. The grass under these palm trees grows actively during the hot, rainy season, capturing the energy of the sun and storing it in molecules of glucose, which are then converted into starch and stored in the grass. Stage 2: Herbivore. These large antelopes, known as wildebeests, consume the grass and transfer some of its stored energy into their own bodies. Stage 3: Carnivore. The lion feeds on wildebeests and other animals, capturing part of their stored energy and storing it in its own body. Stage 4: Scavenger. This hyena and the vultures occupy the same stage in the food chain as the lion. They are also consuming the body of the dead wildebeest, which has been abandoned by the lion. Stage 5: Refuse utilizer. These butterflies, mostly Precis octavia, are feeding on the material left in the hyena’s dung after the food the hyena consumed had passed through its digestive tract. At each of these four levels, only about a third or less of the energy present is used by the recipient. Stage 1: Photosynthesizers Stage 2: Herbivores Stage 3: Carnivore Stage 4: Scavengers Stage 5: Refuse utilizers Fermentation In the absence of oxygen, aerobic metabolism cannot occur, and cells must rely exclusively on glycolysis to pro- duce ATP. Under these conditions, the hydrogen atoms generated by glycolysis are donated to organic molecules in a process called fermentation. Bacteria carry out more than a dozen kinds of fermen- tations, all using some form of organic molecule to ac- cept the hydrogen atom from NADH and thus recycle NAD + : Organic molecule ?→ Reduced organic molecule + NADH + NAD + Often the reduced organic compound is an organic acid— such as acetic acid, butyric acid, propionic acid, or lactic acid—or an alcohol. Ethanol Fermentation Eukaryotic cells are capable of only a few types of fermen- tation. In one type, which occurs in single-celled fungi called yeast, the molecule that accepts hydrogen from NADH is pyruvate, the end product of glycolysis itself. Yeast enzymes remove a terminal CO 2 group from pyru- vate through decarboxylation, producing a two-carbon molecule called acetaldehyde. The CO 2 released causes bread made with yeast to rise, while bread made without yeast (unleavened bread) does not. The acetaldehyde ac- cepts a hydrogen atom from NADH, producing NAD + and ethanol (ethyl alcohol). This particular type of fermenta- tion is of great interest to humans, since it is the source of the ethanol in wine and beer (figure 9.24). Ethanol is a by- product of fermentation that is actually toxic to yeast; as it approaches a concentration of about 12%, it begins to kill the yeast. That explains why naturally fermented wine con- tains only about 12% ethanol. Lactic Acid Fermentation Most animal cells regenerate NAD + without decarboxyla- tion. Muscle cells, for example, use an enzyme called lactate dehydrogenase to transfer a hydrogen atom from NADH back to the pyruvate that is produced by glycolysis. This reaction converts pyruvate into lactic acid and regenerates NAD + from NADH. It therefore closes the metabolic cir- cle, allowing glycolysis to continue as long as glucose is available. Circulating blood removes excess lactate (the ion- ized form of lactic acid) from muscles, but when removal cannot keep pace with production, the accumulating lactic acid interferes with muscle function and contributes to muscle fatigue. In fermentation, which occurs in the absence of oxygen, the electrons that result from the glycolytic breakdown of glucose are donated to an organic molecule, regenerating NAD + from NADH. Chapter 9 How Cells Harvest Energy 181 9.4 Cells can metabolize food without oxygen. Glucose 2 Pyruvate Alcohol fermentation in yeast G L Y C O L Y S I S 2 ATP 2 ADP CO O - CO CH 3 CO O – CH 3 C 2 Ethanol 2 Acetaldehyde 2 Lactate HOH H CH OH CH 3 Glucose 2 Pyruvate Lactic acid fermentation in muscle cells G L Y C O L Y S I S 2 ATP 2 ADP CO O - CO CH 3 2 NAD + OC H CH 3 2 NADH 2 NAD + 2 NADH CO 2 FIGURE 9.24 How wine is made. Yeasts carry out the conversion of pyruvate to ethanol. This takes place naturally in grapes left to ferment on vines, as well as in fermentation vats containing crushed grapes. When the ethanol concentration reaches about 12%, its toxic effects kill the yeast; what remains is wine. Muscle cells convert pyruvate into lactate, which is less toxic than ethanol. However, lactate is still toxic enough to produce a painful sensation in muscles during heavy exercise, when oxygen in the muscles is depleted. 182 Part III Energetics Chapter 9 Summary Questions Media Resources 9.1 Cells harvest the energy in chemical bonds. ? The reactions of cellular respiration are oxidation- reduction (redox) reactions. Those that require a net input of free energy are coupled to the cleavage of ATP, which releases free energy. ? The mechanics of cellular respiration are often dictated by electron behavior, which is in turn influenced by the presence of electron acceptors. Some atoms, such as oxygen, are very electronegative and thus behave as good oxidizing agents. 1. What is the difference between an autotroph and a heterotroph? How does each obtain energy? 2. What is the difference between digestion and catabolism? Which provides more energy? ? In eukaryotic cells, the oxidative respiration of pyruvate takes place within the matrix of mitochondria. ? The electrons generated in the process are passed along the electron transport chain, a sequence of electron carriers in the inner mitochondrial membrane. ? Some of the energy released by passage of electrons along the electron transport chain is used to pump protons out of the mitochondrial matrix. The reentry of protons into the matrix is coupled to the production of ATP. This process is called chemiosmosis. The ATP then leaves the mitochondrion by facilitated diffusion. 3. Where in a eukaryotic cell does glycolysis occur? What is the net production of ATP during glycolysis, and why is it different from the number of ATP molecules synthesized during glycolysis? 4. By what two mechanisms can the NADH that results from glycolysis be converted back into NAD + ? 5. What is the theoretical maximum number of ATP molecules produced during the oxidation of a glucose molecule by these processes? Why is the actual number of ATP molecules produced usually lower than the theoretical maximum? 9.2 Cellular respiration oxidizes food molecules. ? The catabolism of fatty acids begins with H9252-oxidation and provides more energy than the catabolism of carbohydrates. 6. How is acetyl-CoA produced during the aerobic oxidation of carbohydrates, and what happens to it? How is it produced during the aerobic oxidation of fatty acids, and what happens to it then? 9.3 Catabolism of proteins and fats can yield considerable energy. ? Fermentation is an anaerobic process that uses an organic molecule instead of oxygen as a final electron acceptor. ? It occurs in bacteria as well as eukaryotic cells, including yeast and the muscle cells of animals. 7. How do the amounts of ATP produced by the aerobic oxidation of glucose and fatty acids compare? Which type of substance contains more energy on a per-weight basis? 9.4 Cells can metabolize food without oxygen. http://www.mhhe.com/raven6e http://www.biocourse.com ? Exploration: Oxidative Respiration ? Art Activity: Aerobic Cellular Respiration ? Art Activity: Organization of Cristae ? Electron Transport and ATP ? Glycolysis ? Krebs Cycle ? Electron Transport ? Fermentation 183 10 Photosynthesis Concept Outline 10.1 What is photosynthesis? The Chloroplast as a Photosynthetic Machine. The highly organized system of membranes in chloroplasts is essential to the functioning of photosynthesis. 10.2 Learning about photosynthesis: An experimental journey. The Role of Soil and Water. The added mass of a growing plant comes mostly from photosynthesis. In plants, water supplies the electrons used to reduce carbon dioxide. Discovery of the Light-Independent Reactions. Photosynthesis is a two-stage process. Only the first stage directly requires light. The Role of Light. The oxygen released during green plant photosynthesis comes from water, and carbon atoms from carbon dioxide are incorporated into organic molecules. The Role of Reducing Power. Electrons released from the splitting of water reduce NADP + ; ATP and NADPH are then used to reduce CO 2 and form simple sugars. 10.3 Pigments capture energy from sunlight. The Biophysics of Light. The energy in sunlight occurs in “packets” called photons, which are absorbed by pigments. Chlorophylls and Carotenoids. Photosynthetic pigments absorb light and harvest its energy. Organizing Pigments into Photosystems. A photosystem uses light energy to eject an energized electron. How Photosystems Convert Light to Chemical Energy. Some bacteria rely on a single photosystem to produce ATP. Plants use two photosystems in series to generate enough energy to reduce NADP + and generate ATP. How the Two Photosystems of Plants Work Together. Photosystems II and I drive the synthesis of the ATP and NADPH needed to form organic molecules. 10.4 Cells use the energy and reducing power captured by the light reactions to make organic molecules. The Calvin Cycle. ATP and NADPH are used to build organic molecules, a process reversed in mitochondria. Reactions of the Calvin Cycle. Ribulose bisphosphate binds CO 2 in the process of carbon fixation. Photorespiration. The enzyme that catalyzes carbon fixation also affects CO 2 release. L ife on earth would be impossible without photosyn- thesis. Every oxygen atom in the air we breathe was once part of a water molecule, liberated by photosynthesis. The energy released by the burning of coal, firewood, gasoline, and natural gas, and by our bodies’ burning of all the food we eat—all, directly or indirectly, has been cap- tured from sunlight by photosynthesis. It is vitally impor- tant that we understand photosynthesis. Research may en- able us to improve crop yields and land use, important goals in an increasingly crowded world. In the previous chapter we described how cells extract chemical energy from food molecules and use that energy to power their activities. In this chapter, we will examine photosynthesis, the process by which organisms capture energy from sun- light and use it to build food molecules rich in chemical energy (figure 10.1). FIGURE 10.1 Capturing energy. These sunflower plants, growing vigorously in the August sun, are capturing light energy for conversion into chemical energy through photosynthesis. 