17
reduction compared to N 2 reduction. In acetylene reduction, up to 95% of the
electron flux goes to ethylene (depending on experimental conditions) (Newton
2015) compared to the 3:1 ratio of N 2 to H 2 production with N 2 as substrate.
The general reaction is similar in alternative nitrogenases, although details of
alternate substrates and efficiencies differ (Newton 2015). The alternative nitrogenases are much less efficient than the Mo nitrogenase and much less of the electron
flux goes to N 2 (Newton 2015).
The process of N 2 fixation is highly sensitive to O 2 , because the nitrogenase proteins themselves are very rapidly and irreversibly inactivated by O 2 (Artz et al.
2017; Eady et al. 1975; Fay 1992; Gallon 1981, 1992). This presents a paradox,
since O 2 is needed for respiration (oxidative phosphorylation) to supply the energy
for the reaction (Marchal and Vanderleyden 2000; Staal et al. 2003). The position of
the FeS cluster close to the surface of the Fe protein may explain its extreme O 2
sensitivity (Benton et al. 2002). As a result of O 2 , microorganisms have evolved a
number of mechanisms to avoid O 2 , protect active enzyme from being inactivated,
and regulate the expression of genes in response to O 2 (Dixon and Kahn 2004; Fay
1992). Interestingly, the Fe protein itself can reduce O 2 (Thorneley and Ashby
1989), suggested to potentially be a form of “autoprotection” in some organisms
(Bergman et al. 1997).
Many N 2 -fixing microorganisms are anaerobes, such as sulfate reducers, methanogens and spirochaetes, and live in anoxic or low-O 2 environments like sediments
or animal guts, and thereby avoid O 2 . Other microorganisms, such as Klebsiella are
facultative and fix N 2 only under anoxic or microaerophilic conditions (Hill 1988).
Azotobacter fixes N 2 under aerobic conditions, and has a low affinity, but a high K m
cytochrome oxidase which is the basis for respiratory protection (Kennedy and
Bishop 2004). Azotobacter also has a FeSII protein (the Shethna protein) that binds
to and protects Mo nitrogenase from O 2 (Kennedy and Bishop 2004). In some bacteria, the Fe protein is post-translationally reversibly inactivated by ADP-ribosylation
in response to O 2 (Nordlund and Ludden 2004). Cell wall composition, such as the
inclusion of hopanoids, can reduce the diffusion rate of O 2 into the cell (Berry et al.
1993; Cornejo-Castillo and Zehr 2019).
O 2 metabolism in respiration and photosynthesis generates the toxic radical
superoxide, hydrogen peroxide and hydroxyl radicals. N 2 -fixing microorganisms,
including cyanobacteria use O 2 scavenging enzymes such as superoxide dismutase
and catalase to prevent the damaging oxidation effects of intracellular molecular O 2
(Fay 1992). Hydrogenases associated with N 2 -fixing microorganisms may also be
involved in O 2 scavenging (Fay 1992).
Microorganisms also regulate expression of nitrogenase genes in response to O 2
to avoid wasting energy (Robson and Postgate 1980) (see below). In proteobacteria
the nifLA genes respond to O 2 . The NifA protein is an activator involved in normal
expression. In the presence of O 2 , NifL binds to NifA preventing activation of transcription of the nif operon (Dixon 2004), but the specifics of how the regulation
works differs among species (Kennedy and Bishop 2004).
Almost all (except for some unusual symbionts) cyanobacteria have oxygenic
photosynthesis and evolve O 2 , which is incompatible with N 2 fixation at the same
2.3 The Biological Nitrogen Fixation Reaction
reduction compared to N 2 reduction. In acetylene reduction, up to 95% of the
electron flux goes to ethylene (depending on experimental conditions) (Newton
2015) compared to the 3:1 ratio of N 2 to H 2 production with N 2 as substrate.
The general reaction is similar in alternative nitrogenases, although details of
alternate substrates and efficiencies differ (Newton 2015). The alternative nitrogenases are much less efficient than the Mo nitrogenase and much less of the electron
flux goes to N 2 (Newton 2015).
The process of N 2 fixation is highly sensitive to O 2 , because the nitrogenase proteins themselves are very rapidly and irreversibly inactivated by O 2 (Artz et al.
2017; Eady et al. 1975; Fay 1992; Gallon 1981, 1992). This presents a paradox,
since O 2 is needed for respiration (oxidative phosphorylation) to supply the energy
for the reaction (Marchal and Vanderleyden 2000; Staal et al. 2003). The position of
the FeS cluster close to the surface of the Fe protein may explain its extreme O 2
sensitivity (Benton et al. 2002). As a result of O 2 , microorganisms have evolved a
number of mechanisms to avoid O 2 , protect active enzyme from being inactivated,
and regulate the expression of genes in response to O 2 (Dixon and Kahn 2004; Fay
1992). Interestingly, the Fe protein itself can reduce O 2 (Thorneley and Ashby
1989), suggested to potentially be a form of “autoprotection” in some organisms
(Bergman et al. 1997).
Many N 2 -fixing microorganisms are anaerobes, such as sulfate reducers, methanogens and spirochaetes, and live in anoxic or low-O 2 environments like sediments
or animal guts, and thereby avoid O 2 . Other microorganisms, such as Klebsiella are
facultative and fix N 2 only under anoxic or microaerophilic conditions (Hill 1988).
Azotobacter fixes N 2 under aerobic conditions, and has a low affinity, but a high K m
cytochrome oxidase which is the basis for respiratory protection (Kennedy and
Bishop 2004). Azotobacter also has a FeSII protein (the Shethna protein) that binds
to and protects Mo nitrogenase from O 2 (Kennedy and Bishop 2004). In some bacteria, the Fe protein is post-translationally reversibly inactivated by ADP-ribosylation
in response to O 2 (Nordlund and Ludden 2004). Cell wall composition, such as the
inclusion of hopanoids, can reduce the diffusion rate of O 2 into the cell (Berry et al.
1993; Cornejo-Castillo and Zehr 2019).
O 2 metabolism in respiration and photosynthesis generates the toxic radical
superoxide, hydrogen peroxide and hydroxyl radicals. N 2 -fixing microorganisms,
including cyanobacteria use O 2 scavenging enzymes such as superoxide dismutase
and catalase to prevent the damaging oxidation effects of intracellular molecular O 2
(Fay 1992). Hydrogenases associated with N 2 -fixing microorganisms may also be
involved in O 2 scavenging (Fay 1992).
Microorganisms also regulate expression of nitrogenase genes in response to O 2
to avoid wasting energy (Robson and Postgate 1980) (see below). In proteobacteria
the nifLA genes respond to O 2 . The NifA protein is an activator involved in normal
expression. In the presence of O 2 , NifL binds to NifA preventing activation of transcription of the nif operon (Dixon 2004), but the specifics of how the regulation
works differs among species (Kennedy and Bishop 2004).
Almost all (except for some unusual symbionts) cyanobacteria have oxygenic
photosynthesis and evolve O 2 , which is incompatible with N 2 fixation at the same
2.3 The Biological Nitrogen Fixation Reaction