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Nitrogenase Complex
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Article Contents
Paul W Ludden, University of Wisconsin, Madison, Wisconsin, USA
. Introduction
The nitrogenase complex carries out the fixation of nitrogen by reducing molecular
dinitrogen (N2) to ammonium (NH41 ) and also reduces acetylene to ethylene. The two
component proteins are dinitrogenase and dinitrogenase reductase. Although different
metals can form the metal clusters contained in nitrogenase proteins, all nitrogenases have
similar properties. The nitrogenase enzyme system is widely distributed among the
Bacteria and the Archaea, with protein sequences highly conserved across the kingdoms.
. Structure of the Nitrogenase Complex
. Dinitrogenase Reductase
. Dinitrogenase
. Metal Clusters of Dinitrogenase
. Alternate Forms of Dinitrogenase
. Detailed Mechanism for Nitrogen Fixation
. Oxygen Lability of Nitrogenase Proteins
Introduction
Nitrogenases carry out the reduction of molecular
dinitrogen (N2) to ammonium (NH41 ) according to the
reaction shown in eqn [I].
N2 1 nMgATP 1 8e 2 1 10H 1 !2NH41
1 nMgADP 1 H2
[I]
(n 2 per electron)
Although the overall reaction is slightly exothermic, the
very high bond strength of the N–N triple bond
(225 kcal mol 2 1) demands a very high energy of activation
for bond breakage, and thus the reaction is considered to
be one of the more difficult reactions catalysed by a
biological system. The industrial conversion of N2 to
ammonium by the Haber–Bosch process requires both
high temperature and pressure of N2 and H2 gases.
The nitrogenase enzyme system is widely distributed
among Bacteria and the Archaea (Young, 1992), but no
eukaryotic system has been shown to contain a nitrogenase. (Some plants, most notably the legumes, form
symbioses with nitrogen-fixing bacteria and thus nitrogen
fixation is often considered to be a ‘plant process’.
However, the actual nitrogen fixation in these symbioses
is performed by the nitrogenase enzyme complex encoded,
synthesized and localized in the bacterial endophyte of
these symbioses.) The nitrogenases from all studied
systems have very similar properties. Most of the discussion in this article is based on the properties and activities
of some well-studied systems: Azotobacter vinelandii (an
obligate, aerobic soil bacterium), Klebsiella pneumoniae (a
facultative, anaerobic bacterium), Clostridium pasteurianum (an obligate anaerobic bacterium) and Rhodospirillum
rubrum (a phototrophic bacterium). Where germane, the
properties of nitrogenases from other systems will be
noted.
Structure of the Nitrogenase Complex
The nitrogenase enzyme complex consists of two component proteins: dinitrogenase and dinitrogenase reductase.
Dinitrogenase reductase is the unique and specific electron
donor to dinitrogenase. Dinitrogenase accumulates electrons and catalyses the reduction of substrates, including
the biologically and agronomically important substrate,
N2. The properties of the component proteins are
described in the following sections. This article will also
deal primarily with the structural gene products of the nif
(nitrogen fixation) regulon (nifHDK), but will refer to some
of the other gene products of the nif regulon as well.
The properties of the nitrogenase proteins are remarkably conserved in nature. The primary sequence of the
dinitrogenase reductase protein is at least 70% identical
among species and across kingdoms (from the Bacteria to
the Archaea), and the primary sequences of the dinitrogenase subunit proteins are also highly conserved. Current
knowledge suggests that the metal clusters of all nifencoded nitrogenases are identical, and the electron
paramagnetic resonance (EPR) spectra of the various
proteins are remarkably similar. Furthermore, it is possible
to mix component proteins from different organisms (a
dinitrogenase reductase from one organism and a dinitrogenase from another) and form a functional nitrogenase
in almost every case. In the few cases where a functional
mix is not observed (e.g. the dinitrogenase from A.
vinelandii and the dinitrogenase reductase from C.
pasteurianum), the lack of activity is due to formation of
a complex that is too tight, rather than an inability to
interact.
