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Mechanisms for Generating Low Potential Electrons across the Metabolic Diversity of Nitrogen-Fixing Bacteria

American Society for Microbiology
Applied and Environmental Microbiology
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The availability of fixed nitrogen is a limiting factor in the net primary production of all ecosystems. Diazotrophs overcome this limit through the conversion of atmospheric dinitrogen to ammonia. Diazotrophs are phylogenetically diverse bacteria and archaea that exhibit a wide range of lifestyles and metabolisms, including obligate anaerobes and aerobes that generate energy through heterotrophic or autotrophic metabolisms. Despite the diversity of metabolisms, all diazotrophs use the same enzyme, nitrogenase, to reduce N2. Nitrogenase is an O2-sensitive enzyme that requires a high amount of energy in the form of ATP and low potential electrons carried by ferredoxin (Fd) or flavodoxin (Fld). This review summarizes how the diverse metabolisms of diazotrophs utilize different enzymes to generate low potential reducing equivalents for nitrogenase catalysis. These enzymes include substrate-level Fd oxidoreductases, hydrogenases, photosystem I or other light-driven reaction centers, electron bifurcating Fix complexes, proton motive force-driven Rnf complexes, and Fd:NAD(P)H oxidoreductases. Each of these enzymes is critical for generating low potential electrons while simultaneously integrating the native metabolism to balance nitrogenase's overall energy needs. Understanding the diversity of electron transport systems to nitrogenase in various diazotrophs will be essential to guide future engineering strategies aimed at expanding the contributions of biological nitrogen fixation in agriculture.
The electron transport systems (ETS) of free-living and symbiotic aerobic diazotrophs. (a) The ETS in A. vinelandii can use 2 pathways. The first (left side) is a respiratory protection pathway consisting of an uncoupled type II NADH dehydrogenase (NDHII) and terminal oxidase cytochrome bd (Cyt bd). The second path is a traditional proteobacterial electron transport chain with full proton coupled complexes: NADH dehydrogenase (NDHI), succinate dehydrogenase (Succinate DH), cytochromes bc1 (Cyt bc1), and terminal oxidase cytochrome o (cyt o). The production of reduced ferredoxin (Fd) for nitrogenase is also part of the ETS with Fix and Rnf complexes. (b) Electron transport of Rhizobia. A generic look at the electron transport chain of rhizobia primarily based on data from Bradyrhizobium japonicum. Carbon enters the bacteroid mostly as organic acids malate (Mal) and succinate (Suc). NADH produced from the TCA cycle is oxidized by NADH dehydrogenase NDHI and produces quinol (QH2). Succinate dehydrogenase (SucDH) also reduces quinone to QH2. Electrons for nitrogenase are produced through the enzyme complex Fix which bifurcates electrons from NADH to quinone and ferredoxin (Fd). QH2 is oxidized by cytochrome bc1 and reduces cytochrome c (Cytc). There is a diversity of terminal oxidases in rhizobia: under higher oxygen concentrations, cytochrome aa3 is used, while under lower oxygen conditions (like those present for nitrogen fixation), cytochrome cbb3 is used. Two proton translocating quinol oxidases are also present as cytochrome bd and cytochrome bo3.
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Mechanisms for Generating Low Potential Electrons across the
Metabolic Diversity of Nitrogen-Fixing Bacteria
Alexander B. Alleman,a*John W. Petersb
a
Institute of Biological Chemistry, Washington State University, Pullman, Washington, USA
b
Department of Chemistry and Biochemistry, University of Oklahoma, Norman, Oklahoma, USA
ABSTRACT The availability of xed nitrogen is a limiting factor in the net primary pro-
duction of all ecosystems. Diazotrophs overcome this limit through the conversion of
atmospheric dinitrogen to ammonia. Diazotrophs are phylogenetically diverse bacteria
and archaea that exhibit a wide range of lifestyles and metabolisms, including obligate
anaerobes and aerobes that generate energy through heterotrophic or autotrophic meta-
bolisms. Despite the diversity of metabolisms, all diazotrophs use the same enzyme, nitro-
genase, to reduce N
2
. Nitrogenase is an O
2
-sensitive enzyme that requires a high amount
of energy in the form of ATP and low potential electrons carried by ferredoxin (Fd) or a-
vodoxin (Fld). This review summarizes how the diverse metabolisms of diazotrophs utilize
different enzymes to generate low potential reducing equivalents for nitrogenase catalysis.
These enzymes include substrate-level Fd oxidoreductases, hydrogenases, photosystem I
or other light-driven reaction centers, electron bifurcating Fix complexes, proton motive
force-driven Rnf complexes, and Fd:NAD(P)H oxidoreductases. Each of these enzymes is
critical for generating low potential electrons while simultaneously integrating the native
metabolism to balance nitrogenases overall energy needs. Understanding the diversity of
electron transport systems to nitrogenase in various diazotrophs will be essential to guide
future engineering strategies aimed at expanding the contributions of biological nitrogen
xation in agriculture.
KEYWORDS diazotrophs, electron transport, nitrogen xation, physiology
Nitrogen in the form of ammonia, nitrate, or urea is required for primary production in
terrestrial and marine ecosystems (1). Most terrestrial nitrogen is stored in the atmos-
phere as dinitrogen (N
2
) gas and is unavailable to the majority of organisms. However,
some bacteria and archaea, called diazotrophs, can convert N
2
into bioavailable ammonia
(NH
3
) through biological nitrogen xation (BNF). Diazotrophs account for 60% of the xed
N
2
input into the biogeochemical nitrogen cycle (2). Diazotrophs xN
2
via the activity of
nitrogenase, which catalyzes the cleavage of the N
2
triple bond and its reduction to ammo-
nia. BNF can be extraordinarily challenging for diazotrophs, as nitrogenase requires energy
from ATP hydrolysis and low potential electrons and is sensitive to oxygen inactivation (3).
Despite the aforementioned limitations of nitrogenase, diazotrophs are found in most eco-
logical niches having a primary role in a diversity of microbial communities. Diazotrophs re-
side in oligotrophic marine ecosystems, the extreme environments of hot springs, the guts
of termites, and in symbiotic relationships with crops (48). Diazotrophs can occupy these
diverse niches through various physiological adaptations that shape native metabolisms to
protect nitrogenase from oxygen while supplying enough ATP and reducing equivalents.