184 Part III Energetics 10.1 What is photosynthesis? Cuticle Epidermis Mesophyll Vascular bundle Stoma Bundle sheath Chloroplasts Vacuole Nucleus Cell wall Outer membrane Inner membrane Stroma Granum Thylakoid The Chloroplast as a Photosynthetic Machine Life is powered by sunshine. The energy used by most liv- ing cells comes ultimately from the sun, captured by plants, algae, and bacteria through the process of photosynthesis. The diversity of life is only possible because our planet is awash in energy streaming earthward from the sun. Each day, the radiant energy that reaches the earth equals about 1 million Hiroshima-sized atomic bombs. Photosynthesis captures about 1% of this huge supply of energy, using it to provide the energy that drives all life. The Photosynthetic Process: A Summary Photosynthesis occurs in many kinds of bacteria and algae, and in the leaves and sometimes the stems of green plants. Figure 10.2 describes the levels of organization in a plant leaf. Recall from chapter 5 that the cells of plant leaves contain organelles called chloroplasts that actually carry out the photosynthetic process. No other structure in a plant cell is able to carry out photosynthesis. Photosynthe- FIGURE 10.2 Journey into a leaf. A plant leaf possesses a thick layer of cells (the mesophyll) rich in chloroplasts. The flattened thylakoids in the chloroplast are stacked into columns called grana (singular, granum). The light reactions take place on the thylakoid sis takes place in three stages: (1) capturing energy from sunlight; (2) using the energy to make ATP and reducing power in the form of a compound called NADPH; and (3) using the ATP and NADPH to power the synthesis of organic molecules from CO 2 in the air (carbon fixation). The first two stages take place in the presence of light and are commonly called the light reactions. The third stage, the formation of organic molecules from atmos- pheric CO 2 , is called the Calvin cycle. As long as ATP and NADPH are available, the Calvin cycle may occur in the absence of light. The following simple equation summarizes the overall process of photosynthesis: 6 CO 2 + 12 H 2 O + light —→ C 6 H 12 O 6 + 6 H 2 O + 6 O 2 carbon water glucose water oxygen dioxide Inside the Chloroplast The internal membranes of chloroplasts are organized into sacs called thylakoids, and often numerous thylakoids are stacked on one another in columns called grana. The thy- lakoid membranes house the photosynthetic pigments for capturing light energy and the machinery to make ATP. Surrounding the thylakoid membrane system is a semiliq- uid substance called stroma. The stroma houses the en- zymes needed to assemble carbon molecules. In the mem- branes of thylakoids, photosynthetic pigments are clustered together to form a photosystem. Each pigment molecule within the photosystem is capa- ble of capturing photons, which are packets of energy. A lat- tice of proteins holds the pigments in close contact with one another. When light of a proper wavelength strikes a pigment molecule in the photosystem, the resulting excita- tion passes from one chlorophyll molecule to another. The excited electron is not transferred physically—it is the en- ergy that passes from one molecule to another. A crude analogy to this form of energy transfer is the initial “break” in a game of pool. If the cue ball squarely hits the point of the triangular array of 15 pool balls, the two balls at the far corners of the triangle fly off, but none of the central balls move. The energy passes through the central balls to the most distant ones. Eventually the energy arrives at a key chlorophyll mole- cule that is touching a membrane-bound protein. The en- ergy is transferred as an excited electron to that protein, which passes it on to a series of other membrane proteins that put the energy to work making ATP and NADPH and building organic molecules. The photosystem thus acts as a large antenna, gathering the light harvested by many indi- vidual pigment molecules. The reactions of photosynthesis take place within thylakoid membranes within chloroplasts in leaf cells. Chapter 10 Photosynthesis 185 Sunlight Light reactions Organic molecules CO 2 H 2 O O 2 Photosystem ADP NADPH NADP + Stroma Thylakoid Thylakoid Stroma Granum ATP Calvin cycle FIGURE 10.2 (continued) membrane and generate the ATP and NADPH that fuel the Calvin cycle. The fluid interior matrix of a chloroplast, the stroma, contains the enzymes that carry out the Calvin cycle. The Role of Soil and Water The story of how we learned about photosynthesis is one of the most interesting in science and serves as a good intro- duction to this complex process. The story starts over 300 years ago, with a simple but carefully designed experiment by a Belgian doctor, Jan Baptista van Helmont (1577–1644). From the time of the Greeks, plants were thought to obtain their food from the soil, literally sucking it up with their roots; van Helmont thought of a simple way to test the idea. He planted a small willow tree in a pot of soil after weighing the tree and the soil. The tree grew in the pot for several years, during which time van Helmont added only water. At the end of five years, the tree was much larger: its weight had increased by 74.4 kilograms. However, all of this added mass could not have come from the soil, because the soil in the pot weighed only 57 grams less than it had five years earlier! With this experiment, van Helmont demonstrated that the substance of the plant was not produced only from the soil. He incorrectly concluded that mainly the water he had been adding accounted for the plant’s increased mass. A hundred years passed before the story became clearer. The key clue was provided by the English scientist Joseph Priestly, in his pioneering studies of the properties of air. On the 17th of August, 1771, Priestly “accidentally hit upon a method of restoring air that had been injured by the burning of candles.” He “put a [living] sprig of mint into air in which a wax candle had burnt out and found that, on the 27th of the same month, another candle could be burned in this same air.” Somehow, the vegetation seemed to have restored the air! Priestly found that while a mouse could not breathe candle-exhausted air, air “restored” by vegetation was not “at all inconvenient to a mouse.” The key clue was that living vegetation adds something to the air. How does vegetation “restore” air? Twenty-five years later, Dutch physician Jan Ingenhousz solved the puzzle. Working over several years, Ingenhousz reproduced and significantly extended Priestly’s results, demonstrating that air was restored only in the presence of sunlight, and only by a plant’s green leaves, not by its roots. He proposed that the green parts of the plant carry out a process (which we now call photosynthesis) that uses sunlight to split carbon dioxide (CO 2 ) into carbon and oxygen. He suggested that the oxygen was released as O 2 gas into the air, while the carbon atom combined with water to form carbohydrates. His proposal was a good guess, even though the later step was subsequently modified. Chemists later found that the proportions of carbon, oxygen, and hydrogen atoms in car- bohydrates are indeed about one atom of carbon per mole- cule of water (as the term carbohydrate indicates). A Swiss botanist found in 1804 that water was a necessary reactant. By the end of that century the overall reaction for photo- synthesis could be written as: CO 2 + H 2 O + light energy —→ (CH 2 O) + O 2 It turns out, however, that there’s more to it than that. When researchers began to examine the process in more detail in the last century, the role of light proved to be un- expectedly complex. Van Helmont showed that soil did not add mass to a growing plant. Priestly and Ingenhousz and others then worked out the basic chemical reaction. Discovery of the Light-Independent Reactions Ingenhousz’s early equation for photosynthesis includes one factor we have not discussed: light energy. What role does light play in photosynthesis? At the beginning of the previous century, the English plant physiologist F. F. Blackman began to address the question of the role of light in photosynthesis. In 1905, he came to the startling conclu- sion that photosynthesis is in fact a two-stage process, only one of which uses light directly. Blackman measured the effects of different light inten- sities, CO 2 concentrations, and temperatures on photo- synthesis. As long as light intensity was relatively low, he found photosynthesis could be accelerated by increasing the amount of light, but not by increasing the tempera- ture or CO 2 concentration (figure 10.3). At high light in- tensities, however, an increase in temperature or CO 2 concentration greatly accelerated photosynthesis. Black- man concluded that photosynthesis consists of an initial set of what he called “light” reactions, that are largely in- dependent of temperature, and a second set of “dark” re- actions, that seemed to be independent of light but lim- ited by CO 2 . Do not be confused by Blackman’s labels—the so-called “dark” reactions occur in the light (in fact, they require the products of the light reactions); their name simply indicates that light is not directly in- volved in those reactions. Blackman found that increased temperature increases the rate of the dark carbon-reducing reactions, but only up to about 35°C. Higher temperatures caused the rate to fall off rapidly. Because 35°C is the temperature at which many plant enzymes begin to be denatured (the hydrogen bonds that hold an enzyme in its particular catalytic shape begin to be disrupted), Blackman concluded that enzymes must carry out the dark reactions. Blackman showed that capturing photosynthetic energy requires sunlight, while building organic molecules does not. 186 Part III Energetics 10.2 Learning about photosynthesis: An experimental journey. The Role of Light The role of light in the so-called light and dark reactions