The complex between the components has been studied
in a number of ways. Most notably, the crystal structure of
dinitrogenase reductase bound to dinitrogenase has been
elucidated. This structure shows that the dinitrogenase
reductase protein undergoes a significant conformational
change upon binding to dinitrogenase, whereas the
dinitrogenase protein remains relatively unchanged in
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1
Nitrogenase Complex
physiologically and catalytically relevant. The midpoint
potential for the interconversion of oxidized and oneelectron reduced forms of dinitrogenase reductase is about
–250 mV in the absence of nucleotides (ATP); this value is
lowered to approximately –380 mV in the presence of
nucleotides. This decrease of midpoint potential upon
binding MgATP is thought to be significant in the ability of
dinitrogenase reductase to pass electrons to dinitrogenase.
A decrease in midpoint potential is also seen upon binding
of MgADP.
Dinitrogenase reductase binds two molecules of
MgATP at a pair of sites distal from the site of the Fe4S4
cluster, at the approximate locations indicated in the
cartoon in Figure 1 (Seefeldt et al., 1992). Each subunit
binds one molecule of MgATP. Upon binding MgATP,
dinitrogenase reductase undergoes a conformational
change that can be observed by the increased susceptibility
of the Fe4S4 cluster to iron chelators and by a change in the
line shape of the EPR spectrum of the reduced protein. The
Kd for MgATP for the reduced form of dinitrogenase
reductase from A. vinelandii is 300 mmol L 2 1, while the Kd
for MgATP binding to the oxidized form is much lower,
approximately 100 mmol L 2 1. The Kd for binding of
MgADP is near 100 mmol L 2 1 for the reduced enzyme
and again about 40 mmol L 2 1 for the oxidized form of the
enzyme. Each MgATP binding site is capable of binding
MgADP, but other nucleotides are poor ligands for
dinitrogenase reductase and only guanosine 5’-triphosphate (GTP) is capable of supporting poor electron
shape (Schindelin et al., 1997). Several types of complex
have been investigated. A form of posttranslational
regulation of nitrogenase activity that occurs in some
organisms involves the attachment of ADP-ribose at an
arginine residue near the Fe4S4 binding site of dinitrogenase reductase; this ADP-ribosylation prevents the binding
of the two proteins.
Dinitrogenase Reductase
Dinitrogenase reductase (also referred to as the iron
protein, the Fe protein, or NifH) is an a2 dimer of the
nifH gene product and it contains a single Fe4S4 cluster that
bridges the two subunits of the protein at cysteines 98 and
132 of each subunit. The protein contains about 290 amino
acids and the primary sequences of dinitrogenase reductases from even the most divergent species are highly
conserved. The protein has the approximate shape shown
in Figure 1, with the Fe4S4 cluster sitting near the surface at
the c2 axis of protein symmetry. The molecular structures
of crystalline forms of the protein in the ADP-bound form
and in a complex with its electron acceptor, dinitrogenase,
have been determined (Georgiadis et al., 1992).
Dinitrogenase reductase exists in the oxidized
([Fe4S4]2 1 ), one-electron reduced ([Fe4S4] 1 ) and twoelectron reduced ([Fe4S4]0) forms. Of these, the oxidized
and one-electron reduced forms are considered to be
FeMo-co
P
P
e– donor
FeMo-co
Dinitrogenase
1e–
MgATP
FeS
FeS
MgATP
Multiple
cycles of
electron
transfer
with ATP
hydrolysis
2MgATP
Dinitrogenase
reductase
(oxidized)
Dinitrogenase
reductase
(reduced)
C2H2
H+
N2
FeMo-co
P
C2H4
–
2e
reduction
H2
+
P
FeMo-co
2NH4
–
8e
reduction
Dinitrogenase
(reduced)
Figure 1 The path of electron flow in nitrogenase.
