Environmental Engineering Reference
In-Depth Information
The highly conserved nature of the H-cluster is reflected in the fact that different
[FeFe] enzymes have similar FTIR and EPR spectroscopic properties, and the 'H
ox
'
mixed valence [4Fe4S]
2+
(Fe
P
1+
Fe
D
2+
) state, which will bind H
2
, can be used to
fingerprint all [FeFe] hydrogenases analyzed to date. Both the [4Fe4S] and [FeFe]
components of the H-cluster are redox-active, and a particular challenge has been to
understand the states formed under highly reducing conditions. Electrochemical
studies have complemented the structural and spectroscopic characterizations and
comparisons of inhibition by CO and formaldehyde as a function of potential
particularly highlights how the enzyme reactivity can change dramatically at very
low potentials [
66
]. A 'super-reduced'[4Fe4S]
1+
(Fe
P
1+
Fe
D
1+
) is thought to play a
role in the catalytic cycle [
45
].
The [FeFe] hydrogenases are incredibly active, for example in a H
2
production
assay the
Clostridium pasteurianum
enzyme had a measured turnover rate of
3400
mol min
1
mg
1
(in 1 min 1 g of enzyme would produce 83 L of atmo-
spheric pressure H
2
)[
45
]. This high activity correlates with irreversible inhibition
by O
2
, which damages the [4Fe4S] cluster of the active site rather than the [2Fe]
center [
67
]. Although O
2
binding can be blocked by CO inhibition, there is
currently no proposed mechanism for how to re-engineer an [FeFe] hydrogenase
to 'neutralize' inhibitory O
2
and therefore remain active in air.
μ
4.2
[FeFe] Hydrogenase Biosynthesis
The mechanism of [FeFe] hydrogenase biosynthesis is well understood and it is
possible to engineer
E. coli
to produce a fully mature [FeFe] hydrogenase by
transferring into the cell just four genes [
45
]. First,
hydA
encodes the apo-protein
and this will be assembled to contain the [4Fe4S] portion of the H-cluster due to the
action of the standard
E. coli
FeS “housekeeping” proteins. Second, the genes
hydE
,
hydF
, and
hydG
are required because these encode the specific maturases which
synthesize and attach the [2Fe] part of the H-cluster to the [4Fe4S] cubane. The CO
and CN
ligands are synthesized from tyrosine via the action of the radical
S-adenosyl methionine enzyme HydG [
45
,
68
].
4.3
[Fe] Hydrogenases
The [Fe] hydrogenases were only discovered in 1990 and they have also been
labelled as 'H
2
-forming methylenetetrahydromethanopterin dehydrogenases' and
'iron-sulfur cluster-free' hydrogenases [
7
]. These titles reveal key details about
both the function and structure of these enzymes. First, the cytochrome-free
hydrogenotrophic methanogens which express this enzyme use it to catalyze the
reversible transfer of hydride from H
2
to methylenetetrahydromethanopterin
(Figure
8
). This reaction is one step in the overall metabolic reduction of CO
2
to
methane. Secondly, the only metal center in each protein subunit is the single iron
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