was worked out in the 1930s by C. B. van Niel, then a graduate student at Stanford University studying photosynthesis in bacteria. One of the types of bacteria he was studying, the purple sulfur bacteria, does not release oxygen during photosynthesis; instead, they convert hydrogen sulfide (H 2 S) into globules of pure elemental sulfur that accumulate inside themselves. The process that van Niel observed was CO 2 + 2 H 2 S + light energy → (CH 2 O) + H 2 O + 2 S The striking parallel between this equation and Ingenhousz’s equation led van Niel to propose that the generalized process of photosynthesis is in fact CO 2 + 2 H 2 A + light energy → (CH 2 O) + H 2 O + 2 A In this equation, the substance H 2 A serves as an electron donor. In photosynthesis performed by green plants, H 2 A is water, while among purple sulfur bacteria, H 2 A is hydrogen sulfide. The product, A, comes from the splitting of H 2 A. Therefore, the O 2 produced during green plant photosyn- thesis results from splitting water, not car- bon dioxide. When isotopes came into common use in biology in the early 1950s, it became possible to test van Niel’s revolu- tionary proposal. Investigators examined photosynthesis in green plants supplied with 18 O water; they found that the 18 O label ended up in oxygen gas rather than in carbohy- drate, just as van Niel had predicted: CO 2 + 2 H 2 18 O + light energy —→ (CH 2 O) + H 2 O + 18 O 2 In algae and green plants, the carbohydrate typically pro- duced by photosynthesis is the sugar glucose, which has six carbons. The complete balanced equation for photosynthe- sis in these organisms thus becomes 6 CO 2 + 12 H 2 O + light energy —→ C 6 H 12 O 6 + 6 O 2 + 6 H 2 O. We now know that the first stage of photosynthesis, the light reactions, uses the energy of light to reduce NADP (an electron carrier molecule) to NADPH and to manufac- ture ATP. The NADPH and ATP from the first stage of photosynthesis are then used in the second stage, the Calvin cycle, to reduce the carbon in carbon dioxide and form a simple sugar whose carbon skeleton can be used to synthesize other organic molecules. Van Niel discovered that photosynthesis splits water molecules, incorporating the carbon atoms of carbon dioxide gas and the hydrogen atoms of water into organic molecules and leaving oxygen gas. The Role of Reducing Power In his pioneering work on the light reactions, van Niel had further proposed that the reducing power (H + ) generated by the splitting of water was used to convert CO 2 into organic matter in a process he called carbon fixation. Was he right? In the 1950s Robin Hill demonstrated that van Niel was indeed right, and that light energy could be used to generate reducing power. Chloroplasts isolated from leaf cells were able to reduce a dye and release oxygen in response to light. Later experiments showed that the electrons released from water were transferred to NADP + . Arnon and coworkers showed that illuminated chloroplasts deprived of CO 2 accu- mulate ATP. If CO 2 is then introduced, neither ATP nor NADPH accumulate, and the CO 2 is assimilated into organic molecules. These experiments are important for three rea- sons. First, they firmly demonstrate that photosynthesis oc- curs only within chloroplasts. Second, they show that the light-dependent reactions use light energy to reduce NADP + and to manufacture ATP. Thirdly, they confirm that the ATP and NADPH from this early stage of photosynthesis are then used in the later light-independent reactions to reduce carbon dioxide, forming simple sugars. Hill showed that plants can use light energy to generate reducing power. The incorporation of carbon dioxide into organic molecules in the light-independent reactions is called carbon fixation. Chapter 10 Photosynthesis 187 Maximum rate Excess CO 2 ; 35°C Temperature limited Excess CO 2 ; 20°C CO 2 limited Light intensity (foot-candles) Rate of photosynthesis Light limited 500 (b) 1000 1500 2000 2500 Insufficient CO 2 (0.01%); 20°C FIGURE 10.3 Discovery of the dark reactions. (a) Blackman measured photosynthesis rates under differing light intensities, CO 2 concentrations, and temperatures. (b) As this graph shows, light is the limiting factor at low light intensities, while temperature and CO 2 concentration are the limiting factors at higher light intensities. (a) The Biophysics of Light Where is the energy in light? What is there in sunlight that a plant can use to reduce carbon dioxide? This is the mystery of photosynthesis, the one fac- tor fundamentally different from processes such as respiration. To an- swer these questions, we will need to consider the physical nature of light it- self. James Clerk Maxwell had theo- rized that light was an electromagnetic wave—that is, that light moved through the air as oscillating electric and magnetic fields. Proof of this came in a curious experiment carried out in a laboratory in Germany in 1887. A young physicist, Heinrich Hertz, was attempting to verify a highly mathe- matical theory that predicted the exis- tence of electromagnetic waves. To see whether such waves existed, Hertz de- signed a clever experiment. On one side of a room he constructed a powerful spark generator that consisted of two large, shiny metal spheres standing near each other on tall, slender rods. When a very high sta- tic electrical charge was built up on one sphere, sparks would jump across to the other sphere. After constructing this device, Hertz set out to investigate whether the sparking would create invisible electromagnetic waves, so-called radio waves, as predicted by the mathemati- cal theory. On the other side of the room, he placed the world’s first radio receiver, a thin metal hoop on an insulat- ing stand. There was a small gap at the bottom of the hoop, so that the hoop did not quite form a complete circle. When Hertz turned on the spark generator across the room, he saw tiny sparks passing across the gap in the hoop! This was the first demonstration of radio waves. But Hertz noted another curious phenomenon. When UV light was shining across the gap on the hoop, the sparks were produced more readily. This unexpected facilitation, called the photoelectric effect, puzzled investigators for many years. The photoelectric effect was finally explained using a concept proposed by Max Planck in 1901. Planck devel- oped an equation that predicted the blackbody radiation curve based upon the assumption that light and other forms of radiation behaved as units of energy called photons. In 1905 Albert Einstein explained the photoelectric effect uti- lizing the photon concept. Ultraviolet light has photons of sufficient energy that when they fell on the loop, electrons were ejected from the metal surface. The photons had transferred their energy to the electrons, literally blasting them from the ends of the hoop and thus facilitating the passage of the electric spark induced by the radio waves. Visible wavelengths of light were unable to remove the electrons because their photons did not have enough en- ergy to free the electrons from the metal surface at the ends of the hoop. The Energy in Photons Photons do not all possess the same amount of energy (fig- ure 10.4). Instead, the energy content of a photon is in- versely proportional to the wavelength of the light: short- wavelength light contains photons of higher energy than long-wavelength light. X rays, which contain a great deal of energy, have very short wavelengths—much shorter than visi- ble light, making them ideal for high-resolution microscopes. Hertz had noted that the strength of the photoelectric effect depends on the wavelength of light; short wave- lengths are much more effective than long ones in produc- ing the photoelectric effect. Einstein’s theory of the photo- electric effect provides an explanation: sunlight contains photons of many different energy levels, only some of which our eyes perceive as visible light. The highest energy photons, at the short-wavelength end of the electromag- netic spectrum (see figure 10.4), are gamma rays, with wavelengths of less than 1 nanometer; the lowest energy photons, with wavelengths of up to thousands of meters, are radio waves. Within the visible portion of the spectrum, violet light has the shortest wavelength and the most ener- getic photons, and red light has the longest wavelength and the least energetic photons. 188 Part III Energetics 10.3 Pigments capture energy from sunlight. 1 nm 400 nm 0.001 nm 10 nm 1000 nm Increasing wavelength Visible light Increasing energy 0.01 cm 1 cm 1 m Radio wavesInfraredX raysGamma rays UV light 100 m 430 nm 500 nm 560 nm 600 nm 650 nm 740 nm FIGURE 10.4 The electromagnetic spectrum. Light is a form of electromagnetic energy conveniently thought of as a wave. The shorter the wavelength of light, the greater its energy. Visible light represents only a small part of the electromagnetic spectrum between 400 and 740 nanometers. Ultraviolet Light The sunlight that reaches the earth’s surface contains a significant amount of ultraviolet (UV) light, which, because of its shorter wavelength, possesses considerably more en- ergy than visible light. UV light is thought to have been an important source of energy on the primitive earth when life originated. To- day’s atmosphere contains ozone (derived from oxygen gas), which absorbs most of the UV photons in sunlight, but a considerable amount of UV light still manages to pene- trate the atmosphere. This UV light is a po- tent force in disrupting the bonds of DNA, causing mutations that can lead to skin can- cer. As we will describe in a later chapter, loss of atmospheric ozone due to human ac- tivities threatens to cause an enormous jump in the incidence of human skin cancers throughout the world. Absorption Spectra and Pigments How does a molecule “capture” the energy of light? A photon can be envisioned as a very fast-moving packet of energy. When it strikes a molecule, its energy is either lost as heat or absorbed by the electrons of the mol- ecule, boosting those electrons into higher energy levels. Whether or not the photon’s energy is absorbed depends on how much energy it carries (defined by its wavelength) and on the chemical nature of the molecule it hits. As we saw in