T.

Clarke

The Structure and Mechanism of Mo-Dependent

Nitrogenase Complexes
Introduction________________________________________________________________________
____

The incorporation of nitrogen into biological systems is essential to all known forms of life.
Nitrogen is present in multitude of biological compounds, including proteins, and nucleotides.
However, nitrogen fixation poses a major biochemical problem as the only viable nitrogen
source on earth is atmospheric N 2. Dinitrogen (NN) is a highly inert gaseous molecule with a
total bond dissociation energy of ~1000kJ/mol. Each of the three N-N bonds must be broken to
produce biologically available ammonia (NH 3). It was only in the past century that Fritz Haber
and Carl Bosch developed the first synthetic process for ammonia production. The Haber-Bosch
process is widely used in modern industry; however, the reaction requires mixing N 2 and H2
under extreme pressure and temperature in the presence of rare earth catalysts such as
osmium and ruthenium, and is therefore impossible in biological systems. Natures answer to
the problem is enzymatic nitrogen reduction by a group of bacteria known as diazotrophs.
These bacteria are found free-living in soils around the world, and in symbiotic relationships
with leguminous plants. Diazotrophs possess the necessary enzyme complexes to reduce
molecular dinitrogen to ammonia under standard biological conditions. The most important
enzyme complex in this reduction process is nitrogenase. Nitrogenases are hetero-oligomeric
protein complexes containing multiple redox sites and various transition metals including iron,
vanadium, and molybdenum. Nitrogenases catalyze the reduction of dinitrogen to ammonia
based on the reaction below:
N2 +8e- + 8H+ + 16MgATP 2NH3 + H2 + 16MgADP + 16PI
One of the most energy demanding transformations in nature, hydrolyzing sixteen ATP
molecules, nitrogen fixation is a marvel of biochemistry. The enzyme complex has developed a
highly efficient mechanism to catalyze the reduction of dinitrogen, a process with an energy of
activation of 420 kj/mol1.
As the entry point for nitrogen in biological systems, nitrogenases play a key role in the
nitrogen cycle. The production of ammonia is necessary not only for the production of biological
molecules, but is often further oxidized to nitrate and nitrite which act as terminal electron
acceptors in anaerobic respiration. Three recognized forms of anaerobic respiration exist,
denitrification, respiratory ammonification, and ammanox, all of which produce a proton-motive
force across the bacterial membrane for use in ATP synthesis. The standard biological redox
potentials of the nitrate and nitrite anions (0.74 and 0.1 V respectively) make them moderate
electron acceptors, especially when coupled with NADH oxidation (E=0.32).
Although nitrogenases are diverse
in structure and composition, the
most abundant form of the
enzyme
is
the
molybdenum
variant. Early biochemical analysis
of Mo-nitrogenases indicated two
dissociable iron-sulfur proteins,
now called the Fe and MoFe
proteins2 (Figure
1).
Further
crystallographic
evidence
confirmed the structure of both
proteins. Following resolution of
the structures, experiments were
conducted
to
determine
the
mechanism
by
which
the
nitrogenase
complex
reduces
dinitrogen.
Though
current
experimental techniques have

Figure 1: The Nitrogenase complex isolated from A.

vinelandii. The MoFe protein possesses a plane of
symmetry down the centre, allowing for the association
of two Fe-proteins on either side. The MoFe protein also
possesses an active site on each symmetrical face.

proved inadequate to determine the most intricate steps of the reaction process (this is
discussed later), the general reaction mechanism is broken into three general electron transfer
steps: (i) the reduction of the Fe protein by the electron transporters ferredoxin and flavodoxin
(ii) transfer of electrons from the Fe-protein to the MoFe-protein in a MgATP dependent process
(iii) reduction and protonation of dinitrogen bound to the FeMo cofactor of the MoFe-protein.