2
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Nitrogenase Complex
transfer from dinitrogenase reductase to dinitrogenase.
Mg2+ is required for effective binding of adenine nucleotides to the enzyme.
A number of spectroscopic techniques have been applied
to the study of dinitrogenase reductase. The protein shows
a decrease in absorbance at 430 nm when the Fe4S4 cluster
is reduced to the 1 1 state, and this change has been useful
in monitoring the oxidation status of the protein. The 1 1
oxidation state of the enzyme also exhibits a mixed s 5 1/2,
s 5 3/2 spin system, which can be observed by EPR at low
temperatures ( 5 30 K). The oxidized enzyme is EPRsilent. EPR has been a useful tool in establishing the redox
state of the enzyme in freeze-quenched samples. The EPR
spectrum of reduced dinitrogenase reductase changes upon
binding MgATP.
The optimal accumulation of functional dinitrogenase
reductase in vivo is observed in cells that have functional
nifM, nifS and nifU genes. The gene products of nifU and
nifS are involved in the synthesis of the Fe4S4 cluster of the
protein, while the nifM gene product plays an unknown
role in the maturation of dinitrogenase reductase.
Dinitrogenase reductase performs other roles in addition to the transfer of electrons to dinitrogenase. In the
absence of dinitrogenase reductase, the cells fail to
complete the synthesis of the iron–molybdenum cofactor
of the dinitrogenase protein (see below). Furthermore,
dinitrogenase reductase is required for the proper insertion
of the iron–molybdenum cofactor into the dinitrogenase
protein. Thus, the protein has at least three roles in the
nitrogen fixation process. Dinitrogenase reductase that
lacks its Fe4S4 cluster is unable to transfer electrons, but
can still perform its roles in the biosynthesis and insertion
of the iron–molybdenum cofactor.
Dinitrogenase
Dinitrogenase is an a2b2 tetramer of the nifD and nifK gene
products and has a molecular weight of approximately
240 kDa. Dinitrogenase is the site of substrate reduction,
and this protein contains two each of two unique metal
clusters, the P cluster and the iron–molybdenum cofactor
(FeMo-co) (as depicted in the cartoon in Figure 1; the
structures of the two cofactors are shown in Figure 2). No
nucleotide binding sites that are essential to the reduction
of substrates have been reported on the dinitrogenase
protein. The MgATP molecules that bind to dinitrogenase
reductase and are hydrolysed during electron transfer from
dinitrogenase reductase to dinitrogenase do not come into
contact with dinitrogenase. The dinitrogenase tetramer is
essentially a pair of dimers, each of which functions
independently in the reduction of substrates. The distance
between the two FeMo-co dimers is approximately 7 nm,
and it is unlikely that the two cofactor molecules interact.
The distance between the P cluster and the FeMo-co dimer
in an ab dimer of the protein is about 1.4 nm. Electrons are
thought to enter the protein from the Fe4S4 cluster of
dinitrogenase reductase via the P cluster, coming finally to
FeMo-co, which serves as the site of substrate reduction on
the enzyme. The concept that FeMo-co is the site of
substrate reduction relies on the observation that the
substrate specificity and inhibitor susceptibility of nitrogenase are affected when altered forms of FeMo-co are
incorporated into the dinitrogenase protein. Furthermore,
some inhibitors of dinitrogenase, such as carbon monoxide, have direct effects on the EPR spectrum of FeMo-co
in the dinitrogenase protein. Dinitrogenase has no activity
by itself and cannot be reduced to a catalytically competent
form by any reductant other than dinitrogenase reductase.
