Environmental Engineering Reference
In-Depth Information
3.2.1.2 The M
7
and M
8
Intermediates
The M
7
and M
8
intermediates were identified by EPR spectroscopy as the two
common intermediate states upon turnover of the Ile
ʱ
70
/Gln
ʱ
195
double-substituted
MoFe protein by various substrates, such as N
2
, diazene (N
2
H
2
), hydrazine (N
2
H
4
),
and methyldiazene (NH
¼
N-CH
3
)[
74
,
75
]. The M
7
state intermediate, designated
H at the time of discovery, showed a broad EPR signal at low field in the Q-band
EPR spectra. This signal was later determined to arise from an integer-spin system
(where
S
2, with a ground-state non-Kramers doublet) [
75
], which is consistent
with the prediction that the odd-number states of the MoFe protein cycle (i.e., M
n
states where n is an odd number) are likely to be non-Kramer systems [
57
,
76
].
ESEEM analysis of the
15
N-labeled diazene,
15
N-labeled hydrazine, and
2
H-labeled methyldiazene further suggests that this intermediate consists of
only one FeMoco-bound N atom and is most likely a [
NH
2
] moiety (Figure
5
)
[
67
,
68
,
75
]. Contrary to the M
7
intermediate, the M
8
intermediate, designated I at
the time of discovery, displays a strong
S
1/2 EPR signal that is easily accessible
for further analysis by advanced spectroscopic methods [
74
]. In combination
with HYSCORE measurements,
1,2
H, and
15
N ENDOR spectroscopic analyses
deduce that this intermediate consists of a bound [
¼
NH
x
] moiety, where x
¼
2or
3[
65
,
67
,
74
]. Since the [
NH
2
] moiety had been assigned to H (or the M
7
intermediate), it was concluded that the NH
x
moiety in I had an x
¼
3 and that I
would represent the M
8
state of the MoFe protein cycle (Figure
5
).
3.2.2 The Reductive Dihydrogen Elimination Mechanism
The characterization of the structure of the M
4
intermediate has led to the proposal
of a mechanism that involves the activation of N
2
upon the reductive elimination of
H
2
(Figure
6
). This mechanism presumably begins with a transient conversion of
bridging hydrides to terminal hydrides that renders an elevated reactivity of the Fe
atoms of FeMoco toward N
2
. Previous studies involving the synthesis of N
2
-bound
Fe, Co, and other transition metal complexes have demonstrated that N
2
binding
was promoted upon the reductive elimination of H
2
, likely through the reduction of
the metal center that lowered the energy barrier for N
2
activation [
77
-
80
]. In the
case of nitrogenase, the eliminated H
2
is thought to carry away only two of the four
reducing equivalents, allowing the remaining reducing equivalents to be used for
the reduction of N
2
. A bound N
2
H
2
species may be generated in this process through
the coupling of N
2
with the two protons that are possibly bound to the sulfides
(Figure
6
). Since the elimination of hydrides as H
2
would be the key to the binding
and reduction of N
2
, this proposed mechanism could provide an adequate explana-
tion for the obligate formation of H
2
during N
2
reduction (see equation
1
).
Efforts to substantiate this proposed mechanism are currently underway, with
one attempted approach focusing on the micro-reversibility of the H
2
elimination
step. This approach is based on the hypothesis that the reductive elimination of
hydrides can be reversed by the oxidative addition of H
2
and,
in one such
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