Chapter 19
Oxidative phosphorylation
and photophosphorylation
( 氧化磷酸化和光合磷酸化 )
Generation of ATP by using a across-
membrane proton gradient,which is
generated from electron flowing
through a chain of carriers.
1,ATP is synthesized using the same
strategy in oxidative phosphorylation
and photophosphorylation
? Oxidative phosphorylation is the process in which ATP is
generated as a result of electron flow from NADH or
FADH2 to O2 via a series of membrane-bound electron
carriers,called the respiratory chain (reducing O2 to H2O at
the end).
? Photophosphorylation is the process in which ATP (and
NADPH) is synthesized as a result of electron flow from
H2O to NADP+ via a series of membrane-bound electron
carriers (oxidizing H2O to O2 at the beginning).
? Oxidative phosphorylation and photophosphorylation are
mechanistically similar:
– Both involve the flow of electrons through a chain of membrane-
bound carriers.
– The energy released from,downhill” electron flow is first used for
“uphill” pumping of protons to produce a proton gradient ( thus
a transmembrane electrochemical potential) across a biomembrane.
– ATP is then synthesized by a,downhill” transmembrane flow of
protons through a specific protein machinery (ATP synthase).
Oxidative
Phosphorylation
(0n inner membrane
of mitochondria)
Photophosphorylation
(on thylakoid of chloroplasts)
2,Electrons collected in NADH and
FADH2 are released and transported
to O2 via the respiratory chain
? The chain is located on the convoluted inner
membrane (cristae) of mitochondria in eukaryotic
cells (revealed by Eugene Kennedy and Albert
Lehninger in 1948) or on the plasma membrane in
prokaryotic cells.
? A 1.14-volt potential difference (?E`0) between
NADH (-0.320 V) and O2 (0.816 V) drives electron
flow through the chain.
? The respiratory chain consists of four large multi-
protein complexes (I,II,III,and IV; three being
proton pumps) and two mobile electron carriers,
ubiquinone (Q or coenzyme Q,and cytochrome c.
? Prosthetic groups acting in the proteins of
respiratory chain include flavins (FMN,FAD),
hemes (heme A,iron protoporphyrin IX,heme C),
iron-sulfur clusters (2Fe-2S,4Fe-4S),and copper.
Cristae (the convoluted
inner membrane of
mitochondria) is
where the respiratory
chain is located.
Four multi-protein
Complexes (I,II,
III,and IV)
Two mobile
Electron carriers
I II
III
IV
FMN can accept one electron
( and FMNH2 can donate one
electron) to form a semiquinone
radical intermediate.
Heme groups
of cytochrome
proteins
Heme groups
Of cytochromes
Different types of
iron-sulfur centers
?Iron atoms cycle between Fe2+
(reduced) and Fe3+(oxidized).
?At least eight Fe-S proteins
act in the respiratory chain.
4Fe-4S2Fe-2S
A ferredoxin
Reduced cytochromes
has three absorption
bands in the visible
wavelengthsCyt a,~600 nm;
Cyt b,~560 nm;
Cyt c,~550 nm
3,NADH enters the chain at NADH
dehydrogenase (complex I)
? Also named as NADH:ubiquinone oxidoreductase or
NADH-Q reductase.
? A,L” shaped 850 kD multimeric protein complex of 42
different subunits (larger than a ribosome!).
? Polypeptides encoded by both genomes.
? FMN,Fe-S centers act as prosthetic groups.
? Exergonic electron transferring is coupled to endergonic
proton pumping ( with 4 H+ pumped from the matrix side to
intermembrane space per electron pair transferred),with
mechanism unknown.
? Final electron acceptor is ubiquinone (coenzyme Q).
NADH Dehydrogenase
(complex I)
Amytal,rotenone,piericidin A inhibit
electron flow from the Fe-S centers of
complex I to ubiquinone
?Fat soluble
benzoquinone with a
very long isoprenoid side
chain; can accept one or
two electrons,forming
radical semiquinone or
ubiquinol (QH2); QH2
diffuses to the next
complex (III); the only
electron carrier not
bound to a protein.