The nitrogenase proteins exhibit EPR signals due to
unpaired electrons on their metal clusters, and these have
been very useful in determining the path of electrons
through the enzyme. The reduced form of dinitrogenase
reductase exhibits an EPR signal g 5 1.94 that is characteristic of Fe4S4 clusters. Dinitrogenase has a unique
EPR signal in its reduced ‘resting state’ at g 5 3.65. When
the proteins are mixed in the presence of MgATP and a
suitable electron donor (in vitro, the chemical reductant
sodium dithionite is commonly used), substrate reduction
begins; the EPR signal of dinitrogenase protein is then lost
owing to its oxidation, and EPR signal of dinitrogenase at
g 5 3.65 is lost when FeMo-co goes to a more reduced
state.
Substrates for nitrogenase
Nitrogen (N2) is the biologically significant substrate for
nitrogenase, and its reduction to ammonium allows
bacteria that are capable of fixing N2 to grow in the
absence of a fixed nitrogen source. However, nitrogenase is
able to reduce a number of triple- and double-bonded
substrates. Furthermore, in the absence of N2 or other
substrate, nitrogenase reduces protons to H2 at a rate
equivalent to the maximal electron flow when saturating
N2 is available. In fact, it is never possible to isolate the
enzyme in its most reduced form, because the enzyme will
always become reoxidized through the reduction of
protons. Even at great excess of dissolved N2, the enzyme
diverts 25% or more of the electrons that pass through
FeMo-co for the reduction of protons. This is interpreted
as a requirement for the reduction of one pair of protons
(2e 2 ) for each N2 reduced to ammonium (6e 2 ).
Nitrogenase will also perform a number of other
substrate reductions including the two-electron reduction
of acetylene to ethylene, the six-electron reduction of CN 2
to CH4 and NH41 , the two-electron reduction of azide to
N2 and ammonium, and the reduction of carbon dioxide to
carbon monoxide. Of these, the reduction of acetylene is
the most notable, as it provides a widely used, rapid and
simple assay for the enzyme in which an inexpensive gas
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Nitrogenase Complex
Cys
S
Fe
S
S
S
Cys
The Fe4S4 cluster
of dinitrogenase
reductase
Fe
S
Fe
S
Cys
S
Fe
S
Cys
O
O
S
S
cys
S
Fe
S
S
Fe
Fe
FeMo-co of
dinitrogenase
H
Fe
S
O
H
Mo
S
Fe
Fe
Fe
O
S
H
N
his
S
H
H
H
H
S
O
O
Fe
Fe
Fe
Fe
Fe
The P cluster
of dinitrogenase
Fe
Fe
Fe
Figure 2 Metal clusters of nitrogenase proteins.
chromatograph is used to detect the product ethylene. The
acetylene reduction assay also allows the accurate measurement of nitrogenase enzyme activity in vivo; very few
enzymes can be measured in vivo in a manner as easily as
nitrogenase. The acetylene reduction assay is depicted in
the cartoon in Figure 3. Unlike N2, acetylene is able to
completely suppress proton reduction by nitrogenase,
indicating that proton reduction is not required for the
simpler two-electron reduction of acetylene. A correction
for this difference must always be made when using results
4
of the acetylene reduction assay to estimate rates of N2
reduction.
Metal Clusters of Dinitrogenase
As noted above, dinitrogenase contains two unique metal
clusters (Figure 2). The first of these, the P cluster, is shown
in its reduced (PN) form. This form of the cluster is of
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Nitrogenase Complex
Add enzyme or
cells via syringe
Add C2H2
via syringe
(10% C2H2;
90% Ar or He)
C2H2/Ar
Serum stoppered vial,
filled with Ar or He
Incubate
with shaking
for 10 min
Take a
0.5 –1.0 mL
gas sample
via syringe
C2H2/Ar
C2H4
Inject sample
into gas
chromatograph
Recorder
Gas chromatograph
C2H4
C2H2
Activity -C2H4
peak height
Flame ionization
detector
Figure 3 The acetylene reduction assay.
interest because it features a central sulfur atom that is
hexagonally coordinated by six iron atoms. All iron atoms
of the P cluster are in the ferrous state and the cofactor is
thought to undergo oxidation and reduction by the
reversible cleavage of one iron bond to the central sulfur
atom. The P cluster sits at the interface of the ab subunit
pair and is bound to the protein by cysteine residues from
both proteins. The P cluster has not been isolated from the
dinitrogenase protein, and the biosynthetic pathway for
the P cluster is not known at this time. No mutants that
produce a stable form of dinitrogenase lacking the P cluster
have yet been isolated.