The
FeProtein__________________________________________________________________________
The Fe-protein of Mo-nitrogenase is responsible for electron transfer to the MoFe-protein from
several endogenous reductants. The first solved Fe-protein structure was isolated from
Azobacter vinelandii3. The structure was determined to be a homodimer linked by a single
[4Fe:4S] cubane along the dimer interface. The cluster is symmetrically coordinated by residues
Cys97 and Cys132 of each subunit (Figure 2). Each Fe-protein subunit contains an MgATP binding
domain ~15 from the Fe:S cluster. Redox studies on the [4Fe:4S] cluster show three possible
oxidation states, native 2 Ferrous/2
Ferric [4Fe:4S]+2, reduced 3 Ferrous
[4Fe:4S]+1 and all ferrous [4Fe:4S]0
4
.
Reduction of the Fe-protein (the
primary
step
in
nitrogenase
catalysis) is carried out by specific
oxidoreductase enzymes. Initial
experiments
on
anaerobic
Clostridium pasteuranium showed
the endogenous reductase to be
ferredoxin or, under iron deficient
conditions, an inducible flavodoxin 5.
However, in vitro studies of
nitrogenase
isolated
from
Azobacter vinelandii showed no
enzymatic activity without the
addition of artificial reductants such
as
dithionite6.
Subsequent
experiments conducted by Beneman et al. 7 discovered an additional flavoprotein, known as
azotoflavin, responsible for Fe-protein reduction in A. vinelandii.
Resolution of the nitrogenase
Figure 2: The 4Fe:4S cluster of the Fe-Protein. The
crystal structure showed large
cofactor is located on the protein surface along the
surface incompatibility between the
dimer interface. Symmetrical ligation at cys 97 and cys132
native
MoFe
and
Fe-proteins,
provides a covalent link between the two protein
indicating extensive conformational
subunits.
change prior to interaction between the substituent proteins 8. As the Fe-protein has known
nucleotide binding domains, it was theorized that binding of an ATP cofactor would induce
conformational change to favor surface interactions with the MoFe protein. EPR experiments on
reduced Fe-protein species isolated from C. pastueridium showed a change in signal intensity
following MgATP binding, confirming large conformational change in the protein backbone 9. The
nucleotide binding sites in the Fe-protein possess highly conserved residues involved in direct
interaction with the Mg2+ species of MgATP. The Walker A motif (Gly-X-X-X-X-Gly-Lys/Ser) and
Walker B motif (Asp-X-X-Gly) are found in positions 9-16 and 125-128 of each subunit
respectively10. These residues are thought to interact directly with Mg 2+ and the -phosphate of
MgATP bound at the P-loop. Two switch-regions near the P-loop sense changes in the binding
domain through interactions with the Mg 2+ ions, modulating the Fe-protein surface. Residues
lys41 and asp43 of switch-region I detect variations in phosphate composition and Mg 2+
coordination, translating the changes to the protein surface and regulating association with the
MoFe protein. Switch region II communicates changes in the binding domain to the environment
of the [4Fe:4S] cluster 11. The change in [4Fe:4S] environment lowers the cluster reduction
potential by ~100mV, producing the stronger reducing species required for catalysis 12.
Hageman et al.13 showed using pre-steady state enzyme assays that a total of two MgATP
molecules bind to the reduced Fe-protein and are hydrolyzed in the electron transfer process to

the MoFe Protein. Further burst hydrolysis studies by the same group showed a marked lag in H 2
production by the nitrogenase complex not observed when following MgATP hydrolysis in the
same reaction14. The experiments indicate that MgATP hydrolysis is necessary for electron
transfer to the MoFe protein. The hydrolysis process is significant as the majority of biological
redox processes proceed without energy input. It is thus theorized that hydrolysis of MgATP is
required to overcome a kinetic barrier to electron transfer and drive irreversibility of the
reaction3.
The electron transfer process is thought to be a cyclic association and dissociation whereby
MgATP bound Fe-protein binds to the MoFe-protein, carries out electron transfer coupled with
hydrolysis of two MgATP molecules then dissociates to reproduce the oxidized protein. The
electron transfer process to the MoFe protein is carried out eight times until the native
substrate N2 is fully reduced to NH3.