chapter 2, electrons occupy discrete energy levels in their orbits around atomic nuclei. To boost an electron into a different energy level requires just the right amount of energy, just as reach- ing the next rung on a ladder requires you to raise your foot just the right distance. A specific atom can, therefore, absorb only certain photons of light—namely, those that correspond to the atom’s available electron energy levels. As a result, each molecule has a characteristic absorption spectrum, the range and efficiency of photons it is capable of absorbing. Molecules that are good absorbers of light in the visible range are called pigments. Organisms have evolved a vari- ety of different pigments, but there are only two general types used in green plant photosynthesis: carotenoids and chlorophylls. Chlorophylls absorb photons within narrow energy ranges. Two kinds of chlorophyll in plants, chloro- phylls a and b, preferentially absorb violet-blue and red light (figure 10.5). Neither of these pigments absorbs pho- tons with wavelengths between about 500 and 600 nanometers, and light of these wavelengths is, therefore, reflected by plants. When these photons are subsequently absorbed by the pigment in our eyes, we perceive them as green. Chlorophyll a is the main photosynthetic pigment and is the only pigment that can act directly to convert light en- ergy to chemical energy. However, chlorophyll b, acting as an accessory or secondary light-absorbing pigment, com- plements and adds to the light absorption of chlorophyll a. Chlorophyll b has an absorption spectrum shifted toward the green wavelengths. Therefore, chlorophyll b can absorb photons chlorophyll a cannot. Chlorophyll b therefore greatly increases the proportion of the photons in sunlight that plants can harvest. An important group of accessory pigments, the carotenoids, assist in photosynthesis by cap- turing energy from light of wavelengths that are not effi- ciently absorbed by either chlorophyll. In photosynthesis, photons of light are absorbed by pigments; the wavelength of light absorbed depends upon the specific pigment. Chapter 10 Photosynthesis 189 Chlorophyll b Chlorophyll a Relative light absorption Wavelength (nm) 400 450 500 550 600 650 700 Carotenoids FIGURE 10.5 The absorption spectrum of chlorophyll. The peaks represent wavelengths of sunlight that the two common forms of photosynthetic pigment, chlorophyll a (solid line) and chlorophyll b (dashed line), strongly absorb. These pigments absorb predominately violet-blue and red light in two narrow bands of the spectrum and reflect the green light in the middle of the spectrum. Carotenoids (not shown here) absorb mostly blue and green light and reflect orange and yellow light. Chlorophylls and Carotenoids Chlorophylls absorb photons by means of an excitation process analogous to the photoelectric effect. These pigments contain a complex ring structure, called a porphyrin ring, with alternating single and double bonds. At the center of the ring is a magnesium atom. Photons absorbed by the pigment molecule excite electrons in the ring, which are then chan- neled away through the alternating carbon-bond system. Sev- eral small side groups attached to the outside of the ring alter the absorption properties of the molecule in different kinds of chlorophyll (figure 10.6). The precise absorption spectrum is also influenced by the local microenvironment created by the association of chlorophyll with specific proteins. Once Ingenhousz demonstrated that only the green parts of plants can “restore” air, researchers suspected chlorophyll was the primary pigment that plants employ to absorb light in photosynthesis. Experiments conducted in the 1800s clearly verified this suspicion. One such experiment, per- formed by T. W. Englemann in 1882 (figure 10.7), serves as a particularly elegant example, simple in design and clear in outcome. Englemann set out to characterize the action spectrum of photosynthesis, that is, the relative effective- ness of different wavelengths of light in promoting photo- synthesis. He carried out the entire experiment utilizing a single slide mounted on a microscope. To obtain different wavelengths of light, he placed a prism under his micro- scope, splitting the light that illuminated the slide into a spectrum of colors. He then arranged a filament of green algal cells across the spectrum, so that different parts of the filament were illuminated with different wavelengths, and allowed the algae to carry out photosynthesis. To assess how fast photosynthesis was proceeding, Englemann chose to monitor the rate of oxygen production. Lacking a mass spectrometer and other modern instruments, he added aerotactic (oxygen-seeking) bacteria to the slide; he knew they would gather along the filament at locations where oxygen was being produced. He found that the bacteria ac- cumulated in areas illuminated by red and violet light, the two colors most strongly absorbed by chlorophyll. All plants, algae, and cyanobacteria use chlorophyll a as their primary pigments. It is reasonable to ask why these photosynthetic organisms do not use a pigment like retinal (the pigment in our eyes), which has a broad absorption spectrum that covers the range of 500 to 600 nanometers. The most likely hypothesis involves photoefficiency. Al- though retinal absorbs a broad range of wavelengths, it does so with relatively low efficiency. Chlorophyll, in con- trast, absorbs in only two narrow bands, but does so with high efficiency. Therefore, plants and most other photo- synthetic organisms achieve far higher overall photon cap- ture rates with chlorophyll than with other pigments. 190 Part III Energetics Thylakoid membrane Thylakoid Granum Chlorophyll a: R = —CH 3 Chlorophyll b: R = —CHO CO 2 CH 3 CH 2 CH 2 C O CH 2 CH CCH 3 CH 2 CH 2 CH 2 CHCH 3 CH 2 CH 2 CH 2 CHCH 3 CH 2 CH 2 CH 2 CHCH 3 CH 3 CH 2 CH 3 CH 3 H Mg O O NN NN CH 3 CH 3 H H H H Porphyrin head HRCHH 2 C Hydrocarbon tail Chlorophyll molecules embedded in a protein complex in the thylakoid membrane FIGURE 10.6 Chlorophyll. Chlorophyll molecules consist of a porphyrin head and a hydrocarbon tail that anchors the pigment molecule to hydrophobic regions of proteins embedded within the membranes of thylakoids. The only difference between the two chlorophyll molecules is the substitution of a —CHO (aldehyde) group in chlorophyll b for a —CH 3 (methyl) group in chlorophyll a. Carotenoids consist of carbon rings linked to chains with alternating single and double bonds. They can absorb photons with a wide range of energies, although they are not always highly efficient in transferring this energy. Carotenoids assist in photosynthesis by capturing energy from light of wavelengths that are not efficiently absorbed by chlorophylls (figure 10.8; see figure 10.5). A typical carotenoid is β-carotene, whose two carbon rings are connected by a chain of 18 carbon atoms with al- ternating single and double bonds. Splitting a molecule of β-carotene into equal halves produces two molecules of vit- amin A. Oxidation of vitamin A produces retinal, the pig- ment used in vertebrate vision. This explains why carrots, which are rich in β-carotene, enhance vision. A pigment is a molecule that absorbs light. The wavelengths absorbed by a particular pigment depend on the available energy levels to which light-excited electrons can be boosted in the pigment. Chapter 10 Photosynthesis 191 Absorbance Filament of green alga Oxygen-seeking bacteria T.W. Englemann revealed the action spectrum of photosynthesis in the filamentous alga Spirogyra in 1882. Englemann used the rate of oxygen production to measure the rate of photosynthesis. As his oxygen indicator, he chose bacteria that are attracted by oxygen. In place of the mirror and diaphragm usually used to illuminate objects under view in his microscope, he substituted a "microspectral apparatus," which, as its name implies, produced a tiny spectrum of colors that it projected upon the slide under the microscope. Then he arranged a filament of algal cells parallel to the spread of the spectrum. The oxygen-seeking bacteria congregated mostly in the areas where the violet and red wavelengths fell upon the algal filament. FIGURE 10.7 Constructing an action spectrum for photosynthesis. As you can see, the action spectrum for photosynthesis that Englemann revealed in his experiment parallels the absorption spectrum of chlorophyll (see figure 10.5). Oak leaf in summer Oak leaf in autumn FIGURE 10.8 Fall colors are produced by carotenoids and other accessory pigments. During the spring and summer, chlorophyll in leaves masks the presence of carotenoids and other accessory pigments. When cool fall temperatures cause leaves to cease manufacturing chlorophyll, the chlorophyll is no longer present to reflect green light, and the leaves reflect the orange and yellow light that carotenoids and other pigments do not absorb. Organizing Pigments into Photosystems The light reactions of photosynthesis occur in membranes. In bacteria like those studied by van Niel, the plasma mem- brane itself is the photosynthetic membrane. In plants and algae, by contrast, photosynthesis is carried out by or- ganelles that are the evolutionary descendants of photosyn- thetic bacteria, chloroplasts—the photosynthetic mem- branes exist within the chloroplasts. The light reactions take place in four stages: 1. Primary photoevent. A photon of light is captured by a pigment. The result of this primary photoevent is the excitation of an electron within the pigment. 2. Charge separation. This excitation energy is trans- ferred to a specialized chlorophyll pigment termed a reaction center, which reacts by transferring an ener- getic electron to an acceptor molecule, thus initiating electron transport. 3. Electron transport. The excited electron is shut- tled along a series of electron-carrier molecules em- bedded within the photosynthetic membrane. Several of them react by transporting protons across the membrane, generating a gradient of proton concen- tration. Its arrival at the pump induces the transport of a proton across the membrane. The electron is then passed to an acceptor. 