The other unique cofactor of dinitrogenase is the iron–
molybdenum cofactor (FeMo-co) (Shah and Brill, 1977).
FeMo-co consists of MoFe7S9 and the seven-carbon
organic acid, homocitrate (Figure 2); its structure was
deduced when the structure of the dinitrogenase protein
was determined by X-ray diffraction (Kim and Rees, 1992).
FeMo-co is found entirely in the a subunit (nifD product)
of dinitrogenase and is bound to the protein by his442
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5
Nitrogenase Complex
ligation to the molybdenum and cys275 ligation to the iron
atom most distal from the molybdenum atom. FeMo-co
can be isolated by extraction of denatured dinitrogenase
protein with organic solvents such as N-methyl formamide
(NMF) and is stable in anaerobic NMF solution. Isolated
FeMo-co can be used to activate FeMo-co-deficient
dinitrogenase (apodinitrogenase) isolated from mutant
strains unable to synthesize FeMo-co. Apodinitrogenase is
stable in several forms in vivo and in vitro. At the time of
writing, the site of N2 or other substrate binding to FeMoco is unknown.
Isolated FeMo-co is incapable of reduction of N2,
protons or acetylene, and the binding of these substrates to
isolated FeMo-co has not been demonstrated. Isolated
FeMo-co exhibits an s 5 3/2 spin system that is detectable
by EPR; the EPR of isolated FeMo-co is similar to that of
FeMo-co in the protein, but the line shape is significantly
broader. It has not been possible to reduce isolated FeMoco to the EPR-silent state of FeMo-co observed in freezequenched samples of the nitrogenase complex in steady
state turnover. Thus it appears that the dinitrogenase
protein provides a unique environment for the function of
FeMo-co.
Biosynthesis of the metal clusters of
nitrogenase proteins
Biosynthesis of the FeS cluster of dinitrogenase reductase
The nifU and nifS products have been implicated in the
biosynthesis of the Fe4S4 cluster of dinitrogenase reductase. NifS is a pyridoxal phosphate-dependent cysteine
desulfurylase (Zheng and Dean, 1994). This reaction is
thought to provide S2 2 for FeS cluster synthesis. NifU is
isolated as an Fe2S2-cluster-containing protein and is
thought to accumulate iron for cluster synthesis for
dinitrogenase reductase. Homologues of nifS have now
been found in a range of bacteria, plants and animals and
are thought to play a role in FeS cluster synthesis for a wide
range of ferredoxins and FeS proteins.
Biosynthesis of FeMo-co
The biosynthesis of FeMo-co involves at least the
nifQBNEVHX products. NifQ is required only under very
stringent conditions of molybdenum starvation, and its
role is fulfilled either noncatalytically or by other proteins
under molybdenum-sufficient conditions. NifB is responsible for the production of NifB-co, an iron and sulfur
donor to FeMo-co. NifV is homocitrate synthase and uses
a-ketoglutarate and acetyl–CoA to make the homocitrate
component of FeMo-co. The nifN and nifE genes have
sequence similarity to nifD and nifK, and their products
form an a2b2 tetramer with similarity to dinitrogenase.
NifNE is proposed to serve as a scaffold upon which at least
a portion of FeMo-co is built. The role of NifH is not
established, but mutants deficient in nifH do not accumu6
late FeMo-co and NifH (dinitrogenase reductase) is
required for synthesis of FeMo-co in vitro. The role of
NifX is known only from its stimulation of FeMo-co
synthesis in vitro, and there is little or no effect of mutation
of nifX on FeMo-co accumulation in vivo. Note that the
dinitrogenase structural gene products (NifD and NifK)
are not required for FeMo-co synthesis in vivo or in vitro.