This review details how a diversity of diazotrophs use electron transport enzymes in con-
cert with physiological adaptations to support the energetic needs of nitrogenase-depend-
ent reduction of N
2
.
The enzymatic reduction of N
2
has one of the highest energy barriers in biology,
requiring large amounts of reducing chemical energy and intricate metal cofactors to
Editor Jennifer B. Glass, Georgia Institute of
Technology
Copyright © 2023 American Society for
Microbiology. All Rights Reserved.
Address correspondence to John W. Peters,
jw.peters@ou.edu.
*Present address: Alexander B. Alleman,
Department of Biological Sciences, University
of Idaho, Moscow, Idaho, USA.
The authors declare no conict of interest.
Published 8 May 2023
May 2023 Volume 89 Issue 5 10.1128/aem.00378-23 1
MINIREVIEW
perform the reaction (9). Nitrogenase is the only enzyme that can catalyze the reduc-
tion of N
2
to NH
3
and exists in 3 related forms with different metal dependencies: Mo-
dependent, V-dependent, and heterometal-independent forms. These 3 forms of nitro-
genase have been demonstrated to share the same overall mechanism but have
slightly different reaction stoichiometries (10). Mo-dependent nitrogenase is the most
commonly occurring, extensively studied, and most efcient operating with the opti-
mal reaction stoichiometry shown in (Equation 1) (9, 11).
N218e2116 MgATP 18H1
!2NH31H2116 MgADP 116 Pi (1)
Mo-dependent nitrogenase consists of 2 separable component proteins termed the
Fe protein and the MoFe protein to reect the composition of their respective metal-
containing cofactors (9, 12). The Fe protein is a homodimer containing a single [4Fe-
4S] cluster and 2 binding sites for ATP (13). The MoFe protein is an
a
2
b
2
tetramer con-
taining two [8Fe-7S] clusters (P-clusters) and two [7Fe-Mo-9S-C-citrate] FeMo-cofactors
(FeMo-cos) that are located at the sites of substrate binding and reduction (Fig. 1) (14,
15). The metal cofactors are labile to oxygen, and organisms have evolved various
strategies to protect nitrogenase from oxygen inactivation (16, 17).
The Fe protein is reduced by ferredoxin (Fd) or avodoxin (Fld) in vivo (1820). Fd and
Fld are small electron transfer proteins containing either FeS cluster(s) or a avin mononu-
cleotide (FMN), respectively (2123). Diazotrophs typically express several Fds and Flds,
andbothcanserveaselectrondonorstotheFeproteininanindividualorganism(2427).
During catalysis, the Fe protein switches between an ADP-bound and ATP-bound confor-
mational state, modulating the reduction potential of the [4Fe-4S] cluster (28, 29). Fd/Fld
most likely reduces the ADP-bound Fe protein preferentially, requiring Fd/Fld to have a
reduction potential of at least -415 to -430 mV (Fig. 2) (30, 31) (Table 1).
Maintaining a low reduction potential is essential for efcient electron transport to
nitrogenase. When discussing electron transfer mechanisms, we use standard reduction
potentials (E
0
), which is the potential at pH 7, where the concentrations of the oxidized
and reduced forms are equal. In reality, biological processes occur far from equilibrium,
allowing reactions to become thermodynamically favorable, meaning a redox couple with
E
0
of -300 mV might be maintained at -350 mV within the cell (32). Diazotrophs utilize the
disequilibrium of redox couples to sustain the production of ATP and reduce Fd for
nitrogenase.
Diazotroph metabolism differs mainly in the intercellular oxidation-reduction potential
and nature of primary electron carriers. For example, low potential Fd is one of the central
electron carriers in anaerobic bacteria, making Fd readily available for nitrogen xation. In
FIG 1 The nitrogenase enzyme is a heterodimer consisting of 3 subunits per dimer, the Fe protein
(NifH), and the 2 subunits of the MoFe protein (NifDK). The Fe-protein contains 1 FeS cluster that
accepts electrons from Fd or Fld and reduces the P-cluster in the MoFe protein in an ATP-dependent
electron transfer. The P-cluster delivers electrons to the MoFe cofactor allowing electrons to be loaded
for the reduction of N
2
. For complete N
2
reduction, the Fe protein must deliver 8 electrons to the
MoFe protein, requiring 2 ATP per electron transfer. Each metal cofactor is very sensitive to oxygen
damage.
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contrast, aerobic diazotrophs carry most electrons on the NAD(P)
1
/NAD(P)H reduction
potentials at ;2320 mV and need to input energy or maintain favorable redox ratios to
reduce Fd/Fld at ;2400 to 2500 mV. There are 6 known or proposed mechanisms that
generate low potential electrons in the form of Fd or Fld in order to donate to nitrogenase:
(i) substrate-level Fd oxidoreductases, (ii) hydrogenases, (iii) photosystem I or other light-
driven reaction centers, (iv) electron bifurcating Fix complexes, (v) proton motive force
(pmf) driven Rnf complexes, and (vi) Fd:NAD(P)H oxidoreductases (Fig. 2).
OBLIGATE OR FACULTATIVE ANAEROBIC METABOLISM AND SUBSTRATE-LEVEL FD
REDUCTION
During anaerobic metabolism, low potential electrons are generated through sub-
strate-level Fd reduction. For example, obligate anaerobes Clostridium pasteurianum
and Desulfovibrio africanus, and facultative anaerobes like Klebsiella pneumoniae and
FIG 2 Electrochemical potential landscapes of electron transport mechanism to Fd/Fld and the Fe protein
of nitrogenase (Fe Pro) (values in Table 1). (a) Pyruvate ferredoxin oxidoreductase (PFOR) catalyzes the
reduction of Fd from pyruvate to form acetyl-CoA and CO
2
. (b) Bi-directional hydrogenase (H
2
ase) in C.
pasteurianum catalyzes the reduction of Fd directly from H
2
. Fd and the Fe protein of nitrogenase are at a
higher potential in C.pasteurianum than other diazotrophs, facilitating the electron transfer. (c) Light-
coupled Fd reduction in photosystem I or other reaction centers catalyzes the reduction of Fd from
plastocyanin or other cytochromes. (d) Rnf catalyzes the reduction of Fd through the coupling of the
proton motive force (pmf). (e) Fix couples the reduction of quinone with the reduction of Fd using
electron bifurcation. (f) Ferredoxin:NADPH oxidoreductase (FNR) catalyzes Fd reduction by using the redox
ratios of the NADPH and Fd. Heterocyst Fd only has a reduction potential of 2351mV, which is not low
enough for Fe protein reduction. An increase in the concentration of NADPH and reduced Fd will
decrease the E
h
(blue to red color), allowing the FNR reaction to be thermodynamically feasible.