4. Chemiosmosis. The protons that accumulate on one side of the membrane now flow back across the membrane through specific protein complexes where chemiosmotic synthesis of ATP takes place, just as it does in aerobic respiration. Discovery of Photosystems One way to study how pigments absorb light is to measure the dependence of the output of photosynthesis on the in- tensity of illumination—that is, how much photosynthesis is produced by how much light. When experiments of this sort are done on plants, they show that the output of pho- tosynthesis increases linearly at low intensities but lessens at higher intensities, finally saturating at high-intensity light (figure 10.9). Saturation occurs because all of the light-absorbing capacity of the plant is in use; additional light doesn’t increase the output because there is nothing to absorb the added photons. It is tempting to think that at saturation, all of a plant’s pigment molecules are in use. In 1932 plant physiologists Robert Emerson and William Arnold set out to test this hypothesis in an organism where they could measure both the number of chlorophyll molecules and the output of photosynthesis. In their experiment, they measured the oxygen yield of photosynthesis when Chlorella (unicellular green algae) were exposed to very brief light flashes lasting only a few microseconds. Assuming the hypothesis of pig- ment saturation to be correct, they expected to find that as they increased the intensity of the flashes, the yield per flash would increase, until each chlorophyll molecule ab- sorbed a photon, which would then be used in the light re- actions, producing a molecule of O 2 . Unexpectedly, this is not what happened. Instead, satu- ration was achieved much earlier, with only one molecule of O 2 per 2500 chlorophyll molecules! This led Emerson and Arnold to conclude that light is absorbed not by inde- pendent pigment molecules, but rather by clusters of chlorophyll and accessory pigment molecules which have come to be called photosystems. Light is absorbed by any one of the hundreds of pigment molecules in a photosystem, which transfer their excitation energy to one with a lower energy level than the others. This reaction center of the photosystem acts as an energy sink, trapping the excitation energy. It was the saturation of these reaction centers, not individual molecules, that was observed by Emerson and Arnold. Architecture of a Photosystem In chloroplasts and all but the most primitive bacteria, light is captured by such photosystems. Each photosystem is a network of chlorophyll a molecules, accessory pigments, and associated proteins held within a protein matrix on the surface of the photosynthetic membrane. Like a magnify- ing glass focusing light on a precise point, a photosystem channels the excitation energy gathered by any one of its pigment molecules to a specific molecule, the reaction cen- ter chlorophyll. This molecule then passes the energy out 192 Part III Energetics Expected Observed Saturation when all photosystems are in use Intensity of light flashes Output ( O 2 yield per flash) Saturation when all chlorophyll molecules are in use FIGURE 10.9 Emerson and Arnold’s experiment. When photosynthetic saturation is achieved, further increases in intensity cause no increase in output. of the photosystem so it can be put to work driving the syn- thesis of ATP and organic molecules. A photosystem thus consists of two closely linked components: (1) an antenna complex of hundreds of pig- ment molecules that gather photons and feed the cap- tured light energy to the reaction center; and (2) a reac- tion center, consisting of one or more chlorophyll a molecules in a matrix of protein, that passes the energy out of the photosystem. The Antenna Complex. The antenna complex captures photons from sunlight (figure 10.10). In chloroplasts, the antenna complex is a web of chlorophyll molecules linked together and held tightly on the thylakoid membrane by a matrix of proteins. Varying amounts of carotenoid acces- sory pigments may also be present. The protein matrix serves as a sort of scaffold, holding individual pigment mol- ecules in orientations that are optimal for energy transfer. The excitation energy resulting from the absorption of a photon passes from one pigment molecule to an adjacent molecule on its way to the reaction center. After the trans- fer, the excited electron in each molecule returns to the low-energy level it had before the photon was absorbed. Consequently, it is energy, not the excited electrons them- selves, that passes from one pigment molecule to the next. The antenna complex funnels the energy from many elec- trons to the reaction center. The Reaction Center. The reaction center is a trans- membrane protein-pigment complex. In the reaction cen- ter of purple photosynthetic bacteria, which is simpler than in chloroplasts but better understood, a pair of chlorophyll a molecules acts as a trap for photon energy, passing an ex- cited electron to an acceptor precisely positioned as its neighbor. Note that here the excited electron itself is trans- ferred, not just the energy as we saw in pigment-pigment transfers. This allows the photon excitation to move away from the chlorophylls and is the key conversion of light to chemical energy. Figure 10.11 shows the transfer of energy from the reac- tion center to the primary electron acceptor. By energizing an electron of the reaction center chlorophyll, light creates a strong electron donor where none existed before. The chlorophyll transfers the energized electron to the primary acceptor, a molecule of quinone, reducing the quinone and converting it to a strong electron donor. A weak electron donor then donates a low-energy electron to the chloro- phyll, restoring it to its original condition. In plant chloro- plasts, water serves as the electron donor. Photosystems contain pigments that capture photon energy from light. The pigments transfer the energy to reaction centers. There, the energy excites electrons, which are channeled away to do chemical work. Chapter 10 Photosynthesis 193 Electron donor Electron acceptor Photon Reaction center chlorophyll Chlorophyll molecules Photosystem FIGURE 10.10 How the antenna complex works. When light of the proper wavelength strikes any pigment molecule within a photosystem, the light is absorbed by that pigment molecule. The excitation energy is then transferred from one molecule to another within the cluster of pigment molecules until it encounters the reaction center chlorophyll a. When excitation energy reaches the reaction center chlorophyll, electron transfer is initiated. Electron donor Electron acceptor Acceptor reduced Chlorophyll oxidized Acceptor reduced Donor oxidized Excited chlorophyll molecule + – + – FIGURE 10.11 Converting light to chemical energy. The reaction center chlorophyll donates a light-energized electron to the primary electron acceptor, reducing it. The oxidized chlorophyll then fills its electron “hole” by oxidizing a donor molecule. How Photosystems Convert Light to Chemical Energy Bacteria Use a Single Photosystem Photosynthetic pigment arrays are thought to have evolved more than 3 billion years ago in bacteria similar to the sul- fur bacteria studied by van Niel. 1. Electron is joined with a proton to make hydrogen. In these bacteria, the absorption of a photon of light at a peak absorption of 870 nanometers (near infrared, not visi- ble to the human eye) by the photosystem results in the transmission of an energetic electron along an electron transport chain, eventually combining with a proton to form a hydrogen atom. In the sulfur bacteria, the proton is extracted from hydrogen sulfide, leaving elemental sulfur as a by-product. In bacteria that evolved later, as well as in plants and algae, the proton comes from water, producing oxygen as a by-product. 2. Electron is recycled to chlorophyll. The ejection of an electron from the bacterial reaction center leaves it short one electron. Before the photosystem of the sulfur bacteria can function again, an electron must be re- turned. These bacteria channel the electron back to the pigment through an electron transport system similar to the one described in chapter 9; the electron’s passage drives a proton pump that promotes the chemiosmotic synthesis of ATP. One molecule of ATP is produced for every three electrons that follow this path. Viewed overall (fig- ure 10.12), the path of the electron is thus a circle. Chemists therefore call the electron transfer process leading to ATP formation cyclic photophosphorylation. Note, however, that the electron that left the P 870 reac- tion center was a high-energy electron, boosted by the absorption of a photon of light, while the electron that returns has only as much energy as it had before the pho- ton was absorbed. The difference in the energy of that electron is the photosynthetic payoff, the energy that drives the proton pump. For more than a billion years, cyclic photophosphory- lation was the only form of photosynthetic light reaction that organisms used. However, its major limitation is that it is geared only toward energy production, not to- ward biosynthesis. Most photosynthetic organisms incor- porate atmospheric carbon dioxide into carbohydrates. Because the carbohydrate molecules are more reduced (have more hydrogen atoms) than carbon dioxide, a source of reducing power (that is, hydrogens) must be provided. Cyclic photophosphorylation does not do this. The hydrogen atoms extracted from H 2 S are used as a source of protons, and are not available to join to carbon. Thus bacteria that are restricted to this process must scavenge hydrogens from other sources, an inefficient undertaking. Why Plants Use Two Photosystems After the sulfur bacteria appeared, other kinds of bacteria evolved an improved version of the photosystem that over- came the limitation of cyclic photophosphorylation in a neat and simple way: a second, more powerful photosystem using another arrangement of chlorophyll a was combined with the original. In this second photosystem, called photosystem II, molecules of chlorophyll a are arranged with a different geometry, so that more shorter wavelength, higher energy photons are absorbed than in the ancestral photosystem, which is called photosystem