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10.2 Fundamentals
Much is known about the biochemistry and molecular biology of N 2 fixation from
work on terrestrial model bacterial systems such as Clostridium, Azotobacter and
Klebsiella. Nitrogenase has a complex structure with dinitrogenase reductase coordinating a FeS center and binding ATP, and dinitrogenase with P clusters and the
MoFe cofactor at the reaction site.
The nitrogenase enzyme is comprised of two proteins, nitrogenase and nitrogenase reductase encoded by nifD(G)K and nifH, respectively. The most widely studied and broadly distributed nitrogenase, sometimes termed “conventional”
nitrogenase, has a MoFe complex at the reaction site. Alternative nitrogenases contain three proteins encoded by vnfDGK or anfDGK, depending whether Mo is
replaced by V or Fe. The three proteins are evolutionarily related and are also related
to bacteriochlorophyll reductases. The alternative nitrogenases have different characteristics of efficiency. There are 4 phylogenetic clusters of the nitrogenase genes
for the enzyme. Cluster 1 contains genes from typical proteobacteria, Cluster 2
contains the alternative genes, Cluster 3 contains genes from mostly anaerobic
microorganisms. Until recently, cluster 4 contained genes not believed to be involved
in N 2 fixation. The evolution of nitrogenase remains controversial, but it is clearly
an ancient enzyme. However, evolution and diffusion among prokaryotic phyla has
involved substantial horizontal gene transfers.
The Fe protein cycle passes electrons from dinitrogenase reductase to nitrogenase, along with use of 2 ATP per docking through the P cluster to the FeMo cofactor where 2 NH 3 and H 2 are released after 8 cycles.
N 2 fixation involves more than the structural HD(G)K genes. Many of these
genes are highly conserved and necessary for a variety of functions including metal
transport and scaffolding for nitrogenase assembly. There are also additional nonnif genes involved in symbiosis. Most is known about the non-structural genes from
terrestrial free-living and symbiotic systems. The minimal gene set needed includes
nifHDK, EN and B.
The nitrogenase genes are highly regulated by transcriptional and sometimes
post-translational mechanisms. These regulatory mechanisms turn synthesis off and
on in response to environmental factors such as presence of fixed N or O 2 , in order
to avoid the costs of synthesis when nitrogenase is not needed or could be damaged.
Nitrogenase catalyzes the reduction of multiple substrates, including acetylene
whose reduction to ethylene can be used as a measure of N 2 fixation activity. It is
also highly sensitive to inactivation by O 2 . Thus, microorganisms have evolved
many different adaptations for avoiding O 2 , including in O 2 -evolving cyanobacteria.
Many diazotrophs have systems that regulate the transcription of nif genes in
response to O 2 . Some have modified cell wall components to restrict the diffusion of
O 2 from the environment, and others are simply anaerobes that only fix N 2 in anoxic
habitats. Some cyanobacteria develop specialized cells called heterocysts that are
not oxygenic, and are where N 2 fixation takes place.
10 Summary and Conclusions
10.2 Fundamentals
Much is known about the biochemistry and molecular biology of N 2 fixation from
work on terrestrial model bacterial systems such as Clostridium, Azotobacter and
Klebsiella. Nitrogenase has a complex structure with dinitrogenase reductase coordinating a FeS center and binding ATP, and dinitrogenase with P clusters and the
MoFe cofactor at the reaction site.
The nitrogenase enzyme is comprised of two proteins, nitrogenase and nitrogenase reductase encoded by nifD(G)K and nifH, respectively. The most widely studied and broadly distributed nitrogenase, sometimes termed “conventional”
nitrogenase, has a MoFe complex at the reaction site. Alternative nitrogenases contain three proteins encoded by vnfDGK or anfDGK, depending whether Mo is
replaced by V or Fe. The three proteins are evolutionarily related and are also related
to bacteriochlorophyll reductases. The alternative nitrogenases have different characteristics of efficiency. There are 4 phylogenetic clusters of the nitrogenase genes
for the enzyme. Cluster 1 contains genes from typical proteobacteria, Cluster 2
contains the alternative genes, Cluster 3 contains genes from mostly anaerobic
microorganisms. Until recently, cluster 4 contained genes not believed to be involved
in N 2 fixation. The evolution of nitrogenase remains controversial, but it is clearly
an ancient enzyme. However, evolution and diffusion among prokaryotic phyla has
involved substantial horizontal gene transfers.
The Fe protein cycle passes electrons from dinitrogenase reductase to nitrogenase, along with use of 2 ATP per docking through the P cluster to the FeMo cofactor where 2 NH 3 and H 2 are released after 8 cycles.
N 2 fixation involves more than the structural HD(G)K genes. Many of these
genes are highly conserved and necessary for a variety of functions including metal
transport and scaffolding for nitrogenase assembly. There are also additional nonnif genes involved in symbiosis. Most is known about the non-structural genes from
terrestrial free-living and symbiotic systems. The minimal gene set needed includes
nifHDK, EN and B.
The nitrogenase genes are highly regulated by transcriptional and sometimes
post-translational mechanisms. These regulatory mechanisms turn synthesis off and
on in response to environmental factors such as presence of fixed N or O 2 , in order
to avoid the costs of synthesis when nitrogenase is not needed or could be damaged.
Nitrogenase catalyzes the reduction of multiple substrates, including acetylene
whose reduction to ethylene can be used as a measure of N 2 fixation activity. It is
also highly sensitive to inactivation by O 2 . Thus, microorganisms have evolved
many different adaptations for avoiding O 2 , including in O 2 -evolving cyanobacteria.
Many diazotrophs have systems that regulate the transcription of nif genes in
response to O 2 . Some have modified cell wall components to restrict the diffusion of
O 2 from the environment, and others are simply anaerobes that only fix N 2 in anoxic
habitats. Some cyanobacteria develop specialized cells called heterocysts that are
not oxygenic, and are where N 2 fixation takes place.
10 Summary and Conclusions