Thus, the cofactor is synthesized and then inserted into the
FeMo-co-deficient enzyme rather than being constructed
on site.
Using extracts of A. vinelandii or K. pneumoniae, an in
vitro FeMo-co synthesis system has been devised and used
to study steps of the biosynthesis of FeMo-co. Although
not required for FeMo-co synthesis, FeMo-co-deficient
dinitrogenase is included in the assay mixture so that the
completed FeMo-co can be inserted into dinitrogenase and
its activity determined by the acetylene reduction assay.
Isolated NifB-co will bind to NifNE in the absence of any
other factors, but no further processing of the iron of NifBco takes place in the absence of other factors. When
MgATP, MoO24 2 , dinitrogenase reductase (NifH), NifX,
homocitrate and reductant (in the form of dithionite) are
added to the mixture, FeMo-co is synthesized and can be
inserted into dinitrogenase.
Insertion of FeMo-co
The insertion of FeMo-co requires the FeMo-co-deficient
form of dinitrogenase, FeMo-co, NifH (dinitrogenase
reductase), and a non-nif protein called gamma. Strains of
A. vinelandii that cannot produce FeMo-co, but have a
functional nifH gene, produce a hexameric form of FeMoco-deficient dinitrogenase with two subunits each of NifD,
NifK and gamma. This form of FeMo-co-deficient
dinitrogenase can be activated in vitro by the addition of
purified FeMo-co. On the other hand, if the strain that is
unable to make FeMo-co does not contain NifH, then a
tetrameric form of dinitrogenase containing only the NifK
and NifD subunits is isolated and NifH and MgATP are
required for the attachment of gamma to FeMo-codeficient dinitrogenase. Once gamma has performed its
role of inserting FeMo-co, it dissociates from dinitrogenase and is not necessary for function of the enzyme. MoS24 2
is a strong inhibitor of FeMo-co insertion. No stable form
of dinitrogenase that lacks the P cluster has been isolated.
Alternate Forms of Dinitrogenase
When A. vinelandii is grown on medium lacking molybdenum, it is able to produce a vanadium-containing
nitrogenase if vanadium is present and a form of
nitrogenase that contains no transition metal other than
iron if neither molybdenum nor vanadium is present
(Bishop and Premakumar, 1992). The structural genes for
these nitrogenases are distinct from the structural genes for
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Nitrogenase Complex
the molybdenum-containing, nif-encoded nitrogenase.
The vanadium-nitrogenase is encoded by the vnf regulon
and the iron-only nitrogenase is encoded by the anf
regulon. Although genetically distinct, the systems are
significantly similar. For example, the dinitrogenase
reductase of the vnf-encoded system will donate electrons
to the nif-encoded dinitrogenase and it will substitute for
the nif-encoded dinitrogenase reductase in both FeMo-co
synthesis and FeMo-co insertion.
Although the structures of the metal centres of the
components of the vanadium and iron-only nitrogenases
have not been determined by X-ray crystallography, the
FeS centre of dinitrogenase reductase and the P cluster of
dinitrogenase are thought to be identical to those shown in
Figure 2 for the molybdenum system; the active site
cofactors are thought to differ only by the substitution of
vanadium or iron for molybdenum in FeMo-co. Isolated
FeV-co and FeFe-co have been shown to substitute for
FeMo-co in the FeMo-co-deficient dinitrogenase and to
catalyse at least acetylene reduction in that protein
environment. Furthermore, the nifB gene, which encodes
the protein responsible for producing the iron and sulfur
precursor for FeMo-co (NifB-co), is required for all three
systems. The nifV gene, which encodes homocitrate
synthase, is also required for all three systems.