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Bacillus polymyxa produce Fd in CoA-dependent reactions (3340). The half-reaction of
pyruvate oxidation to acetyl-CoA and CO
2
by pyruvate-ferredoxin oxidoreductases
(PFORs) has a reduction potential of 2540 mV, allowing low potential Fds to act as the
electron acceptor (Fig. 2a) - (Equation 2) (4143). PFORs contain FeS clusters sensitive
to oxygen, restricting PFORs from being expressed and functioning only in anaerobic
conditions (44).
pyruvate 1CoASH 12Fdox $acetylCoA 1CO212Fdred 12H1(2)
PFORs are phylogenetically diverse, found throughout all kingdoms, and represent
key steps in the Wood-Ljungdahl and reductive TCA pathways (4547). Many PFORs
are found within the genomes of diazotrophs, but only a few have been shown to sup-
port nitrogenase activity. In K. pneumoniae, a facultative anaerobe that only xes nitro-
gen under anaerobic or microaerobic conditions, the rst characterized electron trans-
port mechanism to nitrogenase was determined to be Fd produced by PFOR (38, 48).
The gene for PFOR was designated nifJ, and an associated avodoxin gene was termed
nifF. Genes homologous to nifJ have been found in multiple diazotrophs, but not all are
associated with nitrogen xation. In the cyanobacterium Anabaena 7120, its PFOR is not
required for diazotrophic growth under standard conditions but is essential under Fe-limi-
tation (49, 50). In the purple nonsulfur bacterium (PNSB) Rhodospirillum rubrum,thereisa
signicant amount of PFOR activity, and puried PFOR supports pyruvate-dependent in
vitro nitrogenase activity with puried R. rubrum Fd (51, 52). PFOR in Rhodobacter capsula-
tus is upregulated under nitrogen-xing conditions, but decreased levels are found in ace-
tate and dark aerobically grown cells (45). However, PFORs overall contribution to nitrogen
xation in the PNSB may be low since other mechanisms, discussed later in the review,
have been found to provide the primary source of low potential electrons (5356). Overall,
PFORs tend to be essential to general anaerobic metabolism and can support nitrogen x-
ation in obligate or facultative anaerobes.
In other anaerobic metabolisms, hydrogenases have been implicated in substrate-
level Fd reduction for nitrogen xation. Two classes of hydrogenase, [FeFe]- and [NiFe]-,
catalyze the reversible oxidation of H
2
(5759). Under standard conditions, the H
1
/H
2
reduction potential is 2414 mV; therefore, reducing Fd (;2400 to 2500 mV) from H
2
is a favorable or a slightly endergonic reaction (60). Nitrogen-xing C. pasteurianum pro-
duces 3 non-bifurcating [FeFe]-hydrogenases that exhibit different catalytic properties.
C. pasteurianum hydrogenase II (CpII) is upregulated 7.5-fold under nitrogen-xing con-
ditions and is proposed to recapture electrons that are produced as H
2
during nitroge-
nase catalysis (59, 61). In C. pasteurianum, the standard reduction potential of Fd is
2412 mV, and the ADP-bound Fe protein standard reduction potential has been
TABLE 1 Reduction potential values and references from Fig. 2
Species Type Potential (mV) Figure 2 Reference
Azotobacter vinelandii Nitrogenase ATP bound 2430 d and e 31
Azotobacter vinelandii Nitrogenase ADP bound 2470 d and e 31
Clostridium pasteurianum Nitrogenase ATP bound 2415 a and b 31
Clostridium pasteurianum Nitrogenase ADP bound 2415 a and b 31
Azotobacter vinelandii Flavodoxin 2483 d and e 21
Azotobacter vinelandii Ferredoxin 2619 d and e 207
Anabaena 7120 Ferredoxin heterocyst 2351 f 208
Anabaena 7120 Ferredoxin vegetative 2384 f 208
Synechocystis PCC 6803 Cytochrome c
6
1320 c 176
General Quinone 4 e 32
General NAD
1
/ NADH (1:1) 2320 e, d, and f 32
General NAD
1
/ NADH (10:1) 2290 e, d, and f 32
General NADP
1
/ NADPH (1:1) 2320 e, d, and f 32
General NADP
1
/ NADPH (1:10) 2350 e, d, and f 32
General NADP
1
/ NADPH (1:100) 2380 e, d, and f 32
General H
2
2420 b 32
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measured at 2415 mV (62, 63). Thus, the reduction potentials of H
2
, Fd, and Fe protein
are within the thermodynamically favorable range allowing for the forward reaction
and the reduction of the Fe protein from H
2
(Fig. 2b).
While hydrogenase may contribute to Fd reduction, many other reactions in anaer-
obic metabolisms result in Fd reduction (64). For example, during nitrogen-xing con-
ditions in C. pasteurianum, 3 distinct PFOR genes are all expressed. Under these condi-
tions, Fld is also expressed 14-fold higher, suggesting the ability of PFORs in C.
pasteurianum to reduce both Fd and Fld (65). Other reactions also produce reduced Fd
through formate oxidation and the reduction of crotonyl-CoA to butyryl-CoA, allowing
multiple pathways to deliver electrons to nitrogenase (66, 67).
Anaerobic metabolisms, either through fermentation or anaerobic respiration, yield
far less ATP compared to aerobic metabolism. For example, the facultative anaerobe K.
pneumoniae xes nitrogen under anaerobic conditions by fermenting glucose (68).
Glucose catabolism and pyruvate degradation through PFOR to acetyl-P via phospho-
transacetylase, then to the nal fermentation product acetate, through acetate kinase,
creates 2 reduced Fds and 1 ATP (69, 70). As nitrogenase requires a 2:1 ATP to reduced
Fd ratio, anaerobic nitrogen xation is limited by ATP synthesis (71). Reductant excess is
balanced by shifting to ethanol or H
2
as a nal fermentation product which consumes
the extra reductant but lowers the overall biomass yields (68). So, while anaerobic
organisms and their reducing environment are ideal for protecting nitrogenase from
oxygen and supplying electrons, overall energy expenditure is limited for optimizing
growth.