Ubiquinone is a mobile
electron/proton carrier
4,FADH2 of flavoproteins also
transfer their electrons to ubiquinone
? Flavoproteins like succinate dehydrogenase
(complex II),fatty acyl-CoA dehydrogenase,and
glycerol 3-phosphate dehydrogenase are associated
to the inner membrane of mitochondria and transfers
their electrons collected on FADH2 to Q to form
QH2.
? The energy released from these electron transferring
is not high enough to promote proton pumping.
Ubiquinone (Q)
accepts electrons
from both NADH
and FADH2 in the
respiratory chain
5,Electrons of QH2 is transferred to
cytochrome c via ubiquinone:cytochrome
c oxidoreductase (complex III)
? Also called cytochrome c reductase or cytochrome
bc1 complex.
? A 250 kD multiprotein complex of 11 subunits.
? Complete 3-D structure was determined in 1997!
? The functional core consists of three subunits,
cytochrome b (with two hemes,bH and bL); an Fe-S
protein; and cytochrome c1 (with the heme group
covalently bound to protein via two thioether bonds).
? Two-electron carrier QH2 passes one electron to the
one-electron carrier Fe-S center,then to the heme C
group in Cyt c1,and finally to the heme C group of
Cyt c; the other electron to bL,bH,and finally to an
Q or Q.- via a so-called,Q cycle”.
? Cytochrome c,a soluble protein located in the
intermembrane space,will move to complex IV.
Cytochrome bc1 complex
(complex III)
The three
core subunits
Electron path
in complex III
The Q
cycle
The Q
cycle
The 1st QH2 The 2nd QH2
6,Electrons of Cyt c are transferred
to O2 on cytochrome oxidase
(complex IV)
? A 204 kD 13-subunit protein complex,with structure
determined in 1996.
? Three subunits are probably critical to the function.
? Three copper ions (2 CuA,1CuB),two heme A groups (a
and a3) act as electron carriers in complex IV.
? Four electrons need to be transferred to reduce one O2
molecule at the,Fe-CuB center” (via peroxy intermediates)
of complex IV to form 2 H2O.
? Four,substrate” protons are consumed from
the N side for every four electrons transferred
to one O2 molecule.
? One proton is pumped out from the N to P side
for each electron to be transferred (thus four
protons for four electrons) by an yet defined
mechanism.
The three critical subunits of
cytochrome oxidase (complex IV)
2CuA
CuB
Heme a
Heme a3 CuA
CuA
The electron
path in
complex IV
A proposed reaction cycle for the four-electron
reduction of O2 by cytochrome oxidase (at the
Heme a3-CuB center)
7,A proton gradient across the inner
membrane of mitochondria is generated
using the electron motive force
? An estimate of 10 protons are pumped for oxidizing one
NADH and 6 for one FADH2 accompanying the electron
flow through complexes I,III and IV.
? Conformational changes induced by electron transferring is
believed to be coupled to proton pumping (however,the
actual mechanisms is yet revealed!).
? In actively respiring mitochondria,the measured ?pH is
about 0.75 and difference in electrical potential (??) is
about 0.15-0.2 V.
? The energy stored in such an H+ gradient can
be used to synthesize ATP or to do other work.
A H+ gradient across the inner membrane
of mitochondria (or plasma membrane of
bacteria) is generated by,uphill” H+
pumping using energy released by the
“downhill” flow of electrons.
8,The order of the many electron
carriers on the respiratory chain have
been elucidated via various studies
? Measurement of the standard reduction potential (?E`0)),
Electrons tend to transfer from low ?E`0 carriers to high
?E`0 carriers (but may deviate from this in real cells).
? Oxidation kinetics studies,Full reduction followed by
sudden O2 introduction; earlier oxidation,closer to the end
of the respiratory chain; using rapid and sensitive
spectrophotometric techniques to follow the oxidation of the
cytochromes,which have different wavelength of maximal
absorption).
? Effects of various specific inhibitors,those
before the blocked step should be reduced and
those after be oxidized.
