Environmental Engineering Reference
In-Depth Information
When the gas phase during growth is switched to 80% N
2
/20% CO
2
before cell
harvesting, another form called MCR
ox1
is generated [
72
]. MCR
ox1
also can be
generated by adding Na
2
S to the growing culture prior to harvest [
71
] or by adding
polysulfide to MCR
red2
in vivo
and
in vitro
[
71
]. MCR
ox1
is much more resistant to
O
2
than MCR
red1
, in agreement with the results of EPR, ENDOR, and HYSCORE
experiments that this is an oxidized form of the enzyme containing a Ni(III)-thiolate
(from CoMS
) in resonance with a high-spin Ni(II)-thiyl radical [
78
,
79
]. Addition
of sodium sulfite to growing cultures or to MCR
red2
in vitro
results in formation of
a light-sensitive state, MCR
ox2
, while addition of O
2
to MCR
red2
generates the
MCR
ox3
state [
67
].
While it has been unambiguously shown that Ni(I) is the active state that initiates
catalysis, it is not clear if the product of reaction between MCR and the two
substrates generates Ni(II) and a methyl radical or an organometallic methyl-
Ni(III) intermediate. To characterize the alkyl-Ni(III) species, MCR
red1
has been
reacted with brominated substrate analogs, like bromopropanesulfonate to generate
Ni(III)-propanesulfonate or methyl iodide to give methyl-Ni(III) [
80
]. Whereas it
had been thought that the alkyl-Ni(III) species would be highly unstable, it was
found to be relatively stable and could be characterized by various spectroscopic
and structural methods, as described next.
Although an organometallic methyl-Ni(III) intermediate has been proposed to be
a catalytic intermediate in methane synthesis [
63
,
64
,
81
], such an intermediate has
never been trapped during the reaction of MCR with native substrates. On the other
hand, reaction of MCR
red1
with methyl halides (even in the absence of CoBSH)
quantitatively generates the methyl-Ni(III) state, often called MCR
Me
.[
82
,
83
].
This reaction likely occurs by a nucleophilic attack of Ni(I) on methyl iodide to
generate the iodide anion and the Ni(III)-CH
3
species. Formation of the methyl-
Ni(III) species was confirmed by EPR spectroscopy and the covalent linkage
between the methyl group and the nickel center was confirmed by high resolution
ENDOR and HYSCORE experiments using different isotopes of methyl iodide
[
82
,
83
]. X-ray absorption spectroscopic and X-ray crystallographic studies of the
alkyl-Ni(III) state of MCR reveal a six-coordinate Ni center with an upper axial
Ni-C bond at 2.04
Å
, four Ni-N bonds at 2.08
Å
, and a lower axial Ni-O interaction
at 2.32
, unambiguously establishing the organometallic nature of the methyl-
Ni(III) species [
74
,
84
]. The MCR
Me
can then react with CoMSH (and other
thiolates) to generate MCR
red1
and CH
3
-SCoM (or other alkyl thioethers). Simi-
larly, reaction of the MCR
Me
species with CoMSH and CoBSH produces methane
at a
k
cat
of 1.1 s
1
, which is similar to the steady-state
k
cat
for methane formation
from natural substrates (4.5 s
1
at 25
C) - consistent with the catalytic intermedi-
acy of the methyl-Ni(III) species [
82
].
The Ni(I) in MCR
red1
can react with a variety of compounds with activated alkyl
groups in the absence of CoBSH. For example, reaction with the potent inhibitor,
bromopropanesulfonate (BPS), generates MCR
PS
[
84
], which, like MCR
Me
,
exhibits UV-visible features that emulate those of the Ni(II) protein and an EPR
spectrum with
g
values at 2.223 and 2.115. On the basis of EPR, ENDOR, and
HYSCORE spectroscopic studies, MCR
PS
was assigned as an organometallic
Å
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