One significant difference between the nif-encoded
nitrogenase system and the alternate nitrogenase systems
is the presence of a third subunit, the VnfG or AnfG
protein, in the dinitrogenase system. Although there is not
significant sequence similarity between VnfG or AnfG and
any protein involved in the nif-encoded nitrogenase
system, VnfG and AnfG are thought to perform a role
analogous to the FeMo-co insertase role played by gamma.
Beyond the insertion of FeV-co and FeFe-co, respectively,
VnfG and AnfG are required for the stability or function of
the completed dinitrogenase proteins.
The distribution of alternate nitrogenase systems vary.
Some organisms, like A. vinelandii, have all three systems.
A close relative, Azotobacter chroococcum, has only the nif
and vnf systems, while the phototrophs, such as Rhodobacter capsulatus and Rhodospirillum rubrum, contain the
genes for the nif and anf systems, but not for the vnf system.
Many organisms appear to have only the molybdenumcontaining, nif-encoded system. All naturally occurring,
nitrogen-fixing organisms isolated to date contain a
molybdenum system.
Detailed Mechanism for Nitrogen
Fixation
The mechanism of the nitrogenase enzyme complex is an
area of continued investigation. As depicted in the cartoon
in Figure 1, dinitrogenase reductase brings electrons, one at
a time, to dinitrogenase and transfers an electron at the
expense of hydrolysis of two molecules of MgATP. The
structure of dinitrogenase has similarities to the structures
of G proteins, and the transfer of electrons is a gated,
nucleotide hydrolysis-dependent event that involves a
significant conformational change in the dinitrogenase
reductase protein. Because electrons are transferred to
dinitrogenase singularly, and because the reduction of N2
plus two protons to yield two molecules of ammonium and
one molecule of H2 is an eight-electron process (see eqn [I]),
eight individual transfers must occur before the dinitrogenase enzyme is competent to reduce its substrate. In a
model proposed by Roger Thorneley and David Lowe,
dinitrogenase exists in states E0, E1, E2, E3, . . ., E8 (Lowe
and Thorneley, 1984). Some of the substrates for
nitrogenase require fewer electrons – for example,
acetylene reduction to ethylene requires only two electrons,
and it is thought that acetylene can bind to and be reduced
by dinitrogenase in the E3 state. The slow step in each cycle
of the transfer of electrons from dinitrogenase reductase to
dinitrogenase is the dissociation of the oxidized dinitrogenase reductase with MgADP bound from dinitrogenase.
The reduction of N2 to ammonium is proposed to occur
by a number of two-electron steps following the twoelectron ‘priming step’ in which a pair of protons is reduced
to H2. N2 is thought to bind to the active site by displacing
the bound H2 entity. The site and mode of substrate
binding to the active site cofactor (FeMo-co) is still a
matter of conjecture.
Oxygen Lability of Nitrogenase Proteins
The nitrogenase components are among the most oxygenlabile proteins known, and organisms have devised a
variety of strategies to protect the enzyme from molecular
oxygen. A number of strict anaerobes, such as Clostridium
pasteurianum, simply do not grow in the presence of
oxygen, and thus the lability of nitrogenase proteins to
oxygen is avoided. In the obligate aerobic Azotobacters at
least two mechanisms of defence have been described.
First, A. vinelandii dramatically increases and uncouples
respiration when growing with N2 as the nitrogen source;
thus oxygen is removed by reaction with cytochrome
oxidase. Second, when oxygen does accumulate in the A.
chroococcum cell, the oxidized nitrogenase components
form a complex with a flavoprotein and the complex is
significantly stable to O2 (Robson, 1979).