ANAEROBIC LIGHT-DRIVEN FD REDUCTION
In phototrophic metabolism, reduced Fd can be generated from inputs of light
energy. Bacterial FeS-type I reaction centers (RCI) found in anaerobic green sulfur bac-
teria and heliobacteria, as well as type I photosystems (PSI) found in cyanobacteria,
reduce Fd (72) (Fig. 2c). Direct electron transfer has been proposed from Fd produced
by RCI/PSI to nitrogenase. Heliobacterium modesticaldum is an active diazotroph with a
minimal photosynthetic RC, containing just 2 subunits PshA and PshB (73). The termi-
nal electron acceptor in PshB contains 2 Fd-like [4Fe-4S] clusters that donate electrons
to Fd and is upregulated under diazotrophic conditions (74, 75). H. modesticaldum
does have PFOR and Ferredoxin:NAD(P)H oxidoreductase (FNR) activity, but reduced
diazotrophic growth rates observed under dark chemotrophic conditions suggest that
RCIs have a role in providing reduced Fd for nitrogenase catalysis (76). Reduction of Fd
through FeS-type RC also occurs in nitrogen-xing green sulfur bacteria Chlorobium
tepidum, where Fd is primarily used in the reductive TCA cycle but could also be used
for nitrogen xation (77, 78). The Fd produced by PSI in cyanobacteria can also trans-
port electrons to nitrogenase, but other electron transport mechanisms in cyanobacte-
ria might be favored (see section on heterocyst metabolism).
NADH OXIDATION IN AEROBIC METABOLISM
Aerobic metabolism avoids using Fd as the primary electron carrier for most redox
reactions due to its sensitivity to oxygen and therefore utilizes nucleotide electron car-
riers NAD(P)
1
/NAD(P)H as the primary electron source. Since nitrogenase can only
accept electrons for Fd or Fld, various mechanisms are required to maintain reduced
Fd/Fld production from the NAD(P)
1
/NAD(P)H pool. Primarily, aerobic diazotrophs cou-
ple the endergonic reduction of Fd from NAD(P)H with an exergonic reaction, such as
electron bifurcation or the pmf. Alternatively, some aerobic diazotrophs can maintain a
low reduction potential by decreasing the NAD(P)
1
/NAD(P)H ratio to overcome the
thermodynamic barrier of Fd reduction in FNR.
There are multiple exergonic reactions to couple with the endergonic reduction of NAD
(P)H and Fd in aerobic metabolism. One of the most studied is the avin-based electron
bifurcating enzyme Fix. The Fix enzyme was initially discovered in a genetic locus on the
Rhizobium meliloti 2011 pSym megaplasmid that was essential for nitrogen xation (79
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81). Some genes in this region have homology to known K. pneumoniae nif genes, but
other genes also required for nitrogen xation have an unknown function. These genes
were given the name x(82). Some of the xgenes, xABCX, have been implicated in nitro-
genase biosynthesis in Azorhizobium caulinodans ORS571, but others have been suggested
to have a role in electron transport as FixX has amino acid sequence homology with Fd
(83, 84). The FixABCX complex was nally determined to be involved in electron transfer to
nitrogenase in R. rubrum,inwhichtheDxABCX strains showed an ;80% decrease of in
vivo nitrogenase activity while maintaining full activity in vitro (54, 55).
FixABCX is a membrane-bound oxidoreductase enzyme that generates low potential
electrons Fd or Fld (Fig. 2e). FixAB shares similarities to the family of electron transfer a-
voproteins (ETF) found in all life, including the ETF ubiquinone oxidoreductase, which
transfers electrons to the quinone pool in the membrane (85). FixABCX employs avin-
based electron bifurcation (FBEB) to overcome the energy barrier of the endergonic
reduction of Fd/Fld via NADH oxidation (86, 87). FBEB couples a thermodynamically
favorable exergonic reaction to drive a thermodynamically unfavorable endergonic reac-
tion (88). Using FBEB, FixABCX couples the exergonic reduction of quinone (10 mV) and
the endergonic reduction of Fd (2460 mV) to the oxidation of 2 NADH (2320 mV) in an
overall thermodynamically feasible reaction (Fig. 2e) (Equation 3) (89). Recently the struc-
ture of FixABCX homolog termed EtfABCX from Thermotoga maritima was determined
by cryo-electron microscopy providing insights into the energy landscape of this class of
avin-based electron bifurcating enzymes (90).
2NADH 1CoQ 12Fd=Fldox !2NAD11CoQH212Fd=FldRed (3)
Another mechanism to drive the endergonic reduction of Fd by NADH is harnessing
the proton motive force. Seven genes in R. capsulatus were discovered to be required
for nitrogen xation called Rhodobacter nitrogen xation or rnf (91, 92). The R. capsula-
tus genes rnfABCDGEH are similar to the respiratory Na
1
-dependent NADH:ubiquinone
oxidoreductase (Na
1
-NQR) (93, 94). While Rnf has been implicated in Fd reduction for
nitrogen xation in R. capsulatus (91, 92), Pseudomonas stutzeri (95), and A. vinelandii
(89, 96, 97), the biochemical characterization of Rnf has been in the directionality of Fd
oxidation and H
1
/Na
1
pumping. In Acetobacterium woodii, purication and characteri-
zation of Rnf has shown Na
1
-dependent reversible reduction of NAD
1
with Fd (Fig. 2d)
(Equation 4) (98101). In general, rnf genes are widely distributed in bacteria and
required in some methanogenic archaea (102).
NADH 12Fdox 1DlH1=Na1
$NAD112Fdred 1H1(4)
Recently, cryo-EM structures of Rnf from A. vinelandii and Clostridium tetanomor-
phum implicate strict electron and proton transfer coupling through multiple avins
and FeS clusters (103, 104). This coupling is controlled by signicant conformational
changes to combine the endergonic reduction of Fd with the energy available in the
H
1
/Na
1
motive force.
Many aerobic heterotrophs store electrons in the NAD
1
/NADH electron couple with
a reduction potential of -320 mV and funnel these electrons to oxidative phosphoryla-
tion in the electron transport chain (ETC). In order to produce low potential electrons
in Fd, they must balance the use of Fix or Rnf with the production of ATP while main-
taining a low oxygen environment. One of the most extensively studied diazotrophs is
the obligate aerobic gamma-proteobacteria,A. vinelandii (105). The ability of A. vinelan-
dii to x nitrogen in fully saturated oxygen media is due to multiple mechanisms; from
the creation of an extracellular alginate barrier to limit O
2
diffusion, production of
superoxide dismutase and catalase to consume oxygen radicals, and as a nal barrier
of protection, reversible inactivation, and protection of nitrogenase via the FeSII/
Shethna protein (106109). While the above mechanism provides some level of oxy-
gen protection, the key mechanism that supports nitrogen xation under high oxy-
gen tensions is respiratory protection, where a terminal oxidase consumes oxygen
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at a high rate at the cell surface, preventing the accumulation of oxygen in the cyto-
plasm (110).
