第 32章
生物固氮
自然界中的氮循环（ The Nitrogen Cycle ）
The total amount of nitrogen fixed annually in the biosphere
exceeds 1011 kg,
固氮生物
固氮
微生物
共生型固
氮微生物
自生型固
氮微生物
豆科植物的根瘤菌
非豆科植物 (木麻黄属 )
的根瘤菌
厌氧的巴氏梭菌,需氧固氮菌,
厌氧和光合自养的蓝藻,需氧
和光合自养的细菌
Cyanobacteria and Rhizobia can fix N2
into ammonia
Rhizobia exist in
nodules of
leguminous plants
固氮反应
?N2 + 8H+ +8e- + 16ATP + 16H2O ?
? 2NH3 + H2 + 16ADP + 16Pi
The reduction of N2 to NH3 is thermodynamically
favorable,
? N2 + 3H2 ? 2NH3 ?G`o=-33.5kJ/mol
But kinetically unfavorable,the bond energy for the
triple bond in N2 is 942 kJ/mol,
固氮酶的结构
? 固氮酶复合物包括固氮酶和固氮酶还原酶。
? 固氮酶（ dinitrogenase ）,
两种肽链组成的四聚体,α2β2,活性中心在 α链,
含有多个 4Fe-4S centers 和两个 Mo-Fe clusters,
称铁钼蛋白。
■ 固氮酶还原酶（ dinitrogenase reductase ）：由
两个相同的亚基组成，每个亚基含有一个 Fe4-S4
redox center。 又称铁蛋白。
? The nitrogenase complex is highly conserved among
different diazotrophs,
The nitrogenase complex consists of dinitrogenase and dinitrogenase redutase
dinitrogenase (tetramer)
or FeMo protein dinitrogenase
reductase (dimer)
or Fe protein
dinitrogenase
reductase (dimer)
or Fe protein
e-
4Fe-4S
4Fe-4S
(P-cluster)
Fe-Mo cofactor
4Fe-4S
(P-cluster) 4Fe-4S
ADP
ADP
Fe-Mo cofactor?
ADP
ADP
Molybdenum
N2 is believed to be reduced at
the Fe-Mo cofactor
Fe
Fe Fe
Fe
Fe Fe
Fe
S
S
S
S
S
S
S
S S
Mo
还原一分子 N2需要 8个电子
? 在固氮酶复合物上，消耗 8个电子，将一分
子 N2还原为 2分子 NH3,同时 2个 H+被还原
为 H2,
? 电子来自于铁硫氧还蛋白 (reduced
ferredoxin,from photophosphorylation) 或
黄素蛋白 (reduced flavodoxin,from
oxidative phosphorylation)。
? 每传递一个电子需消化 2个 ATP,所释放的
能量用于改变固氮酶还原酶的构象，促进
电子的传递。
? 固氮酶的作用机制还不清楚，如固氮的同
时产生氢气的问题？
Electrons are transferred
to N2 bound in the
active site of dinitrogenase
via ferredoxin/flavodoxin
and dinitrogenase reductase
固氮酶复合物对氧非常敏感
?Some diazotrophs exist only anaerobically,
?Some cyanobacterial cells develop thick
walls to prevent O2 from entering,
?The bacteria in root nodules are isolated
from O2 by being bathed in a solution of the
oxygen-binding protein leghemoglobin,
Leghemoglobin,produced in legume plants,
has a high affinity to O2 and protects the
nitrogenase complex in rhizobia
固氮酶产生的氨转化为谷氨酰胺和谷氨酸
? First NH4+ is added to the side chain of glutamate to
form glutamine in an ATP-dependent reaction
catalyzed by glutamine synthetase,
? Then the side chain amino group of Gln is further
transferred to a-ketoglutarate to form Glu in a
reaction catalyzed by glutamate synthase,an enzyme
only present in bacteria and plants,not in animals,
? The amide group of Gln is a source of nitrogen in the
synthesis of a variety of compounds,such as
carbamoyl phosphate,Trp,His,glucosamine-6-P,
CTP,and AMP,
? The amino groups of most other amino acids are
derived from glutamate via transamination,
谷氨酰胺合成酶的调节
? 原核生物谷氨酰胺合成酶由 12个亚基组
装成两个六边形的环状结构,
? 该酶至少有 8个别构抑制剂,
? 该酶还受可逆共价调节,每个亚基的 Tyr307
侧链可被可逆腺苷酸化修饰。
The 20 amino acids are
synthesized from
intermediates of
glycolysis,the citric acid
cycle,or pentose
phosphate pathway
氨基酸可转化为许多生理活性物质