A number of filamentous cyanobacteria have the
problem of producing O2 by the water-splitting reaction
of photosynthesis. To avoid producing O2 at the site of
nitrogen fixation, these cyanobacteria differentiate approximately one cell out of every ten to a thick-walled, gasimpermeable cell called a heterocyst. Heterocysts perform
cyclic electron flow for ATP synthesis, but the water
splitting reactions associated with photosystem 2 and the
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Nitrogenase Complex
noncyclic electron flow do not occur in heterocysts. Other
nonheterocystous cyanobacteria carry out a diurnal cycle
of photosynthesis and nitrogen fixation so that nitrogen
fixation is not attempted during the oxygenic photosynthetic phase of the cycle. The various species of Rhizobium,
Bradyrhizobium and Sinorhizobium are obligate aerobes,
yet they must protect their nitrogenase from oxygen as
well. Here, the plant participates by providing an environment that is rich in bound oxygen to support respiration by
the endophytic symbiont, but in which there is little free,
dissolved O2. This is accomplished by the massive
production by the host plant of a haem-containing, O2binding protein called leghaemoglobin in the cells of
symbiotic tissue (called nodules). Leghaemoglobin ensures
the availability of O2 for respiration, but free O2 does not
accumulate in the nodule tissue and destroy nitrogenase.
Oxygen destroys the nitrogenase components by reacting with the metal clusters of the enzyme. In the case of
dinitrogenase reductase, the Fe4S4 cluster is fairly exposed
to solvent and dissolved O2 readily interacts with the
cluster to oxidize all of the iron atoms to the 3 1 (ferric)
oxidation state – a form of the cluster not stable in the
protein. An interesting feature of the reaction of oxygen
with dinitrogenase reductase is the polymerization of the
resulting protein subunits, perhaps by formation of
intersubunit dithiol bonds between the cysteinate residues
that had served as ligands to the FeS cluster. The results of
reaction of the FeMo-co and P clusters of dinitrogenase
with oxygen are less studied, but it is clear that exposure to
O2 leads to loss of enzyme activity and loss of the
spectroscopic features of those two centres. The dinitrogenase protein is less labile to O2 than is dinitrogenase
reductase, the latter having a half-life of only 30 s when
solutions in buffer are exposed to air. Researchers studying
nitrogenases routinely use glove boxes filled with atmospheres containing 1 ppm or less O2 for handling nitrogenase proteins. Anaerobic buffers used in preparation of
nitrogenases also contain the chemical reductant sodium
dithionite to scavenge any O2 that leaks into the system.
References
Bishop PE and Premakumar R (1992) Alternative nitrogen fixation
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Nitrogen Fixation, pp. 736–762. New York: Chapman & Hall.
8
Georgiadis MM, Komiya H, Chakrabarti P, Woo D, Kornuc JJ and
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Further Reading
Burgess BK and Lowe DJ (1996) Mechanism of molybdenum
nitrogenase. Chemistry Review 96: 2983–3011.
Burris RH (1975) The acetylene-reduction technique. In: Nitrogen
Fixation by Free-living Micro-organisms, pp. 249–257. Cambridge:
Cambridge University Press.
Eady RR (1996) Structure–function relationships of alternative
nitrogenases. Chemistry Review 96: 3013–3030.
Elmerich C, Kondorosi A and Newton W (1998) Biological Nitrogen
Fixation for the 21st Century. Dordrecht: Kluwer Academic.
Howard JB and Rees DC (1994) Nitrogenase: a nucleotide-dependent
molecular switch. Annual Review of Biochemistry 63: 235–264.
Ludden PW and Roberts GP (1989) Regulation of nitrogenase activity
by reversible ADP-ribosylation. Current Topics in Cellular Regulation
30: 23–55.
Peters JW, Stowell MHB, Soltis SM, Finnegan MG, Johnson MK and
Rees DC (1997) Redox dependent structural changes in the
nitrogenase P-cluster. Biochemistry 36: 1181–1187.
Seefeldt LC and Dean DR (1997) Role of nucleotides in nitrogenase
catalysis. Accounts of Chemical Research 30: 260–266.
Stacey G, Burris R and Evans H (eds) (1992) Biological Nitrogen
Fixation. New York: Chapman & Hall.
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