I. As in the ancestral photo- system, energy is transmitted from one pigment molecule to another within the antenna complex of these photosys- tems until it reaches the reaction center, a particular pig- ment molecule positioned near a strong membrane-bound electron acceptor. In photosystem II, the absorption peak (that is, the wavelength of light most strongly absorbed) of the pigments is approximately 680 nanometers; therefore, the reaction center pigment is called P 680 . The absorption peak of photosystem I pigments in plants is 700 nanome- ters, so its reaction center pigment is called P 700 . Working together, the two photosystems carry out a noncyclic elec- tron transfer. When the rate of photosynthesis is measured using two light beams of different wavelengths (one red and the 194 Part III Energetics Electron acceptor Photon Energy of electrons Plastocyanin Photosystem ATP ADP pC Ferredoxin b 6 -f complex b 6 -f complex Fd e – e – e – Excited reaction center Reaction center P 870 FIGURE 10.12 The path of an electron in purple sulfur bacteria. When a light-energized electron is ejected from the photosystem reaction center (P 870 ), it passes in a circle, eventually returning to the photosystem from which it was ejected. other far-red), the rate was greater than the sum of the rates using individual beams of red and far-red light (fig- ure 10.13). This surprising result, called the enhancement effect, can be explained by a mechanism involving two photosystems acting in series (that is, one after the other), one of which absorbs preferentially in the red, the other in the far-red. The use of two photosystems solves the problem of ob- taining reducing power in a simple and direct way, by har- nessing the energy of two photosystems. The scheme shown in figure 10.14, called a Z diagram, illustrates the two electron-energizing steps, one catalyzed by each pho- tosystem. The electrons originate from water, which holds onto its electrons very tightly (redox potential = +820 mV), and end up in NADPH, which holds its electrons much more loosely (redox potential = –320 mV). In sulfur bacteria, excited electrons ejected from the reaction center travel a circular path, driving a proton pump and then returning to their original photosystem. Plants employ two photosystems in series, which generates power to reduce NADP + to NADPH with enough left over to make ATP. Chapter 10 Photosynthesis 195 Far-red light on Both lights on Red light on Off Off Time Relative rate of photosynthesis Off FIGURE 10.13 The “enhancement effect.” The rate of photosynthesis when red and far-red light are provided together is greater than the sum of the rates when each wavelength is provided individually. This result baffled researchers in the 1950s. Today it provides the key evidence that photosynthesis is carried out by two photochemical systems with slightly different wavelength optima. Proton gradient formed for ATP synthesis Water-splitting enzyme Photon Photon Energy of electrons Plastocyanin Ferredoxin Plastoquinone Photosystem II NADP + + H + b 6 -f complex NADP reductase b 6 -f complex Photosystem I NADP reductase e – e – H 2 O 2H + + H11002O 2 e – H + e – e – pC Q P 700 P 680 NADPH Fd 1 2 Reaction center Excited reaction center Reaction center Excited reaction center FIGURE 10.14 A Z diagram of photosystems I and II. Two photosystems work sequentially. First, a photon of light ejects a high-energy electron from photosystem II; that electron is used to pump a proton across the membrane, contributing chemiosmotically to the production of a molecule of ATP. The ejected electron then passes along a chain of cytochromes to photosystem I. When photosystem I absorbs a photon of light, it ejects a high-energy electron used to drive the formation of NADPH. How the Two Photosystems of Plants Work Together Plants use the two photosystems dis- cussed earlier in series, first one and then the other, to produce both ATP and NADPH. This two-stage process is called noncyclic photophosphory- lation, because the path of the elec- trons is not a circle—the electrons ejected from the photosystems do not return to it, but rather end up in NADPH. The photosystems are re- plenished instead with electrons ob- tained by splitting water. Photosystem II acts first. High-energy electrons generated by photosystem II are used to synthesize ATP and then passed to photosystem I to drive the production of NADPH. For every pair of elec- trons obtained from water, one mole- cule of NADPH and slightly more than one molecule of ATP are pro- duced. Photosystem II The reaction center of photosystem II, called P 680 , closely resembles the reac- tion center of purple bacteria. It con- sists of more than 10 transmembrane protein subunits. The light-harvesting antenna complex consists of some 250 molecules of chlorophyll a and acces- sory pigments bound to several protein chains. In photosystem II, the oxygen atoms of two water molecules bind to a cluster of manganese atoms which are embedded within an enzyme and bound to the reaction center. In a way that is poorly under- stood, this enzyme splits water, removing electrons one at a time to fill the holes left in the reaction center by departure of light-energized electrons. As soon as four electrons have been removed from the two water molecules, O 2 is released. The Path to Photosystem I The primary electron acceptor for the light-energized elec- trons leaving photosystem II is a quinone molecule, as it was in the bacterial photosystem described earlier. The re- duced quinone which results (plastoquinone, symbolized Q) is a strong electron donor; it passes the excited electron to a proton pump called the b 6 -f complex embedded within the thylakoid membrane (figure 10.15). This complex closely resembles the bc 1 complex in the respiratory electron trans- port chain of mitochondria discussed in chapter 9. Arrival of the energetic electron causes the b 6 -f complex to pump a proton into the thylakoid space. A small copper-containing protein called plastocyanin (symbolized pC) then carries the electron to photosystem I. Making ATP: Chemiosmosis Each thylakoid is a closed compartment into which pro- tons are pumped from the stroma by the b 6 -f complex. The splitting of water also produces added protons that contribute to the gradient. The thylakoid membrane is impermeable to protons, so protons cross back out almost exclusively via the channels provided by ATP synthases. These channels protrude like knobs on the external sur- face of the thylakoid membrane. As protons pass out of 196 Part III Energetics NADPH Photosystem II Photosystem Ib 6 -f complex Photon Stroma Thylakoid space H 2 O 2H + H + H + + NADP + NADP reductase Thylakoid membrane Antenna complex Plastoquinone Water-splitting enzyme Photon Proton gradient Plastocyanin Ferredoxin Fd pC Q H11002O 2 1 2 FIGURE 10.15 The photosynthetic electron transport system. When a photon of light strikes a pigment molecule in photosystem II, it excites an electron. This electron is coupled to a proton stripped from water by an enzyme and is passed along a chain of membrane-bound cytochrome electron carriers (red arrow). When water is split, oxygen is released from the cell, and the hydrogen ions remain in the thylakoid space. At the proton pump (b 6 -f complex), the energy supplied by the photon is used to transport a proton across the membrane into the thylakoid. The concentration of hydrogen ions within the thylakoid thus increases further. When photosystem I absorbs another photon of light, its pigment passes a second high-energy electron to a reduction complex, which generates NADPH. the thylakoid through the ATP syn- thase channel, ADP is phosphorylated to ATP and released into the stroma, the fluid matrix inside the chloroplast (figure 10.16). The stroma contains the enzymes that catalyze the reac- tions of carbon fixation. Photosystem I The reaction center of photosystem I, called P 700 , is a transmembrane complex consisting of at least 13 protein sub- units. Energy is fed to it by an antenna complex consisting of 130 chlorophyll a and accessory pigment molecules. Pho- tosystem I accepts an electron from plastocyanin into the hole created by the exit of a light-energized electron. This arriving electron has by no means lost all of its light-excited energy; al- most half remains. Thus, the absorp- tion of a photon of light energy by photosystem I boosts the electron leav- ing the reaction center to a very high energy level. Unlike photosystem II and the bacterial photosystem, photo- system I does not rely on quinones as electron acceptors. Instead, it passes electrons to an iron-sulfur protein called ferredoxin (Fd). Making NADPH Photosystem I passes electrons to ferredoxin on the stromal side of the membrane (outside the thylakoid). The reduced ferredoxin carries a very-high- potential electron. Two of them, from two molecules of reduced ferredoxin, are then donated to a molecule of NADP + to form NADPH. The reaction is cat- alyzed by the membrane-bound enzyme NADP reductase. Because the reaction occurs on the stromal side of the membrane and involves the uptake of a proton in forming NADPH, it contributes further to the proton gradient es- tablished during photosynthetic electron transport. Making More ATP The passage of an electron from water to NADPH in the noncyclic photophosphorylation described previously gen- erates one molecule of NADPH and slightly more than one molecule of ATP. However, as you will learn later in this chapter, building organic molecules takes more energy than that—it takes one-and-a-half ATP molecules per NADPH molecule to fix carbon. To produce the extra ATP, many plant species are capable of short-circuiting photosystem I, switching photosynthesis into a cyclic photophosphorylation mode, so that the light-excited electron leaving photosystem I is used to make ATP instead of NADPH. The energetic electron is simply passed back to the b 6 -f complex rather than passing on to NADP + . The b 6 -f complex pumps out a proton, adding to the proton gradient driving the chemios- motic synthesis of ATP. The relative proportions of cyclic and noncyclic photophosphorylation in these plants deter- mines the relative amounts of ATP and NADPH available for building organic molecules. The electrons that photosynthesis strips from water molecules provide the energy to form ATP and NADPH. The residual oxygen atoms of the water molecules combine to form oxygen gas. Chapter 10 Photosynthesis 197 Photosystem II b 6 -f complex ATP synthase H + H + H + H + H + H + H + H + Photon Stroma Thylakoid space ATPADP Q Chloroplast Plant cell H11002O 2 1 2 H 2 O 2 FIGURE 10.16 Chemiosmosis in a chloroplast. The b 6 -f complex embedded in the thylakoid membrane pumps protons into the interior of the thylakoid. ATP is produced on the outside surface of the membrane (stroma side), as protons diffuse back out of the thylakoid through ATP synthase channels. The Calvin Cycle Photosynthesis is a way of making organic molecules from carbon dioxide (CO 2 ). These organic molecules contain many C—H bonds and are highly reduced compared with CO 2 . To build organic molecules, cells use raw materials provided by the light reactions: 1. Energy. ATP (provided by cyclic and noncyclic pho- tophosphorylation) drives the endergonic reactions. 