? 卟啉环的合成；
? 肌酸的合成；
? 谷胱甘肽的合成；
? 5－羟色氨和组氨等生理活性物质的合成 ；
? NO的合成,
? 嘌呤环和嘧啶环的合成。
Newly fixed nitrogen
in the form of NH4+
is first incorporated
into glutamate to
form glutamine
N A D P H + H
+
NADP
+
Then the side chain amino group of Gln is further
transferred to a-ketoglutarate to form Glu in a reaction
catalyzed by glutamate synthase
Gln
synthetase Glu
synthase
All organisms can convert
ammonia (NH3) to organic
nitrogen compounds
Glutamate
dehydrogenase
Asparagine
synthetase
Carbamoyl
Phosphate
Synthetase
The bacterial glutamine synthetase
has 12 subunits arranged as two
rings of hexamers
Active
sites
Tyr397
(adenylylation site)
The glutamine
synthetase is
accumulatively
inhibited by at least 8
allosteric effectors,
mostly end products
of glutamine
metabolism
Bifunctional adenylyltransferase (AT)催化谷氨酰胺合成酶的
腺苷酸化和脱腺苷酸,UMP和 PII是该酶的调节因子。
Biosynthesis of
protoporphyrin IX,
the porphyrin of
hemoglobin and
myoglobin,
甘氨酸和琥珀酰辅
酶是卟啉环的前体
Creatine is derived
from Gly,Arg,
and Met,
Glutathione (the
“redox buffer”)
is made from
Glu,Cys,and Gly
Many neurotransmitters are derived
from amino acids like Tyr,Glu,Trp,
Tyr Glu Trp
decarboxylation
decarboxylation
monooxygenation
monooxygenation
decarboxylation
monooxygenation
methylation
Nitric oxide (NO) is made from Arg and O2 in a reaction
catalyzed by nitric oxide synthase (NOS),a
monooxygenase containing five coenzymes,NADP,FMN,
FAD,tetrahydrobiopterin,and a Fe 3+-heme,
(Robert F,Furchgott,Louis J,Ignarro,and Ferid Murad were
awarded the 1998 Nobel Prize in Medicine for revealing the
role of NO in the cardiovascular（ 心血管 ） systems)
monooxygenation
nitric oxide
Synthase nitric oxide Synthase
origins of the ring
atoms of purines
(numbered is the
order of addition)
Ribose 5-P
1
2 3
4
5 6
7
生物固氮
自然界中的氮循环（ The Nitrogen Cycle ）
The total amount of nitrogen fixed annually in the biosphere
exceeds 1011 kg,
固氮生物
固氮
微生物
共生型固
氮微生物
自生型固
氮微生物
豆科植物的根瘤菌
非豆科植物 (木麻黄属 )
的根瘤菌
厌氧的巴氏梭菌,需氧固氮菌,
厌氧和光合自养的蓝藻,需氧
和光合自养的细菌
Cyanobacteria and Rhizobia can fix N2
into ammonia
Rhizobia exist in
nodules of
leguminous plants
固氮反应
?N2 + 8H+ +8e- + 16ATP + 16H2O ?
? 2NH3 + H2 + 16ADP + 16Pi
The reduction of N2 to NH3 is thermodynamically
favorable,
? N2 + 3H2 ? 2NH3 ?G`o=-33.5kJ/mol
But kinetically unfavorable,the bond energy for the
triple bond in N2 is 942 kJ/mol,
固氮酶的结构
? 固氮酶复合物包括固氮酶和固氮酶还原酶。
? 固氮酶（ dinitrogenase ）,
两种肽链组成的四聚体,α2β2,活性中心在 α链,
含有多个 4Fe-4S centers 和两个 Mo-Fe clusters,
称铁钼蛋白。
■ 固氮酶还原酶（ dinitrogenase reductase ）：由
两个相同的亚基组成，每个亚基含有一个 Fe4-S4
redox center。 又称铁蛋白。
? The nitrogenase complex is highly conserved among
different diazotrophs,
The nitrogenase complex consists of dinitrogenase and dinitrogenase redutase
dinitrogenase (tetramer)
or FeMo protein dinitrogenase
reductase (dimer)
or Fe protein
dinitrogenase
reductase (dimer)
or Fe protein
e-
4Fe-4S
4Fe-4S
(P-cluster)
Fe-Mo cofactor
4Fe-4S
(P-cluster) 4Fe-4S
ADP
ADP
Fe-Mo cofactor?
