BCM261: Theme 3: Metabolism of Amino
Acids
The Nitrogen Cycle
Importance of Nitrogen in Biochemistry
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Biochemistry of Molecular Nitrogen
Atmosphere is 80% N2 but non-useful form
- N2 chemically inert
- N2 + 3 H2 2 NH
Even though G′° = –33.5 kJ/mol… breaking a triple bond has high activation energy.
- can be accomplished using nonbiological processes:
o N2 and O2 NO via lightning
o N2 and H2 NH3 via the industrial Haber process - requires T>400°C, P>200 atm
Some Organisms Can “Fix” N2 to Useful Forms
- Most are single-celled prokaryotes (archaea).
- Some live in symbiosis with plants – plants provide ATP from ETC (e.g.,
proteobacteria with legumes such as peanuts, beans)
- A few live in symbiosis with animals. (e.g., spirochaete with termites)
- They have enzymes that overcome the high activation energy by binding and
hydrolyzing ATP
Broader Impact of Understanding the Nitrogen Fixation
Industrial synthesis of NH3 via the Haber process is one of mankind’s most significant chemical processes.
- made chemical fertilizer possible!
- yields over 100 million tons of fertilizer annually
- sustains life of over one-third of human population on Earth
- consumes non-renewable energy (1–2% of total annual energy)
Mimicking biological nitrogen fixation (biomimetic nitrogen fixation) may yield significant energy
savings, or allow use of renewable energy sources
,The Nitrogen Cycle
Chemical transformations maintain a balance between N2 and biologically useful forms of nitrogen.
1. Fixation. Bacteria reduce N2 to NH3 /NH4 + .
2. Nitrification. Bacteria oxidize ammonia into nitrite (NO2 – ) and nitrate (NO3 – ).
3. Assimilation. Plants and microorganisms reduce NO2 – and NO3 – to NH3 via nitrite reductases and nitrate reductases.
- NH3 is incorporated into amino acids, and so on.
- Organisms die, returning NH3 to soil.
- Nitrifying bacteria again convert NH3 to nitrite and nitrate.
4. Denitrification. Nitrate is reduced to N2 under anaerobic conditions.
- NO3 – is the ultimate electron acceptor instead of O2.
The Nitrogen Web
Two Important Enzymes in Nitrogen Assimilation
1. Nitrate reductase NO3 – + 2 e– NO2 –
- large, soluble protein
- contains novel Mo cofactor
- e – from NADH (e donor)
2. Nitrite reductase NO2 – + 6 e– NH4 +
- found in chloroplasts in plants: e – comes from ferredoxin
- in non-photosynthetic microbes: e – comes from NADPH
,Nitrogen Fixation Is Carried Out by the Nitrogenase Complex
N2 + 3 H2 = 2 NH3
- exergonic (G = –33.5 kJ/mol) but very slow due to the triple bond’s high activation energy
The nitrogenase complex uses ATP to overcome the activation energy – from plant
Passes electrons to N2 and catalyzes a step-wise reduction of N2 to NH3
- About 16 ATP molecules are consumed per one N2
Enzymes and Cofactors in the Nitrogenase Complex
- Source of e – varies between organisms - often pyruvate ferredoxin
- ATP hydrolysis and ATP binding help overcome the high activation energy.
- Has regions homologous to GTP-binding proteins used in signaling
- Has novel FeMo cofactor (or V in some organisms)
- The holoenzyme consists of two identical dinitrogenase reductase molecules (green),
each with a 4Fe-4S redox center and binding sites for two ATP, and two identical
dinitrogenase heterodimers (purple and blue), each with a P cluster (Fe-S center) and
an FeMo cofactor. In this structure, ADP is bound in the ATP site, to make the
crystal more stable.
, The Fe-Mo Cofactor in the Dinitrogenase Subunit
Consist of
- 7 Fe atoms
- 9 S atoms
- 1 Mo atom
- 1 bound homocitrate
The nitrogen binds to the center of the Mo-FeS cage and is coordinated to the molybdenum atom.
Electrons are passed to the molybdenum-bound nitrogen via the iron-sulfur complex.
Nitrogen Fixation by the Nitrogenase Complex
1. Pyruvate passes e – to ferredoxin or flavodoxin.
2. Ferredoxin or flavodoxin pass e – to dinitrogenase reductase.
3. The reductase passes e – to dinitrogenase.
4. Dinitrogenase passes e – to nitrogen (or to protons) to make NH3.
5. Formation of H2 appears an obligatory side reaction
Redox Reactions in Dinitrogenase
- The net reaction of the nitrogenase complex:
- Dinitrogenase reductase catalyzes:
o transfer of 8 e – to dinitrogenase
o hydrolysis of ATP with release of protons
- Dinitrogenase catalyzes:
o transfer of 6 e – to nitrogen: formation of NH3
o transfer of 2 e – to protons: formation of H2
- Mechanism of dinitrogenase is poorly understood:
o extremely complex redox reaction that involves several metal
atoms as cofactors and/or electron transporters
o two plausible mechanisms that involve the Fe-Mo cofactor binding
directly to N
Nitrogen-Fixing Bacteria in Root Nodules of Legumes
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Acids
The Nitrogen Cycle
Importance of Nitrogen in Biochemistry
-
-
-
o
o
o
o
o
Biochemistry of Molecular Nitrogen
Atmosphere is 80% N2 but non-useful form
- N2 chemically inert
- N2 + 3 H2 2 NH
Even though G′° = –33.5 kJ/mol… breaking a triple bond has high activation energy.