2. Reducing power. NADPH (provided by photosys- tem I) provides a source of hydrogens and the energetic electrons needed to bind them to carbon atoms. Much of the light energy captured in photosynthesis ends up invested in the energy-rich C—H bonds of sugars. Carbon Fixation The key step in the Calvin cycle—the event that makes the reduction of CO 2 possible—is the attachment of CO 2 to a very special organic molecule. Photosynthetic cells produce this molecule by reassembling the bonds of two intermedi- ates in glycolysis, fructose 6-phosphate and glyceraldehyde 3-phosphate, to form the energy-rich five-carbon sugar, ribulose1,5-bisphosphate (RuBP), and a four-carbon sugar. CO 2 binds to RuBP in the key process called carbon fixation, forming two three-carbon molecules of phospho- glycerate (PGA) (figure 10.17). The enzyme that carries out this reaction, ribulose bisphosphate carboxylase/oxygenase (usually abbreviated rubisco) is a very large four-subunit enzyme present in the chloroplast stroma. This enzyme works very sluggishly, processing only about three mole- cules of RuBP per second (a typical enzyme processes about 1000 substrate molecules per second). Because it works so slowly, many molecules of rubisco are needed. In a typical leaf, over 50% of all the protein is rubisco. It is thought to be the most abundant protein on earth. Discovering the Calvin Cycle Nearly 100 years ago, Blackman concluded that, because of its temperature dependence, photosynthesis might involve enzyme-catalyzed reactions. These reactions form a cycle of enzyme-catalyzed steps similar to the Krebs cycle. This cycle of reactions is called the Calvin cycle, after its dis- coverer, Melvin Calvin of the University of California, Berkeley. Because the cycle begins when CO 2 binds RuBP to form PGA, and PGA contains three carbon atoms, this process is also called C 3 photosynthesis. 198 Part III Energetics 10.4 Cells use the energy and reducing power captured by the light reactions to make organic molecules. H 2 C Rubisco 2 molecules of phosphoglycerate (PGA) CO O HC OH HC OH P H 2 C O Ribulose 1,5-bisphosphate (RuBP) P H 2 C C O – O O HC OH P CO 2 + H 2 O H 2 C C O – O O HC OH P FIGURE 10.17 The key step in the Calvin cycle. Melvin Calvin and his coworkers at the University of California worked out the first step of what later became known as the Calvin cycle. They exposed photosynthesizing algae to radioactive carbon dioxide ( 14 CO 2 ). By following the fate of a radioactive carbon atom, they found that it first binds to a molecule of ribulose 1,5-bisphosphate (RuBP), then immediately splits, forming two molecules of phosphoglycerate (PGA). One of these PGAs contains the radioactive carbon atom. In 1948, workers isolated the enzyme responsible for this remarkable carbon-fixing reaction: rubisco. The Energy Cycle The energy-capturing metabolisms of the chloroplasts studied in this chapter and the mitochondria studied in the previous chapter are intimately related. Photosynthesis uses the products of respiration as starting substrates, and respi- ration uses the products of photosynthesis as its starting substrates (figure 10.18). The Calvin cycle even uses part of the ancient glycolytic pathway, run in reverse, to produce glucose. And, the principal proteins involved in electron transport in plants are related to those in mitochondria, and in many cases are actually the same. Photosynthesis is but one aspect of plant biology, al- though it is an important one. In chapters 37 through 43, we will examine plants in more detail. We have treated photosynthesis here, in a section devoted to cell biology, because photosynthesis arose long before plants did, and all organisms depend directly or indirectly on photosynthesis for the energy that powers their lives. Chloroplasts put ATP and NADPH to work building carbon-based molecules, a process that essentially reverses the breakdown of such molecules that occurs in mitochondria. Taken together, chloroplasts and mitochondria carry out a cycle in which energy enters from the sun and leaves as heat and work. Chapter 10 Photosynthesis 199 ATP Photo- system I NADP + ADP ATP Calvin cycle ATP ATP NADPH Sunlight Glucose Chloroplast Mitochondrion Heat Pyruvate Krebs cycle Electron transport system NAD + NADH Photo- system II CO 2 H 2 O O 2 FIGURE 10.18 Chloroplasts and mitochondria: Completing an energy cycle. Water and oxygen gas cycle between chloroplasts and mitochondria within a plant cell, as do glucose and CO 2 . Cells with chloroplasts require an outside source of CO 2 and water and generate glucose and oxygen. Cells without chloroplasts, such as animal cells, require an outside source of glucose and oxygen and generate CO 2 and water. Reactions of the Calvin Cycle In a series of reactions (figure 10.19), three molecules of CO 2 are fixed by rubisco to produce six molecules of PGA (containing 6 × 3 = 18 carbon atoms in all, three from CO 2 and 15 from RuBP). The 18 carbon atoms then undergo a cycle of reactions that regenerates the three molecules of RuBP used in the initial step (containing 3 × 5 = 15 carbon atoms). This leaves one molecule of glyceraldehyde 3- phosphate (three carbon atoms) as the net gain. The net equation of the Calvin cycle is: 3 CO 2 + 9 ATP + 6 NADPH + water —→ glyceraldehyde 3-phosphate + 8 P i + 9 ADP + 6 NADP + With three full turns of the cycle, three molecules of carbon dioxide enter, a molecule of glyceraldehyde 3- phosphate (G3P) is produced, and three molecules of RuBP are regenerated (figure 10.20). We now know that light is required indirectly for differ- ent segments of the CO 2 reduction reactions. Five of the Calvin cycle enzymes—including rubisco—are light acti- vated; that is, they become functional or operate more effi- ciently in the presence of light. Light also promotes trans- port of three-carbon intermediates across chloroplast membranes that are required for Calvin cycle reactions. And finally, light promotes the influx of Mg ++ into the chloro- plast stroma, which further activates the enzyme rubisco. 200 Part III Energetics THE CALVIN CYCLE 123 3 3 CO 2 6 3-phosphoglycerate P The Calvin cycle begins when a carbon atom from a CO 2 molecule is added to a five-carbon molecule (the starting material). The resulting six-carbon molecule is unstable and immediately splits into three-carbon molecules. Then, through a series of reactions, energy from ATP and hydrogens from NADPH (the products of the light reactions) are added to the three- carbon molecules. The now-reduced three-carbon molecules either combine to make glucose or are used to make other molecules. Most of the reduced three-carbon molecules are used to regenerate the five-carbon starting material, thus completing the cycle. 5 Glyceraldehyde 3-phosphate 3 RuBP 3 P 6 6 6 Glyceraldehyde 3-phosphate Glucose NADPH 3-phosphoglycerate ATP ATP P 6 Glyceraldehyde 3-phosphate P 1 P (Starting material) RuBP (Starting material) FIGURE 10.19 How the Calvin cycle works. Output of the Calvin Cycle The glyceraldehyde 3-phosphate that is the product of the Calvin cycle is a three-carbon sugar that is a key intermedi- ate in glycolysis. Much of it is exported from the chloro- plast to the cytoplasm of the cell, where the reversal of sev- eral reactions in glycolysis allows it to be converted to fructose 6-phosphate and glucose 1-phosphate, and from that to sucrose, a major transport sugar in plants (sucrose, common table sugar, is a disaccharide made of fructose and glucose). In times of intensive photosynthesis, glyceraldehyde 3- phosphate levels in the stroma of the chloroplast rise. As a consequence, some glyceraldehyde 3-phosphate in the chloroplast is converted to glucose 1-phosphate, in an anal- ogous set of reactions to those done in the cytoplasm, by reversing several reactions similar to those of glycolysis. The glucose 1-phosphate is then combined into an insolu- ble polymer, forming long chains of starch stored as bulky starch grains in chloroplasts. Plants incorporate carbon dioxide into sugars by means of a cycle of reactions called the Calvin cycle, which is driven by the ATP and NADPH produced in the light reactions which are consumed as CO 2 is reduced to G3P. Chapter 10 Photosynthesis 201 3 molecules of 1 molecule of Glyceraldehyde 3-phosphate (3C) (G3P) Glucose and other sugars 3 molecules of 6 molecules of 6 molecules of 6 molecules of 5 molecules of Ribulose 1,5-bisphosphate (RuBP) (5C) 3-phosphoglycerate (3C) (PGA) Glyceraldehyde 3-phosphate (3C) Glyceraldehyde 3-phosphate (3C) (G3P) PGA kinase Rubisco Reforming RuBP Reverse of glycolysis G3P dehydrogenase Carbon fixation Stroma of chloroplast Carbon dioxide (CO 2 ) 6 NADPH 3 ATP 3 ADP 6 ATP 6 ADP 6 NADP + P i 6 P i 2 1,3-bisphosphoglycerate (3C) FIGURE 10.20 The Calvin cycle. For every three molecules of CO 2 that enter the cycle, one molecule of the three-carbon compound, glyceraldehyde 3- phosphate (G3P), is produced. Notice that the process requires energy stored in ATP and NADPH, which are generated by the light reactions. This process occurs in the stroma of the chloroplast. Photorespiration Evolution does not necessarily result in optimum