ADP
ADP
Molybdenum
N2 is believed to be reduced at
the Fe-Mo cofactor
Fe
Fe Fe
Fe
Fe Fe
Fe
S
S
S
S
S
S
S
S S
Mo
还原一分子 N2需要 8个电子
? 在固氮酶复合物上，消耗 8个电子，将一分
子 N2还原为 2分子 NH3,同时 2个 H+被还原
为 H2,
? 电子来自于铁硫氧还蛋白 (reduced
ferredoxin,from photophosphorylation) 或
黄素蛋白 (reduced flavodoxin,from
oxidative phosphorylation)。
? 每传递一个电子需消化 2个 ATP,所释放的
能量用于改变固氮酶还原酶的构象，促进
电子的传递。
? 固氮酶的作用机制还不清楚，如固氮的同
时产生氢气的问题？
Electrons are transferred
to N2 bound in the
active site of dinitrogenase
via ferredoxin/flavodoxin
and dinitrogenase reductase
固氮酶复合物对氧非常敏感
?Some diazotrophs exist only anaerobically,
?Some cyanobacterial cells develop thick
walls to prevent O2 from entering,
?The bacteria in root nodules are isolated
from O2 by being bathed in a solution of the
oxygen-binding protein leghemoglobin,
Leghemoglobin,produced in legume plants,
has a high affinity to O2 and protects the
nitrogenase complex in rhizobia
固氮酶产生的氨转化为谷氨酰胺和谷氨酸
? First NH4+ is added to the side chain of glutamate to
form glutamine in an ATP-dependent reaction
catalyzed by glutamine synthetase,
? Then the side chain amino group of Gln is further
transferred to a-ketoglutarate to form Glu in a
reaction catalyzed by glutamate synthase,an enzyme
only present in bacteria and plants,not in animals,
? The amide group of Gln is a source of nitrogen in the
synthesis of a variety of compounds,such as
carbamoyl phosphate,Trp,His,glucosamine-6-P,
CTP,and AMP,
? The amino groups of most other amino acids are
derived from glutamate via transamination,
谷氨酰胺合成酶的调节
? 原核生物谷氨酰胺合成酶由 12个亚基组
装成两个六边形的环状结构,
? 该酶至少有 8个别构抑制剂,
? 该酶还受可逆共价调节,每个亚基的 Tyr307
侧链可被可逆腺苷酸化修饰。
The 20 amino acids are
synthesized from
intermediates of
glycolysis,the citric acid
cycle,or pentose
phosphate pathway
氨基酸可转化为许多生理活性物质
? 卟啉环的合成；
? 肌酸的合成；
? 谷胱甘肽的合成；
? 5－羟色氨和组氨等生理活性物质的合成 ；
? NO的合成,
? 嘌呤环和嘧啶环的合成。
Newly fixed nitrogen
in the form of NH4+
is first incorporated
into glutamate to
form glutamine
N A D P H + H
+
NADP
+
Then the side chain amino group of Gln is further
transferred to a-ketoglutarate to form Glu in a reaction
catalyzed by glutamate synthase
Gln
synthetase Glu
synthase
All organisms can convert
ammonia (NH3) to organic
nitrogen compounds
Glutamate
dehydrogenase
Asparagine
synthetase
Carbamoyl
Phosphate
Synthetase
The bacterial glutamine synthetase
has 12 subunits arranged as two
rings of hexamers
Active
sites
Tyr397
(adenylylation site)
The glutamine
synthetase is
accumulatively
inhibited by at least 8
allosteric effectors,
mostly end products
of glutamine
metabolism
Bifunctional adenylyltransferase (AT)催化谷氨酰胺合成酶的
腺苷酸化和脱腺苷酸,UMP和 PII是该酶的调节因子。
Biosynthesis of
protoporphyrin IX,
the porphyrin of
hemoglobin and
myoglobin,
甘氨酸和琥珀酰辅
酶是卟啉环的前体
Creatine is derived
from Gly,Arg,
and Met,
Glutathione (the
“redox buffer”)
is made from
Glu,Cys,and Gly
Many neurotransmitters are derived
from amino acids like Tyr,Glu,Trp,
Tyr Glu Trp
decarboxylation
decarboxylation
monooxygenation
monooxygenation
decarboxylation
monooxygenation
methylation
Nitric oxide (NO) is made from Arg and O2 in a reaction
catalyzed by nitric oxide synthase (NOS),a
monooxygenase containing five coenzymes,NADP,FMN,
FAD,tetrahydrobiopterin,and a Fe 3+-heme,
(Robert F,Furchgott,Louis J,Ignarro,and Ferid Murad were
awarded the 1998 Nobel Prize in Medicine for revealing the
role of NO in the cardiovascular（ 心血管 ） systems)
monooxygenation
nitric oxide
Synthase nitric oxide Synthase
origins of the ring
atoms of purines
(numbered is the
order of addition)
Ribose 5-P
1
2 3
4
5 6
7
