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contrast, to accompany the [3Fe4S] and [4Fe4S] clusters which sit medial and distal
from the [NiFe] active site, O
2
-tolerant Group 1 hydrogenases contain a novel
[4Fe3S] site which is closest ('proximal') to the H
2
reaction center [
46
,
47
]. It is
thought that the ability of the [4Fe3S] to access three different redox states ensures
that electrons can be rapidly provided to 'neutralize' inhibitory O
2
via a four-
electron four-proton reduction to yield two H
2
O (equation
9
). The O
2
-tolerant
Group 1 [NiFe] hydrogenases therefore only form the rapidly reactivating “Ni-B”
OH
-bound inhibited Ni
3+
state following exposure to O
2
in the presence of H
2
.
Protein film electrochemistry has been a particularly valuable tool with which to
dissect the O
2
tolerance mechanism of these enzymes. Recent experiments using this
technique have also shown that a Group 1 hydrogenase which functions in high O
2
will become an efficient H
2
producer below pH 4 [
48
]. This is a transformative result
because O
2
-tolerant Group 1 [NiFe] hydrogenases show very little H
2
production
activity at neutral pH, therefore to date all biotechnological applications have used
the enzymes as H
2
-oxidizing catalysts. The ideas developed in generating optimized
enzyme-carbon electrodes for membrane-free H
2
/O
2
fuel cells, including cross
linking to 3-D carbon nanotube surfaces [
49
,
50
], can now be applied in developing
new Group 1 O
2
-tolerant solar H
2
enzyme devices for operation at pH
4.
The 'Group 5' sub-class of [NiFe] hydrogenases were first categorized in 2010
and in human terms these are therefore the newest [NiFe] enzymes. They were
originally identified in
Streptomyces
species as enzymes with the unique ability to
oxidize atmospheric H
2
, e.g., they are functional in 20 % O
2
with an apparent
Michaelis constant for H
2
of less than 100 ppm by volume [
15
,
51
,
52
]. The aerobic
functionality of these enzymes is so impressive that the term 'O
2
-insensitive' rather
than 'O
2
-tolerant' has been used to describe these hydrogenases [
53
]. From the
amino acid sequence, these enzymes are again expected to contain three FeS
clusters, but only [4Fe4S] centers are predicted [
53
]. The distal cluster is thought
to be coordinated by three Cys and one histidine, which is also standard for a Group
1 [NiFe] hydrogenase. More usually for a [4Fe4S] site, the medial cluster is
predicted to be ligated by four Cys. The proximal cluster shows no evidence of
additional Cys to stabilize a [4Fe3S] site, and instead a [4Fe4S] center coordinated
by three Cys and one aspartate is proposed. There are currently no crystal structures
of these enzymes, and no EPR, FTIR or electrochemical data, and these enzymes
will doubtless be a hot topic of hydrogenase research in the coming years.
In Group 3 [NiFe] hydrogenases, the physiologically bidirectional enzymes, there
areexamplesofhydrogenaseswhicharealsofunctionalinO
2
[
54
]. In parallel with
the Group 1 [NiFe] hydrogenases, the O
2
-tolerance mechanism again originates from
the ability of the enzyme to act as an oxidase, reducing O
2
to H
2
OorH
2
O
2
[
54
]. Elucidating the mechanism of this reactivity is complicated because the
hydrogenase forms a super-complex with a second enzyme to generate a bifunctional
unit capable of catalyzing H
2
activation or evolution under physiological conditions,
with concerted binding and reduction or oxidation of additional soluble substrates
like coenzyme F420 or NAD(P)H [
55
]. In the Group 3 hydrogenases from
Synechocystis
sp. PCC6803 and
R. eutropha
there are 8 FeS clusters including 2Fe
sites, therefore deconvoluting the reactivity of these centers is not trivial [
54
,
56
].
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