The
MoFeProtein_______________________________________________________________________
The MoFe protein isolated from A.
vinelandii is a symmetric 22
heterotetramer and is known to be
the site of N2 reduction in the
nitrogenase
complex.
Each
symmetrical face of the protein
possesses a separate active site
containing two metal-sulfur clusters
crucial to catalytic activity, the Pcluster and the FeMo-cluster.
Several pieces of experimental
evidence
implicate
the
FeMo
cluster as the active site cofactor in
the complex. Diazotroph strains
deficient in FeMo biosynthetic
pathways
produce
inactive
nitrogenases which are induced
through the addition of exogenous
FeMo
clusters15.
Furthermore,
compositionally
altered
FeMo
cofactors produce variant substrate
reduction
properties16.
It
is
therefore theorized that electron
transfer from the Fe-protein results
in reduction of the FeMo cofactor
through the mediating p-cluster.
Recent crystallographic experiments have shown that the MoFe protein possess a highly rigid
backbone structure which binds the
Figure 3: The various docking orientations of the FeFe-protein at three docking sites17.
protein when nucleotide free, ADP bound, and AMPPCP
Crystal structures of the complex
bound. Crystallographic evidence has shown that the
with nucleotide-free, ADP bound,
MgATP analogue bound Fe-protein docks with its FeS
and AMPPCP bound Fe-protein
cluster ~5 closer to the MoFe P-cluster than the
showed
different
docking
nucleotide free or ADP bound forms. Note: The ADP
conformations depending on the
bound form is less rigid than the other forms and is
cofactor (AMPPCP is a nontherefore represented as a range17.
hydrolyzable analogue of ATP). The
AMPPCP bound Fe-protein was shown to dock to the MoFe protein with its cubane cluster ~5
closer to the MoFe P-cluster than either the ADP or nucleotide-free forms (Figure 3). The
interaction of AMPPCP bound Fe-protein with the MoFe surface brings the p-cluster equidistant
to the 4Fe:4S cluster of the Fe-protein and the FeMo cofactor, implicating the role of the Pcluster as a mediator for electron transfer to the active site 17.

The
PCluster____________________________________________________________________________
The p-cluster is an 8Fe-7S cofactor found along the pseudosymmetric axis of the MoFe
protein and is ligated by six cysteine residues from the and subunits (Figure 4).
Experiments analyzing the structure of the P-cluster in variable oxidation states have implied
the existence of a native state (PN) and several oxidized states (Pox). As a p-cluster reduced
beyond the native state has not been experimentally observed, it is likely that the cofactor
carries out electron transfer through its higher oxidation states. A proposed mechanism is
reduction of the FeMo-cluster by the P-cluster upon Fe-protein binding. This is followed by quick
reduction of the p-cluster by the Fe-protein during the cyclic association process. Experimental
confirmation for this mechanism is provided by stopped flow UV/VIS and EPR studies indicating
oxidation of the p-cluster upon N2 reduction18. Furthermore, the p-cluster redox potential is
estimated at -300mV, which coincides well with the potential of the MgATP-bound Fe-protein
(Eo=-420mV), indicating thermodynamic favorability of downstream electron transfer to the
FeMo cofactor.
Recent
crystallographic
experiments on the MoFeprotein
have
shown
a
rearrangement of the P-cluster
ligands
upon
change
in
oxidation state. The native pcluster exhibits a bridging
sulfur ligated to six Iron
species
in
a
distorted
octahedron, whereas oxidized
forms of the P-cluster show a
tetraligated
central
sulfur
linking distinct 4Fe:4S and
4Fe:3S clusters19 (Figure 5) As
redox dependent structural
changes are rare in Fe:S
clusters it is suggested that the
P-cluster not only mediates
electron transfer to the FeMo
cofactor, but also acts in proton transfer to newly reduced substrates. Further experimentation
showed a pH dependence on the midpoint potential of the p-cluster, supporting the cofactors
role in proton coupled electron transfer20.
The exact process by which electrons are transferred from the p -cluster to the MoFe-cluster is
yet to be fully understood, but site directed mutagenesis studies of tyr 98 (a residue located on
the alpha helix separating the two clusters) has provided evidence to a potential electron
transfer pathway. Mutation of tyr 98 to his perturbed the electron transfer process to the FeMo
cofactor, reducing nitrogenase activity by nearly 50% 21. A mechanism is proposed whereby the
P-cluster reduces tyr98 which then transfers electrons to its hydrogen bond partner homocitrate
of the FeMo cluster. This mechanism requires further validation as mutant proteins containing
phe and leu residues in place of tyr 98 exhibit similar activity to the wild-type protein without
the potential for hydrogen bonds to homocitrate.
Subsequent
mutatagenesis Figure 4: The resting state p-cluster of the MoFe protein. The
studies showed the importance cluster is located near the protein surface at the -dimer
that cys ligands play in p- interface. Shown in the figure is tyr 98 which is thought to
cluster activity. Substitution of mediate electron transfer between the P-cluster and FeMocoordinating cys ligands with cluster.
Ala or Ser resulted in complete
enzyme inactivity or even failure to form the MoFe
tetramer; this also implies the importance of the
p-cluster in structural integrity of the protein
complex22.