solutions. Rather, it favors workable solutions that can be derived from others that already exist. Photosynthesis is no excep- tion. Rubisco, the enzyme that catalyzes the key carbon- fixing reaction of photosynthesis, provides a decidedly sub- optimal solution. This enzyme has a second enzyme activity that interferes with the Calvin cycle, oxidizing ribulose 1,5- bisphosphate. In this process, called photorespiration, O 2 is incorporated into ribulose 1,5-bisphosphate, which un- dergoes additional reactions that actually release CO 2 . Hence, photorespiration releases CO 2 —essentially undoing the Calvin cycle which reduces CO 2 to carbohydrate. The carboxylation and oxidation of ribulose 1,5-bispho- sphate are catalyzed at the same active site on rubisco, and compete with each other. Under normal conditions at 25°C, the rate of the carboxylation reaction is four times that of the oxidation reaction, meaning that 20% of photo- synthetically fixed carbon is lost to photorespiration. This loss rises substantially as temperature increases, because the rate of the oxidation reaction increases with temperature far faster than the carboxylation reaction rate. Plants that fix carbon using only C 3 photosynthesis (the Calvin cycle) are called C 3 plants. In C 3 photosynthesis, ribulose 1,5-bisphosphate is carboxylated to form a three- carbon compound via the activity of rubisco. Other plants use C 4 photosynthesis, in which phosphoenolpyruvate, or PEP, is carboxylated to form a four-carbon compound using the enzyme PEP carboxylase. This enzyme has no oxidation activity, and thus no photorespiration. Further- more, PEP carboxylase has a much greater affinity for CO 2 than does rubisco. In the C 4 pathway, the four-carbon compound undergoes further modification, only to be de- carboxylated. The CO 2 which is released is then captured by rubisco and drawn into the Calvin cycle. Because an or- ganic compound is donating the CO 2 , the effective concen- tration of CO 2 relative to O 2 is increased, and photorespi- ration is minimized. The loss of fixed carbon as a result of photorespiration is not trivial. C 3 plants lose between 25 and 50% of their photosynthetically fixed carbon in this way. The rate de- pends largely upon the temperature. In tropical climates, especially those in which the temperature is often above 28°C, the problem is severe, and it has a major impact on tropical agriculture. The C 4 Pathway Plants that adapted to these warmer environments have evolved two principal ways that use the C 4 pathway to deal with this problem. In one approach, plants conduct C 4 photosynthesis in the mesophyll cells and the Calvin cycle in the bundle sheath cells. This creates high local levels of CO 2 to favor the carboxylation reaction of ru- bisco. These plants are called C 4 plants and include corn, sugarcane, sorghum, and a number of other grasses. In the C 4 pathway, the three-carbon metabolite phospho- enolpyruvate is carboxylated to form the four-carbon molecule oxaloacetate, which is the first product of CO 2 fixation (figure 10.21). In C 4 plants, oxaloacetate is in turn converted into the intermediate malate, which is trans- ported to an adjacent bundle-sheath cell. Inside the bundle- sheath cell, malate is decarboxylated to produce pyruvate, releasing CO 2 . Because bundle-sheath cells are imperme- able to CO 2 , the CO 2 is retained within them in high con- centrations. Pyruvate returns to the mesophyll cell, where two of the high-energy bonds in an ATP molecule are split to convert the pyruvate back into phosphoenolpyru- vate, thus completing the cycle. The enzymes that carry out the Calvin cycle in a C 4 plant are located within the bundle-sheath cells, where the increased CO 2 concentration decreases photorespiration. Because each CO 2 molecule is transported into the bundle- sheath cells at a cost of two high-energy ATP bonds, and because six carbons must be fixed to form a molecule of glucose, 12 additional molecules of ATP are required to form a molecule of glucose. In C 4 photosynthesis, the ener- getic cost of forming glucose is almost twice that of C 3 photosynthesis: 30 molecules of ATP versus 18. Neverthe- less, C 4 photosynthesis is advantageous in a hot climate: photorespiration would otherwise remove more than half of the carbon fixed. 202 Part III Energetics CO 2 Phosphoenol- pyruvate (PEP) Oxaloacetate Pyruvate Malate Bundle- sheath cell Mesophyll cell PP i + AMP ATP P i + Calvin cycle Glucose CO 2 MalatePyruvate FIGURE 10.21 Carbon fixation in C 4 plants. This process is called the C 4 pathway because the starting material, oxaloacetate, is a molecule containing four carbons. The Crassulacean Acid Pathway A second strategy to decrease photorespiration in hot re- gions has been adopted by many succulent (water-storing) plants such as cacti, pineapples, and some members of about two dozen other plant groups. This mode of initial carbon fixation is called crassulacean acid metabolism (CAM), after the plant family Crassulaceae (the stonecrops or hens-and-chicks), in which it was first dis- covered. In these plants, the stomata (singular, stoma), specialized openings in the leaves of all plants through which CO 2 enters and water vapor is lost, open during the night and close during the day. This pattern of stomatal opening and closing is the reverse of that in most plants. CAM plants open stomata at night and initially fix CO 2 into organic compounds using the C 4 pathway. These or- ganic compounds accumulate throughout the night and are decarboxylated during the day to yield high levels of CO 2 . In the day, these high levels of CO 2 drive the Calvin cycle and minimize photorespiration. Like C 4 plants, CAM plants use both C 4 and C 3 pathways. They differ from C 4 plants in that they use the C 4 pathway at night and the C 3 pathway during the day within the same cells. In C 4 plants, the two pathways take place in different cells (figure 10.22). Photorespiration results in decreased yields of photosynthesis. C 4 and CAM plants circumvent this problem through modifications of leaf architecture and photosynthetic chemistry that locally increase CO 2 concentrations. C 4 plants isolate CO 2 production spatially, CAM plants temporally. Chapter 10 Photosynthesis 203 C 4 plants Mesophyll cell Bundle- sheath cell CO 2 Calvin cycle Glucose C 4 pathway CO 2 CAM plants Mesophyll cell Night Day CO 2 Calvin cycle C 4 pathway Glucose CO 2 FIGURE 10.22 A comparison of C 4 and CAM plants. Both C 4 and CAM plants utilize the C 4 and the C 3 pathways. In C 4 plants, the pathways are separated spatially: the C 4 pathway takes place in the mesophyll cells and the C 3 pathway in the bundle-sheath cells. In CAM plants, the two pathways are separated temporally: the C 4 pathway is utilized at night and the C 3 pathway during the day. 204 Part III Energetics Chapter 10 Summary Questions Media Resources 10.1 What is photosynthesis? ? Light is used by plants, algae, and some bacteria, in a process called photosynthesis, to convert atmospheric carbon (CO 2 ) into carbohydrate. 1. Where do the oxygen atoms in the O2 produced during photosynthesis come from? ? A series of simple experiments demonstrated that plants capture energy from light and use it to convert the carbon atoms of CO 2 and the hydrogen atoms of water into organic molecules. 2. How did van Helmont determine that plants do not obtain their food from the soil? 10.2 Learning about photosynthesis: An experimental journey. ? Light consists of energy packets called photons; the shorter the wavelength of light, the more its energy. When photons are absorbed by a pigment, electrons in the pigment are boosted to a higher energy level. ? Photosynthesis channels photon excitation energy into a single pigment molecule. In bacteria, that molecule then donates an electron to an electron transport chain, which drives a proton pump and ultimately returns the electron to the pigment. ? Plants employ two photosystems. Light is first absorbed by photosystem II and passed to photosystem I, driving a proton pump and bringing about the chemiosmotic synthesis of ATP. ? When the electron arrives at photosystem I, another photon of light is absorbed, and energized electrons are channeled to a primary electron acceptor, which reduces NADP + to NADPH. Use of NADPH rather than NADH allows plants and algae to keep the processes of photosynthesis and oxidative respiration separate from each other. 3. How is the energy of light captured by a pigment molecule? Why does light reflected by the pigment chlorophyll appear green? 4. What is the function of the reaction center chlorophyll? What is the function of the primary electron acceptor? 5. Explain how photosynthesis in the sulfur bacteria is a cyclic process. What is its energy yield in terms of ATP molecules synthesized per electron? 6. How do the two photosystems in plants and algae work? Which stage generates ATP and which generates NADPH? 10.3 Pigments capture energy from sunlight. ? The ATP and reducing power produced by the light reactions are used to fix carbon in a series of reactions called the Calvin cycle. ? RuBP carboxylase, the enzyme that fixes carbon in the Calvin cycle, also carries out an oxidative reaction that uses the products of photosynthesis, a process called photorespiration. ? Many tropical plants inhibit photorespiration by expending ATP to increase the intracellular concentration of CO 2 . This process, called the C 4 pathway, nearly doubles the energetic cost of synthesizing glucose. 7. In a C 3 plant, where do the light reactions occur? Where does the Calvin cycle occur? 8. What is photorespiration? What advantage do C 4 plants have over C 3 plants with respect to photorespiration? What disadvantage do C 4 plants have that limits their distribution primarily to warm regions of the earth? 10.4 Cells use the energy and reducing power captured by the light reactions to make organic molecules. http://www.mhhe.com/raven6e http://www.biocourse.com ? Art Activity: Chloroplast Structure ? Chloroplast ? Energy Conversion ? Photosynthesis ? Light Dependent Reactions ? Light Independent Reactions ? Light and Pigmentation ? The Calvin Cycle ? Photorespiration ? Art Activity: Electromagnetic Spectrum