Respiratory protection is facilitated by a unique branch of the ETC, including a
decoupled NADH-dehydrogenase II (NDHII) and a quinol terminal oxidase called cyto-
chrome bd (111113). This abbreviated pathway is partially coupled to the membrane,
only translocating 1 H
1
/e-, allowing for a high respiration rate (113, 114). As a result,
accumulation of cytochrome bd during high aeration and nitrogen xation is observed,
and knockouts of bd oxidase can no longer grow under diazotrophic conditions (115).
A. vinelandii simultaneously expresses a fully proton coupled ETC under nitrogen-xing
conditions with an NDH-I, cytochrome bc
1
-oxidase, and cytochrome-ooxidases similar
to complex I-IV seen in other proteobacteria and mitochondria (Fig. 3a) (115118). The
implementation of respiratory protection occurs at an energetic cost reducing the bio-
mass yield of A. vinelandii and is sometimes termed a wastingmechanism. It has
recently been proposed that this respiratory protection mechanism may be elicited in
response to a high carbon and oxygen environment (119121).
A. vinelandii possesses both Fix and Rnf mechanisms for generating low potential
electrons for nitrogen xation. The rnf gene cluster is located upstream from the small
nif cluster and is regulated by nifA, the nif gene transcriptional activator. The xgene
cluster is not a part of the major or minor nif gene clusters in A. vinelandii but is upreg-
ulated under nitrogen-xing conditions (122, 123). The Rnf and Fix complexes create
partial redundancy for electron transport to nitrogenase, and deletion of either rnf or
xhas little effect on diazotrophic growth, but the double mutant, Drnf1Dxis unable
to grow diazotrophically (89). Other diazotrophs that encode for Rnf rarely have genes
encoding for Fix and vice versa, making A. vinelandii unusual in having 2 ways to
reduce Fd from NAD
1
/NADH (17, 124). Recently our lab has investigated the roles of
both Rnf and Fix in differing conditions and has shown that while they are redundant
in standard conditions of high carbon and oxygen, Fix is required for efcient growth
under low oxygen conditions (125).
Other aerobic heterotrophic diazotrophs form symbiotic relationships with plants
that provide carbon and a microaerobic environment in exchange for xed nitrogen.
These organisms, which belong to the alpha- and betaproteobacteria, are generally
called rhizobia and are critical for agriculture (126, 127). Root nodules of leguminous
plants house specialized symbiotic rhizobia (bacteroids) within infected plant cells
enclosed in plant-derived membranes called the symbiosome membrane, allowing for
the exchange of nutrients and protection from oxygen diffusion (128). Rhizobia are a
diverse category of bacteria with various hosts with specialized metabolism to support
nitrogen xation. Here, we generalize the production of energy and electron transport
to nitrogenase in a nodule environment.
The carbon source for rhizobia is predominately C4-dicarboxylic acids, primarily suc-
cinate and malate (129). Dicarboxylates are consumed by an NAD
1
-dependent malic
enzyme paired with PEP-carboxykinase to produce acetyl-CoA, which can be used for
carbon storage or consumption in the TCA cycle (130132). Rhizobia express FixABCX
encoded on the Sym plasmid and immediately upstream and regulated by nifA (133,
134). Metabolic changes are required to maintain redox balance during nitrogen xa-
tion, including carbon storage in polyhydroxy-3-butyrate (PHB), lipids, or glycogen
(135, 136). Amino acid export from the rhizobium and carbon sources from the plant
also regulate redox balance in the cell with dicarboxylates creating a highly reduced
environment requiring a higher oxygen demand but increasing the supply of reduc-
ing equivalents to nitrogenase (137). This balance between carbon storage, amino
acid export, nitrogenase demand, and oxygen demand is critical for efcient ammo-
nia export to the plant. Recent studies have shown that R. leguminosarum DxAB
mutants accumulated an order of magnitude more PHB and glycogen, suggesting
balancing the redox homeostasis in the rhizobia by shuttling unused electrons to
PHB or glycogen (133).
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Rhizobia also have complex respiratory chains with multiple terminal oxidases (Fig.
3b). Oxygen levels in the nodules are maintained at 10 to 40 nM, protecting nitroge-
nase from oxygen damage (138). Bradyrhizobium japonicum encodes 8 terminal oxi-
dases, including 2 bd-type oxidases and 6 heme-copper oxidases, which can be further
FIG 3 The electron transport systems (ETS) of free-living and symbiotic aerobic diazotrophs. (a) The ETS in A. vinelandii can use 2 pathways. The rst (left
side) is a respiratory protection pathway consisting of an uncoupled type II NADH dehydrogenase (NDHII) and terminal oxidase cytochrome bd (Cyt bd).
The second path is a traditional proteobacterial electron transport chain with full proton coupled complexes: NADH dehydrogenase (NDHI), succinate
dehydrogenase (Succinate DH), cytochromes bc1 (Cyt bc1), and terminal oxidase cytochrome o(cyt o). The production of reduced ferredoxin (Fd) for
nitrogenase is also part of the ETS with Fix and Rnf complexes. (b) Electron transport of Rhizobia. A generic look at the electron transport chain of rhizobia
primarily based on data from Bradyrhizobium japonicum. Carbon enters the bacteroid mostly as organic acids malate (Mal) and succinate (Suc). NADH
produced from the TCA cycle is oxidized by NADH dehydrogenase NDHI and produces quinol (QH
2
). Succinate dehydrogenase (SucDH) also reduces
quinone to QH
2
. Electrons for nitrogenase are produced through the enzyme complex Fix which bifurcates electrons from NADH to quinone and ferredoxin
(Fd). QH
2
is oxidized by cytochrome bc
1
and reduces cytochrome c (Cytc). There is a diversity of terminal oxidases in rhizobia: under higher oxygen
concentrations, cytochrome aa
3
is used, while under lower oxygen conditions (like those present for nitrogen xation), cytochrome cbb
3
is used. Two proton
translocating quinol oxidases are also present as cytochrome bd and cytochrome bo
3
.