? Isolation and characterization of each of the
multiprotein complexes,specific electron
donors and acceptors can be determined for
portions of the chain.
Electron carriers may have an order of increasing E`0
Various inhibitors generate various
patterns of reduced/oxidized carriers
Reduced Oxidized
Reduced Oxidized
Reduced
9,Electron transfer to O2 was found
to be coupled to ATP synthesis from
ADP + Pi in isolated mitochondria
? ATP would not be synthesized when only ADP and Pi are
added in isolated mitochondria suspensions.
? O2 consumption,an indication of electron flow,was
detected when a reductant (e.g.,succinate) is added,
accompanied by an increase of ATP synthesis.
? Both O2 consumption and ATP synthesis were suppressed
when inhibitors of respiratory chain (e.g.,cyanide,CO,or
antimycin A) was added.
? ATP synthesis depends on the occurrence of electron flow in
mitochondria.
? O2 consumption (thus electron flow) was
neither observed if ADP was not added to the
suspension,although a reductant is provided!
? The O2 consumption was also not observed in the
presence of inhibitors of ATP synthase (e.g.,
oligomycin or venturicidin).
? Electron flow also depends on ATP synthesis!
Electron transfer was found to be
obligatorily coupled to ATP Synthesis
in isolated mitochondria suspensions,
neither occurs without the other.
10,It was widely believed that ATP
synthesis occurs by chemical coupling
? High energy intermediates similar to 1,3-
bisphophoglycerate (which is formed in the glycolytic
pathway and transfers an phosphoryl group to ADP to form
ATP) was once proposed to be produced first from the
electron flows on both the mitochondrial and chloroplast
membranes.
? Phophorylated protein intermediates (as those formed in the
action of phosphoglycerate mutase and
phosphoglucomutase) were also hypothesized.
? But neither were ever revealed despite intense efforts by
many investigators over many years.
11,The chemiosmotic model was
proposed to explain the coupling of
electron flow and ATP synthesis
? First proposed in 1961 by Peter Mitchell (a British).
? Energy released from electron transferring is hypothesized
to be first used to pump protons from the mitochondrial
matrix to the intermembrane space (or from stroma to
thylakoid lumen in chloroplasts),thus generating a proton
gradient across the inner membrane; such a proton-motive
force then drives ATP synthesis by moving protons back
into the matrix via the ATP synthase.
? The model was initially opposed by virtually all researchers
working in oxidative phosphorylation and photosynthesis.
? The chemiosmotic theory explains the dependence of
electron flow on ATP synthesis in mitochondria,the
electron-motive force does not provide enough energy for
pumping the protons when the cost is too high due to the
build-up of the proton gradient without being consumed by
ATP synthesis.
The chemiosmotic
model of Mitchell
12,The supporting evidences for the
chemiosmotic coupling were collected
? A closed membrane system is essential for ATP synthesis
but not for the electron flow (tested with detergent or
physical shearing).
? Hydrophobic weak acids (DNP and FCCP) and ionophores
(valinomycin) were found to be able to uncouple ATP
synthesis from electron transferring.
? The transmembrane proton pumping has been
experimentally detected,pH in the intermembrane space
was found to decrease when electron flow occurs (more
protons are pumped when NADH,rather than succinate,is
utilized as reductant).
? An artificially imposed electrochemical gradient across the
chloroplast thylakoid membrane and inner mitochondrial
membrane alone (both were performed using sub-organelle
vesicles) were found to drive ATP synthesis (with the ATP
synthase present).
? The across-membrane proton gradient was thus finally
accepted as the driving force for ATP synthesis,the
chemiosmotic model was accepted as a theory!
? The chemiosmotic theory unified the apparently disparate
energy transduction processes as oxidative phosphorylation,
photophosphorylation,active transport across membrane
and the motion of bacterial flagella.
ATP synthesized
DNP,a hydrophobic weak acid,uncouples
ATP synthesis from electron flow
DNP and CCCP
are able to
dissipate the
proton gradient
The artificially
imposed proton
gradient alone
(in the absence of
an oxidizable
substrate) was
found to be able to
drive ATP synthesis!