Environmental Engineering Reference
In-Depth Information
In contrast to FeS-mediated O
2
tolerance, the Group 2b [NiFe] hydrogenase H
2
sensor enzymes are able to control the transcription of hydrogenase genes in aerobic
H
2
-oxidizing bacteria because they contain a narrow gas channel which blocks the
access of O
2
to the active site [
46
]. This was proved by decreasing the O
2
tolerance
of
R. eutropha
enzyme variants containing wider gas channels. Attempts to engi-
neer O
2
tolerance in an O
2
-sensitive Group 1 enzyme via mimicry of this blocking
mechanism were partially successful [
57
] and the same approach has been taken to
try and make [FeFe] hydrogenases less sensitive to O
2
inhibition [
58
]. In addition to
gas channels, it is also possible that water and proton channels may assist in
controlling O
2
tolerance mechanisms in hydrogenases which reduce inhibitory O
2
to H
2
O[
46
]. This idea arises from amino acid and structural comparisons which
show that conserved residues appear to form these channels.
3.1.2
[NiFeSe] Hydrogenases
When grown on a selenium-containing medium, some sulfate-reducing or
methanogenic microorganisms are able to construct hydrogenases which encode a
selenocysteine (SeCys) rather than a standard S-Cys as a ligand to the Ni in the
active site of Group 1 or Group 3 [NiFe] hydrogenases [
59
]. These enzymes are
designated [NiFeSe] hydrogenases. In the absence of Se, the [NiFeSe]-producing
microbes will generate a 'normal' Cys-containing [NiFe] hydrogenase homologue,
but when Se is available the genetic regulation instead favors expression of the
seleno H
2
enzyme, suggesting a biochemical advantage to expressing the [NiFeSe]
enzyme. Corresponding to this result, in dye assays the activity of [NiFeSe]
hydrogenases are higher than for [NiFe] hydrogenases.
Electrochemical studies of Group 1 [NiFeSe] hydrogenases have also shown a
greater catalytic bias towards H
2
production over H
2
oxidation, which is unusual for
a Group 1 [NiFe] enzyme [
60
]. The H
2
-producing activity is also sustained in the
presence of O
2
, although the H
2
uptake activity is not [
60
]. The [NiFeSe] H
2
enzymes have therefore been applied in solar H
2
-producing devices but not H
2
/O
2
fuel cells [
34
,
37
]. Relative to their [NiFe] counterparts, the Group 1 [NiFeSe]
hydrogenases contain a different electron transport relay as well as a different
active site. However, the enzymatic reactivity is unchanged when [NiFe] hydrog-
enase variants are constructed which contain the same FeS cluster content as a
[NiFeSe] enzyme (three [4Fe4S] sites) [
61
]. This suggests that the Se plays a
predominant role in tuning the novel activity of [NiFeSe] hydrogenases.
3.2 Biosynthesis
The maturation of [NiFe] hydrogenases has been most extensively studied for the
membrane-bound hydrogenase (MBH) enzyme from
R. eutropha
and hydrogenase-1
from
E. coli
, both of which are Group 1 O
2
-tolerant enzymes. The fact that the
E. coli
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