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divided into 2 quinol oxidases and 4 cytochrome c oxidases (139). Of these, 2 are pre-
dominant, the cbb
3
-type oxidase is encoded by the xNOQP genes, and the caa
3
-type
oxidase is encoded by coxBACF. The cbb
3
-type oxidases have a high afnity for oxygen
(K
M
= 7 nM) and are essential in symbiosis and transport 0.5 H
1
/e- protons across the
membrane (140142). The cytochrome caa
3
seems to not be essential under low oxy-
gen concentrations in B. japonicum and R. leguminosarum but is the most prominent
terminal oxidase in aerobic conditions (139, 143, 144).
Recent work has led to the proposal of combining the co-catabolism of succinate
and arginine and FixABCXs electron bifurcation mechanism to maintain ux through
the electron transport chain (145). The acidication of the symbiosomes may make the
driving force of NADH dehydrogenase no longer sufcient, requiring FixABCX as the
major NADH regenerating pathway. This proposed path, called CATCH-N, suggests a
co-catabolism of 2 equivalents of succinates and 2 arginine leads to the export of 2 ala-
nine and 6 ammonia. This pathway, which nets ;2 to 3 ATPs per cycle, depends on
using FixABCX as the primary NADH consumer (145). The intricacy of the symbiotic me-
tabolism of plants and rhizobia is still being investigated, but the energy-saving bifur-
cation of the Fix mechanism allows for maximal ammonia production in highly acidic
and low oxygen environments of the symbiosome (146).
ANOXYGENIC PHOTOTROPHS
Purple nonsulfur bacteria (PNSB) are metabolically versatile, growing in aerobic or anaer-
obic environments as heterotrophs or autotrophs. They can also generate energy through
respiration, photosynthesis, and in some cases, fermentation (147, 148). However, PNSB can
only x nitrogen anaerobically in the light, creating ATP through anoxygenic photosynthetic
cyclic electron transport and maintaining carbon and electrons supply from organic sub-
strates, such as butyrate (149). To produce ATP, PNSB use light energy absorbed by reaction
center 1 (RC1) to reduce quinone to quinol using electrons donated from cytochrome c.
Cytochrome bc1 oxidizes quinol and reduces cytochrome c while translocating protons to
complete the cyclic electron transport. The generated pmf canbeusedtoproduceATPand
NADH. Generation of NADH is achieved through the reverse reaction of NADH dehydrogen-
ase, and consumption of pmf for NADH production is shown to be required for photoauto-
trophic growth in R. capsulatus and Rhodobacter sphaeroides (150, 151). During anaerobic
phototrophic growth, PNSB must use the Calvin-Benson-Bassham (CBB) cycle as an electron
sink, with the benet of increasing biomass yield (152, 153). However, when cells become
nitrogen-limited, nitrogenase becomes the electron sink, and the CBB cycle is no longer
needed (154). Therefore, the cyclic electron transport of anoxygenic photosynthesis and
reducing environment is ideal for nitrogen xation.
PNSB have been shown to possess Rnf or Fix as mechanisms for transporting elec-
trons to nitrogenase. In Rhodopseudomonas palustris and R. rubrum, FixABCX is located
directly downstream of the nif genes, regulated by nifA, and essential for nitrogen xa-
tion (27, 54, 155) (Fig. 4a). In contrast, R. capsulatus and R. sphaeroides only possess Rnf
(92, 149, 156158) (Fig. 4b). The contrast of 2 different mechanisms for Fd reduction
within the same metabolic model of photoheterotrophy offers a potential comparison
between Fix and Rnf. Different routes to generate biosynthetic precursors from ace-
tate assimilation also determine the balance of electrons in PNSB. R. sphaeroids uses
the reductive ethylmalonyl-CoA pathway, while R. palustris uses the oxidative glyoxy-
late shunt (154, 159). In R. sphaeroids which relies on Rnf for Fd production, the
reductive ethylmalonyl-CoA pathway consumes reductant in the initial assimilation
of acetate to malate and negates the use of the CBB cycle as an electron sink (159). In
contrast, R. palustris, which relies on the Fix complex for Fd production, uses the glyoxy-
late shunt to produce net NADH and requires an electron sink (160). The contrasting ace-
tate assimilation and Fd production pathways might suggest the co-evolution of Fix and
Rnf within corresponding PNSB metabolism. In R. palustris the use of Fix means for every
Fd produced and utilized as an electron sink in nitrogenase, another pair of electrons is
recycled to quinol and, eventually, NADH. The less-reducing environment of R.
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sphaeroids uses Rnf to consume pmf, and there is no corresponding excess reductant.
Each enzyme may play a role in balancing electron ow in certain PNSB bacteria and
depend on the broader redox homeostasis of the specic metabolism.
OXYGENIC PHOTOTROPHS
While substrate-level reduction of Fd, such as that carried out by PFORs in anaero-
bic metabolism, is thermodynamically favorable, reducing Fd from NADH is thermody-
namically unfavorable. As previously covered, enzymes overcome this by coupling Fd
reduction with a thermodynamically favorable reaction, such as using the pmf in Rnfs,
redistributing exothermic reactions with electron bifurcation in Fix, or the input of light
energy in RC/PSI. Reducing Fd directly from NAD(P)H, however, is possible via FNR
under high NADPH/NADP
1
ratios (Equation 5) (Fig. 2f).
FIG 4 Electron transport to nitrogenase in PNSB. a) A generalization of Fd and ATP production in R. palustris and R.
rubrum with cyclic electron transport between the rection center 1 (RC), which reduces quinone to quinol (QH
2
) and
oxidation of QH
2
by cytochrome bc
1
and while reducing cytochrome c (Cytc) and translocating protons, Cytc is then
oxidized by RC to complete the cycle. NADH is produced by the reverse reaction of NADH dehydrogenase (NDHI). Fix
catalyzes the production of Fd in R. palustris and R. rubrum through electron bifurcation where half of the electrons are
directed to the reduction of quinone, either contributing to cyclic electron transport of reduction of NAD
1
. b) Fd and ATP
production in R. capsulatus and R. sphaeroides is very similar to the ETC of R. palustris and R. rubrum but does not contain
Fix but only contains Rnf. Rnf consumes pmf and competes with ATP synthase and NDH-I for pmf.
