Environmental Engineering Reference
In-Depth Information
The observation that N
2
H
4
,N
2
H
2
and N
2
are all substrates of nitrogenase suggests
that N
2
H
2
and N
2
H
4
may represent intermediates generated along the alternating
pathway [
65
,
67
,
68
]; however, there is currently no evidence demonstrating that
N
2
H
2
or N
2
H
4
is indeed formed during the reduction of N
2
by Mo-nitrogenase
[
85
,
86
]. The observation of two common intermediates during the turnover of
N
2
H
4
,N
2
H
2
,andN
2
was also used to argue in favor of the alternating pathway;
yet, these common intermediates - identified lately as the M
7
and M
8
states by
spectroscopic characterization - are shared by both the distal and alternating pathways
and, therefore, cannot be used to exclude one pathway from another (Figure
5
,
Scheme
2
b). Clearly, further efforts are required to distinguish the two proposed
pathways; in particular, the capture and characterization of the nitrogenous interme-
diates bound at stages M
4
-M
6
will likely provide definitive evidence in this regard.
4 The Distinct Structural and Catalytic Features
of V-Nitrogenase
Discovered some 30 years ago, the V-nitrogenase is usually expressed in the absence
of Mo as an “alternative” form of its more efficient Mo-counterpart [
5
]. Like the
Mo-nitrogenase, the V-nitrogenase is a binary system comprising an iron (Fe) protein
component and a vanadium iron (VFe) protein component, each containing
metallocluster(s) analogous to those found in their respective Mo-counterpart.
Catalysis by V-nitrogenase also involves the association/dissociation between the
Fe protein and the VFe protein, which facilitates the ATP-dependent, inter-protein
electron transfer from the former to the latter and the subsequent substrate reduction
at the active cofactor site [
5
,
22
,
87
]. Despite their similarities, the V-nitrogenase
displays some unique structural and catalytic features that are clearly distinct from its
Mo-counterpart. These features will be discussed in detail below.
4.1 The Structural Features
4.1.1 The Fe Protein
The Fe protein (encoded by
vnfH
) of the
A. vinelandii
V-nitrogenase shares ~91 %
of sequence identity with its counterpart (encoded by
nifH
) in the Mo-nitrogenase
[
5
,
22
,
87
]. Like NifH, VnfH is a
60 kDa homodimer that contains 4 Fe atoms and
4 acid-labile S atoms per protein. The cysteinyl ligands of the [Fe
4
S
4
] cluster, as
well as the Walker A motif, are all perfectly conserved in the primary sequences of
VnfH and NifH [
88
]. In the dithionite-reduced state, both VnfH and NifH display
the same, mixed
S
1/2
signals of VnfH are 10 % and 27 %, respectively, more intense than those of NifH
[
89
,
90
]. In addition, VnfH displays a midpoint potential comparable to that of NifH
¼
3/2 :
S
¼
1/2 EPR signals, although the
S
¼
3/2 and
S
¼
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