- can be accomplished using nonbiological processes:
o N2 and O2 NO via lightning
o N2 and H2 NH3 via the industrial Haber process - requires T>400°C, P>200 atm
Some Organisms Can “Fix” N2 to Useful Forms
- Most are single-celled prokaryotes (archaea).
- Some live in symbiosis with plants – plants provide ATP from ETC (e.g.,
proteobacteria with legumes such as peanuts, beans)
- A few live in symbiosis with animals. (e.g., spirochaete with termites)
- They have enzymes that overcome the high activation energy by binding and
hydrolyzing ATP
Broader Impact of Understanding the Nitrogen Fixation
Industrial synthesis of NH3 via the Haber process is one of mankind’s most significant chemical processes.
- made chemical fertilizer possible!
- yields over 100 million tons of fertilizer annually
- sustains life of over one-third of human population on Earth
- consumes non-renewable energy (1–2% of total annual energy)
Mimicking biological nitrogen fixation (biomimetic nitrogen fixation) may yield significant energy
savings, or allow use of renewable energy sources
,The Nitrogen Cycle
Chemical transformations maintain a balance between N2 and biologically useful forms of nitrogen.
1. Fixation. Bacteria reduce N2 to NH3 /NH4 + .
2. Nitrification. Bacteria oxidize ammonia into nitrite (NO2 – ) and nitrate (NO3 – ).
3. Assimilation. Plants and microorganisms reduce NO2 – and NO3 – to NH3 via nitrite reductases and nitrate reductases.
- NH3 is incorporated into amino acids, and so on.
- Organisms die, returning NH3 to soil.
- Nitrifying bacteria again convert NH3 to nitrite and nitrate.
4. Denitrification. Nitrate is reduced to N2 under anaerobic conditions.
- NO3 – is the ultimate electron acceptor instead of O2.
The Nitrogen Web
Two Important Enzymes in Nitrogen Assimilation
1. Nitrate reductase NO3 – + 2 e– NO2 –
- large, soluble protein
- contains novel Mo cofactor
- e – from NADH (e donor)
2. Nitrite reductase NO2 – + 6 e– NH4 +
- found in chloroplasts in plants: e – comes from ferredoxin
- in non-photosynthetic microbes: e – comes from NADPH
,Nitrogen Fixation Is Carried Out by the Nitrogenase Complex
N2 + 3 H2 = 2 NH3
- exergonic (G = –33.5 kJ/mol) but very slow due to the triple bond’s high activation energy
The nitrogenase complex uses ATP to overcome the activation energy – from plant
Passes electrons to N2 and catalyzes a step-wise reduction of N2 to NH3
- About 16 ATP molecules are consumed per one N2
Enzymes and Cofactors in the Nitrogenase Complex
- Source of e – varies between organisms - often pyruvate ferredoxin
- ATP hydrolysis and ATP binding help overcome the high activation energy.
- Has regions homologous to GTP-binding proteins used in signaling
- Has novel FeMo cofactor (or V in some organisms)
- The holoenzyme consists of two identical dinitrogenase reductase molecules (green),
each with a 4Fe-4S redox center and binding sites for two ATP, and two identical
dinitrogenase heterodimers (purple and blue), each with a P cluster (Fe-S center) and
an FeMo cofactor. In this structure, ADP is bound in the ATP site, to make the
crystal more stable.
, The Fe-Mo Cofactor in the Dinitrogenase Subunit
Consist of
- 7 Fe atoms
- 9 S atoms
- 1 Mo atom
- 1 bound homocitrate
The nitrogen binds to the center of the Mo-FeS cage and is coordinated to the molybdenum atom.
Electrons are passed to the molybdenum-bound nitrogen via the iron-sulfur complex.
Nitrogen Fixation by the Nitrogenase Complex
1. Pyruvate passes e – to ferredoxin or flavodoxin.
2. Ferredoxin or flavodoxin pass e – to dinitrogenase reductase.
3. The reductase passes e – to dinitrogenase.
4. Dinitrogenase passes e – to nitrogen (or to protons) to make NH3.
5. Formation of H2 appears an obligatory side reaction
Redox Reactions in Dinitrogenase
- The net reaction of the nitrogenase complex:
- Dinitrogenase reductase catalyzes:
o transfer of 8 e – to dinitrogenase
o hydrolysis of ATP with release of protons
- Dinitrogenase catalyzes:
o transfer of 6 e – to nitrogen: formation of NH3
o transfer of 2 e – to protons: formation of H2
- Mechanism of dinitrogenase is poorly understood:
o extremely complex redox reaction that involves several metal
atoms as cofactors and/or electron transporters
o two plausible mechanisms that involve the Fe-Mo cofactor binding
directly to N
Nitrogen-Fixing Bacteria in Root Nodules of Legumes
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-
-
-
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