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NADP112Fdred 1H1
$NADPH 12Fdox (5)
FNR was rst isolated from pea thylakoids and was shown to be the nal step in
photosynthetic electron transport from Fd to NADP
1
(161, 162). FNRs ubiquity among
living organisms and its diversity of cellular roles highlight the requirement for this
simple electron transfer mechanism. While thermodynamically favorable in the direc-
tion of Fd oxidation and NAD(P)
1
reduction, many FNRs serve primarily in the reverse
Fd reduction direction (163). So how does FNR overcome the energy barrier of Fd
reduction and avoid added input energy mechanisms seen in Rnf, Fix, or PSI?
While there is a diversity of FNRs and well characterized FNRs in photosynthetic
electron transfer, we will focus on FNRs involved in nitrogen xation (see reviews [164,
165] for more on other FNRs). The involvement of FNR in nitrogen xation is well char-
acterized in the lamentous cyanobacteria Anabaena sp. PCC 7120, which forms speci-
alized heterocyst cells that create a microaerobic environment to protect nitrogenase
from oxygen. Heterocysts depend on vegetative cells for electrons and carbon, and
vegetative cells depend on xed nitrogen from the heterocysts, making them interde-
pendent (166, 167).
In Anabaena, 2 separate transcriptional start sites produce 2 isoforms of FNR from a
single gene (petH). There is an ;34 kDa short FNR (FNR
S
) and a longer ;46 kDa FNR
(FNR
L
), which contains an extra N-terminal domain (168, 169). This N-terminal domain
allows interaction with cyanobacteria light-harvesting complexes called phycobili-
somes (PBS), where interaction in the FNR
L
-PBS complex increases the activity toward
NADP
1
reduction (170172). In contrast, the FNR
S
isoform accumulates in the hetero-
cyst under nitrogen and Fe-decient conditions and is not associated with the thyla-
koid membrane (173). The cellular localization suggests specic roles for the isoforms
in either the forward reaction to reduce NADP
1
for FNR
L
or the reverse reaction of Fd
reduction for FNR
S
. While subcellular localization and protein interaction may help
facilitate the reversibility of the reaction, other studies have shown that cofactor envi-
ronment and protein surface residues tune the reduction potential of the FMN cofactor
(reviewed in reference [165]). While tuning the cofactors allows for reversibility, reac-
tion rates still play a signicant role in the overall directionality of the reactions. Within
the heterocyst, the NADP
1
/NADPH ratio is usually found in a reductive state of ;0.01,
powered by the fast glucose-6-phosphate dehydrogenase and oxidative pentose phos-
phate pathway (OPP) (32). In vitro studies with NADPH, FNR, oxidized Fd, and nitroge-
nase showed maximal nitrogenase activity with a ratio of ;0.01 NADP
1
/NADPH (174,
175). The reduction potential of Fd also affects the overall favorability and kinetics of
the reaction. Heterocysts have a specic Fd (HtFd), expressed during nitrogen starva-
tion, that has a higher reduction potential at 2351 mV than its vegetative counterpart
(VFd) at 2384 mV (Fig. 2f) (176). The HtFd is important for nitrogen xation, but other
redundant electron carriers supplement electron transport when deleting the HtFd
gene (177). Overall, at equilibrium, the reduction of Fd directly from NADPH is thermo-
dynamically unfavorable; however, cell physiology, cofactor environment, and cellular
localization can modulate the reaction parameters to overcome the thermodynamic
barriers.
Heterocyst metabolism specializes in protecting nitrogenase from oxygen while
maintaining ATP and Fd for nitrogen xation. First, photosystem II and its water oxidiz-
ing activity is limited, while PSI is upregulated, and cyclic electron transport is used to
create pmf for ATP synthesis (178). Carbon xation through rubisco is not active, and
heterocysts do not contain carboxysomes (179, 180). Nitrogen metabolism also shifts
as the glutamine oxoglutarate aminotransferase enzymes are not expressed in hetero-
cysts, leaving glutamine synthetase to incorporate ammonia from nitrogenase and
transport glutamine back to the vegetative cells (181). An increase in respiration also
occurs through 2 specialized cytochrome c-type terminal oxidases (182).
Energy in the form of carbohydrates, in particular sucrose, is imported to the heter-
ocysts from the photosynthetic vegetative cells and metabolized through the OPP
(183). Deriving the right balance of low potential electrons and ATP for nitrogen
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xation from the OPP pathway requires delivering electrons from NADPH to Fd, as well
as to linear electron transport in the thylakoid membrane to produce ATP. Original
experiments in crude extracts showed that nitrogenase activity could be measured
with the addition of NADP
1
, glucose-6-phosphate, and a renewable ATP source, dem-
onstrating the use of the OPP pathway to produce Fd (184). In addition, nitrogenase
was active both in the light and the dark but had higher activity in the light, indicating
the possibility of directly reducing nitrogenase through NADPH by FNR in the dark and
the thylakoid membrane supporting nitrogen xation under light conditions (184,
185). Within the ocean environment, nitrogen xation can occur at night, even if at a
much lower rate (185, 186). Thylakoid membrane reduction of Fd also contributes to
nitrogenase as dibromothymoquinone (DBMIB) which blocks the plastoquinone reduc-
tion of Cytb6/f, inhibits nitrogen xation (187). DBMIB inhibition suggests that NADPH
electrons enter linear electron transport at plastoquinone, and that PSI can produce Fd
for nitrogenase.
Thylakoid membranes of cyanobacteria contain an NAD(P)H:quinone oxidoreduc-
tase named NDH-1, similar to the complex I found in mitochondria (188, 189). The
NDH-1 complex, along with ATP synthase and PSI complexes, are more abundant in
heterocysts cells than in vegetative cells (190, 191). Recently, Fd has been implicated
as the electron donor to NDH-1 as cyanobacterial NDH-1 lacks NADH binding subunits
and should be considered a ferredoxin:plastoquinone oxidoreductases (192194). This
new insight places Fd as the central electron carrier in heterocysts where Fd produced
either by FNR or PSI is responsible for nitrogen xation, linear electron transport, and
terminal oxidase activity (Fig. 5).