The FeMo-Cluster_____________________________
The FeMo cofactor is a molybdenum containing metal-sulfur cluster with a general chemical
composition MoFe7S8Homocitrate. It is buried in
the subunit and is composed of a 4Fe:3S cubane Figure 5: The redox dependent structural
ligated at the terminal iron by cys 275, and a changes of the p-cluster. Sulfur atoms are
3Fe:3S:Mo species containing molybdenum in shown in light grey and iron in dark grey.
octahedral arrangement with his442 and a Iron atoms are labeled 1-8 for reference.
N
bidentate homocitrate ligand facing the p-cluster The relaxed p-cluster (P ) exhibits a 6(Figure 6). Recent crystallographic studies have coordinate central sulfur atom with an
19
provided evidence for the existence of a light unusually long bond to Iron 6 .
atom octahedrally coordinated inside the FeMo cluster 23. Though the central ligand has been
difficult to characterize initial theories settles on carbon, oxygen, or nitrogen. Early studies into
the nature of the atom provided evidence through ESEEM (electron spin echo envelope
modulation) that nitrogen was the most likely central ligand. However, this was contradicted by
ENDOR studies (Electron nuclear double resonance) which failed to detect the presence of
nitrogen in the cofactor 24. Recent high-resolution and crystallographic studies in conjunction
with ESEEM spectroscopy indicate the presence of a carbon as the central ligand 25. In Silico
studies on inorganic analogues of the FeMo protein assuming a central carbon indicate an
elongation of a Fe-C bond upon N 2 bonding. It is therefore proposed that the Fe-C bondlength is
important in modulating substrate binding in the active site 26.
Due to the physical properties
and nature of electron transfer
in the FeMo cofactors it proved
immensely
difficult
to
characterize
the
exact
mechanism
for
substrate
reduction at the active site. The
FeMo
cluster
only
binds
substrates
upon
several
reduction
cycles,
requiring
isolation
of
reduced
intermediates
to
analyze
substrate binding. Furthermore,
the native state of the FeMo
cofactor exhibits an EPR active
S=3/2 signal; however, upon
reduction several EPR silent
oxidation states of the cofactor
are accessed, making traditional Figure 6: The FeMo-Cluster of Mo-Nitrogenase. The Mo atom is
spectroscopic tools such as EPR octahedrally coordinated with his442 and a bidentate homocitrate.
difficult to implement. Attempts Not shown in the figure is the central ligand of the cluster, likely
to freeze-trap reduced EPR to be a carbon. Through various substrate binding experiments it
active
FeMo
cofactor is theorized that acetylene and N 2 substrates bind to the FeMo
intermediates
have
proved face comprising Irons 2,3,6, and 7.
problematic. In the absence of
suitable substrates, nitrogenase complexes will reduce
protons, making the isolation of reduced MoFe species
difficult without the use of inhibitors such as CO.
Furthermore, experiments attempting to isolate substrate
bound MoFe proteins often resulted in low active-site
occupancy and substrates binding in various motifs 27. The
question therefore remained unsolved as to which 4Fe:4S
face N2 binds, and which intermediates are involved in the
process.
Recently, Dos Santos et al.27 utilized the ability of
nitrogenases
to
reduce
exogenous
substrates, Figure 7: Two proposed mechanisms for
N2
reduction
by
Mo-nitrogenases.
Pathway D is proposed through kinetic
studies, whereas pathway A is the result
of
crystallographic
and
EPR
experiments29.

particularly acetylene derivatives, to elucidate part of the substrate binding mechanism.

Isolation of acetylene resistant MoFe Proteins from mutant A. vinelandii species indicated a
mutation in -Gly69 to serine. The residue is thought to regulate the conformational flexibility of
the proximal -val70 which restricts substrate access to the active site. Site directed
mutagenesis of val to smaller gly or ala varied the size of the active site hole, allowing access
of acetylene derivatives with hydrogen bonding substituents capable of locking into the active
site. Using the acetylene species propargyl alcohol, binding to the FeMo cofactor resulted in an
intermediate with a spin state of . Using radiolabeled carbon and deuterium, ENDOR
spectroscopy elucidated a potential covalent intermediate in acetylene reduction, with the
substrate bound to FeMo face composing Fe atoms 2,3, 6, and 7. As mutagenesis experiments
confirmed acetylene binding to a specific face of the FeMo cofactor, experiments analyzing the
face of N2 binds were attempted. Mutation of -val 70 to a larger Ile lowered the reduction of
acetylene and N2 (>120 times increase in Km) however, proton reduction was unaffected. From
the experiments it was determined that mutations in -val 70 do not perturb reduction of the
FeMo cofactor, and that acetylene and N2 bind along the same face of the active site cofactor.
The same procedure was used in subsequent experimentation to freeze-trap nitrogen
intermediates in the MoFe protein. Studying the freeze-trapped proteins using ENDOR with
radiolabeled nitrogen indicated the presence of three distinct intermediates in N 2 reduction28,
resulting in two proposed mechanisms 29(Figure 7). Mechanism A is largely the product of kinetic
studies, whereas D is proposed from ENDOR/ESEEM studies.

The
Future:_____________________________________________________________________________
In light of the recent developments in nitrogenase mechanism, the exact process by which the
complex reduces N2 has remained elusive. Currently, Hoffman et al have proposed a new
experimental protocol to elucidate the final mystery of nitrogenase catalyzed chemistry. Freezetrapped enzyme intermediates must have a variable accumulation of electrons on the FeMo
cofactor (n). It is therefore hypothesized that n could be revealed by following the relaxation of
the MoFe protein through counting H2 equivalents produced. This procedure requires techniques
which halt intercomponent electron transfer between the Fe and MoFe protein; this will likely be
achieved by manipulation of the association-dissociation cycle required for electron transfer to
the P-cluster. Abolition of this cyclic process would be achieved through freeze-trapping at 77K
with periodic cooling to 2K for the purpose of EPR experiments. In previous experiments on
monooxygenases it is known that electron transfer does occur at these temperatures, and this
process may prove useful in the absolute determination of the Mo-nitrogenase mechanism in
future studies.
1. Modak J. (2002). Haber process for ammonia synthesis. Resonance,
9(7), 6977.
2. Mortenson L. E., Morris J. A., & Jeng D. Y. (1967): Purification, metal
composition and properties of molybdo-ferredoxin and azoferredoxin, two of
the
components
of
the
nitrogen-fixing
system
of
Clostridium
pasteurianum. Biochimica et Biophysica Acta (BBA)-General Subjects, 141.3,
516-522.
3. Georgiadis M. M., Komiya H., Chakrabarti P., Woo D., Kornuc, J. J., &
Rees, D. C. (1992). Crystallographic structure of the nitrogenase
iron protein from Azotobacter vinelandii. Science (New York, N.Y.),
257, 16531659
4. Angove H. C., Yoo S. J., Mnck, E., & Burgess, B. K. (1998). An allferrous state of the Fe protein of nitrogenase. Interaction with
nucleotides and electron transfer to the MoFe protein. The Journal
of biological chemistry, 273, 2633026337
5. Knight E. H. R. (1967). Flavodoxins: Biological Properties. The
Journal of biological chemistry, 242, 13701374.

6. Bulen W. (1966). The Nitrogenase System From Azotobacter: TwoEnzyme Requirement for N2 Reduction, ATP-Dependant H2
Evolution, and ATP Hydrolysis. Proceedings of the National
Academy of Sciences of the United States of America, 56, 979986.
7. Benemann J. R., Yoch D. C., Valentine R. C., & Arnon D. I. (1969).
The electron transport system in nitrogen fixation by Azotobacter. I.
Azotoflavin as an electron carrier. Proceedings of the National
Academy of Sciences of the United States of America, 64(3), 1079
86.
8. Schindelin H., Kisker C., Schlessman J., & Howard J., D. C. (1997).
Structure of ADP-AlF4- Stabilized Nitrogenase Complex and Its
Implications for Signal Transduction. Nature, 387, 370377.
9. Zumft W. G., Cretney W. C., Huang T. C., Mortenson L. E., & Palmer
G. (1972). On the structure and function of nitrogenase from
Clostridium pasteurianum W5. Biochemical and biophysical
research communications, 48(6), 1525-1532.
10.Howard, J. B., & Rees, D. C. (1994). Nitrogenase: a nucleotidedependent molecular switch. Annual review of biochemistry, 63,
235-264.
11.Seefeldt L. C., & Dean D. R. (1997). Role of Nucleotides in
Nitrogenase Catalysis. Accounts of Chemical Research, 30(6), 260
266.
12.Morgan T. V., Mortenson L. E., McDonald J. W., & Watt G. D. (1988).
Comparison of redox and EPR properties of the molybdenum iron
proteins of Clostridium pasteurianum and Azotobacter vinelandi
initrogenases. Journal of Inorganic Biochemistry, 33(2), 111-120.
13.Hageman R. V. (1978). Nitrogenase and nitrogenase reductase
associate and dissociate with each catalytic cycle. Proceedings of
the National Academy of Sciences of the United States of America,
75(6), 26992702.
14.Hageman R., William V., Orme-Johnson H., & Burris R. H. (1980)Role of
magnesium adenosine 5'-triphosphate in the hydrogen evolution reaction
catalyzed by nitrogenase from Azotobacter vinelandii. Biochemistry, 19,
11, 2333-2342.
15.Christiansen J., Goodwin P. J., Lanzilotta W. N., Seefeldt L. C., & Dean D. R.
(1998). Catalytic and Biophysical Properties of a Nitrogenase Apo-MoFe
Protein Produced by an ifB-Deletion Mutant of Azotobacter vinelandii.
Biochemistry, 37(36), 12611-12623.
16.Eady Robert R. (1996)Structure-function relationships of alternative
nitrogenases. Chemical reviews, 96.7, 3013-3030.
17.Tezcan F. A., Kaiser J. T., Mustafi D., Walton M. Y., Howard J. B., &
Rees D. C. (2005). Nitrogenase complexes: multiple docking sites
for a nucleotide switch protein. Science (New York, N.Y.), 309(5739),
137780.
18.Lowe,D., Fisher K., & Thorneley R. (1993) Klebsiella pnuenomiae
nitrogenase: pre-steady-state absorbance changes show that redox
changes occur in the MoFe protein that depend on substrate component
protein ratio; a role for P-centres in reducing dinitrogen? Biochemical
Journal, 292, 93-98.
19.Peters J. W., Stowell M. H., Soltis S. M., Finnegan M. G., Johnson M. K., &
Rees D. C. (1997) Redox-dependent structural changes in the nitrogenase
P-cluster. Biochemistry, 36(6), 1181-1187.