While heterocysts maintain a microaerobic environment through morphological adap-
tations, consumption of excess O
2
within the heterocysts is required for nitrogenase pro-
tection. Flavodiiron protein (FDP), which reduces O
2
directly to H
2
O using Fd as an elec-
tron source, is responsible for light-induced O
2
uptake in heterocysts of Anabaena sp. PCC
7120 (195). Multiple terminal oxidases contribute to oxygen consumption, including the
heme-copper-type cox1 and cox2 genes encoding 2 aa
3
-type terminal oxidases and a cox3
gene cluster encoding a quinol oxidase. During nitrogen xation, only cox2 and cox3 are
expressed in heterocysts along with a cytochrome c
6
that delivers electrons specically to
the caa
3
-type Cox2 terminal oxidase and is required for nitrogen xation (196). Uptake hy-
drogenase (Hup) also plays a crucial role in maintaining redox status in the cell. Although
they do not directly produce Fd in the heterocyst, Hups recycle H
2
from nitrogenase back
into quinone (197). Heterocysts must maintain the ux of electrons to nitrogenase and
through the respiratory chain to produce ATP. Then, electrons also must be prioritized in
terminal oxidases and FDPs to protect nitrogenase from any excess oxygen. The total bal-
ance of these reactions has not been fully investigated.
More work is needed to elucidate the electron transport mechanism to nitrogenase
in heterocysts, but a main electron transport network can be proposed. First, carbon
from vegetative cells enters as sucrose and is hydrolyzed to hexoses or hexose phos-
phates, then metabolized through the OPP pathway to create NADPH, and this NADPH
reduces Fd using the FNR
S
isoform. From here, reduced Fd can be delivered directly to
nitrogenase or used in linear electron ow to produce pmf and ATP. Electrons in
reduced Fd can also be used to consume O
2
and protect nitrogenase through multiple
terminal oxidases and FDPs, which limits O
2
in the cytoplasm (Fig. 5) (195, 196). As
stated in the section above, FNR
S
activity primarily depends on the NADPH/NADP
1
and Fd
red
/Fd
ox
ratios. Maintaining high NADPH and low reduced Fd concentrations
could also be controlled by the thylakoid membranes and varying terminal electron
acceptors (198).
TRANSFER OF NITROGENASE TO NON-DIAZOTROPHIC ORGANISMS
As shown above, bacterial metabolisms have been able to adapt their metabolisms to
support the strict criteria of nitrogenase. These complex adaptations require specicelec-
tron transport enzymes but also a total rebalance of metabolism and redox state. Efforts
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to transfer nitrogenase into non-diazotrophic organisms have become a promising strat-
egy to overcome the disadvantages of industrial nitrogen fertilizers in agriculture (199
201). Signicant advances have been made in synthesizing nitrogenase subunits or its
biosynthetic enzymes in yeast, plant mitochondria, and non-diazotrophic cyanobacteria
(202205). In addition to the challenges of synthesizing a working nitrogenase in non-
diazotrophic organisms, the metabolic context around nitrogenase must be considered.
Yang et al. tested plant FNRs and Fds ability to reduce nitrogenase in an E. coli construct.
The authors rst showed that Klebsiella oxytoca PFOR reduced chloroplast and root-plas-
tid Fds, and these reduced plant Fds could support nitrogenase activity in vivo (206).
FIG 5 Electron transport in heterocyst of Anabaena sp PCC 7120. (1) Carbon enters the heterocyst from the vegetative
cells, usually as sucrose, entering the oxidative pentose phosphate (OPP) pathway through fructose-6-phosphate (F6P) to
produce NADPH. (2) NADPH is oxidized by the short isoform of ferredoxin:NADPH oxidoreductase (FNR
S
). Reduced Fd
then has multiple paths; the main pathway (3) would be the reduction of the Fe protein in nitrogenase for nitrogen
xation. Another path (4) for Fd is the cyclic electron transport starting by reducing plastoquinone to plastiquinol (PQH
2
)
in the ferredoxin:plastoquinone oxidoreductase (NDH-1). Plastiquinone is oxidized by cytochrome b
6
/ftranslocating
protons across the membrane, and plastocyanin (PC) or cytochrome c
6
(CytC
6
) is reduced (5). Finally, photosystem 1 (PSI)
oxidizes PC and uses energy from photons to reduce Fd (6), completing the cyclic electron transport and producing pmf
for ATP synthase to produce ATP. Heterocysts have a diversity of terminal oxidases capable of reducing O
2
from multiple
electron sources. First, avodiiron proteins (FDP) can reduce O
2
directly from Fd (7). Second, 3 terminal oxidases are
found in the thylakoid membrane of heterocysts. First, ubiquinol-oxidase bd (Cytbd) and Cox3 (8), while less characterized,
are not essential for nitrogen xation but contribute to respiration. Cox2 (9) exclusively accepts electrons from Cytc
6
and
translocate protons for ATP synthase.
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Further, they tested the ability of plant FNRs and plant Fd to supply nitrogenase with
electrons and saw a restoration of ;45% of nitrogenase activity compared to K. oxytoca
PFOR and Fld. This review highlights that many mechanisms can provide low potential
reducing equivalents for nitrogen xation, but each mechanism is adapted for a specic
role in each diazotroph. Total physiology, such as maintenance of NADP
1
/NADPH ratios,
Fd reduction potentials, and oxygen protection mechanisms, must be considered if the
goal is to support nitrogen xation with plant FNRs in plant organelles such as root
plastids.
Conclusion. The reduction of N
2
by nitrogenase is one of the most energy-intensive
and complex reactions in biology. A large amount of energy is required, and nitroge-
nases susceptibility to oxygen damage limits its potential in aerobic environments.
Even with the intense evolutionary pressure of nitrogen limitation, life has not been
able to reduce N
2
through any other enzymatic mechanism, nor has it made the
requirements for the nitrogenase reaction conditions less stringent. Diazotrophs utilize
specic electron transport enzymes depending on the mode of metabolism to supply
low potential electrons for nitrogenase catalysis such as substrate-level Fd oxidoreduc-
tases, hydrogenases, photosystem I or other light-driven reaction centers, electron
bifurcating Fix complexes, proton motive force-driven Rnf complexes, and Fd:NAD(P)H
oxidoreductases. Despite the diversity in pathways and enzymes, each diazotroph has
the same criteria of maintaining a microaerobic environment, producing low potential
electrons, and providing a high amount of ATP to maximize ux to nitrogenase.
ACKNOWLEDGMENTS
This work was supported by the U.S. DOE, Ofce of Science, Ofce of Basic Energy
Sciences, under award DE-SC0018143 to J.W.P. Partial salary for J.W.P. was supported by
the United States Department of Agriculture National Institute of Food and Agriculture,
Hatch umbrella project #1015621.
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