20.Lanzilotta W. N., Christiansen J., Dean D. R., & Seefeldt L. C. (1998)

Evidence for coupled electron and proton transfer in the [8Fe-7S] cluster
of nitrogenase. Biochemistry, 37(32), 11376-11384.
21.Peters J. W., Fisher K., Newton W. E., & Dean D. R. (1995) Involvement of
the P cluster in intramolecular electron transfer within the nitrogenase
MoFe protein. Journal of Biological Chemistry, 270(45), 27007-27013.
22.Govezensky D., & Zamir A. (1989)Structure-function relationships in the
alpha subunit of Klebsiella pneumoniae nitrogenase MoFe protein from
analysis of nifD mutants. Journal of bacteriology 171.10, 5729-5735.
23.Einsle O., Tezcan F. A., Andrade S. L. a, Schmid B., Yoshida M.,
Howard J. B., & Rees D. C. (2002). Nitrogenase MoFe-protein at
1.16 A resolution: a central ligand in the FeMo-cofactor. Science
(New York, N.Y.), 297(5587), 1696700.
24.Yang T. C., Maeser N. K., Laryukhin M., Lee H. I., Dean D. R., Seefeldt L.
C., & Hoffman B. M. (2005). The interstitial atom of the nitrogenase
FeMo-cofactor: ENDOR and ESEEM evidence that it is not a
nitrogen. Journal of the American Chemical Society, 127(37), 1280412805.
25.Spatzal T., Aksoyoglu M., Zhang L., Andrade S. L., Schleicher E., Weber
S., & Einsle O. (2011). Evidence for interstitial carbon in nitrogenase
FeMo cofactor. Science, 334(6058), 940-940.
26.Rittle J, & Peters J.(2013) FeN2/CO complexes that model a possible role
for the interstitial C atom of FeMo-cofactor (FeMoco). Proceedings of the
National Academy of Sciences 110.40, 15898-15903.
27.Dos Santos P. C., Igarashi R. Y., Lee H. I., Hoffman B. M., Seefeldt L. C., &
Dean D. R. (2005). Substrate interactions with the nitrogenase active
site. Accounts of chemical research, 38(3), 208-214.
28.Barney B. M., Lukoyanov D., Yang T. C., Dean D. R., Hoffman B. M., &
Seefeldt L. C. (2006). A methyldiazene (HN N CH3)-derived species
bound to the nitrogenase active-site FeMo cofactor: Implications for
mechanism. Proceedings of the National Academy of Sciences, 103(46),
17113-17118.
29.Hoffman Brian M., Dennis R., and Seefeldt L.C. (2009)Climbing
nitrogenase:
toward
a
mechanism
of
enzymatic
nitrogen
fixation. Accounts of chemical research 42.5 609-619.