directly adsorbed on a nanostructured TiO 2 electrode. ATR-FTIR spectroscopy was
used to confirm structural integrity of the adsorbed hydrogenase. However, the
quantification of the total amount of immobilized hydrogenase suggested that a high
percentage of enzyme molecules were not participating on the electrocatalytic
process [37]. This result could be due to incorrect orientation of hydrogenase
molecules for DET with the electrode, or to protein inactivation during adsorption.
Another strategy developed has been the coating of a TiO 2 with electrospun polyacrylonate fibres, to which the membrane-bound NiFe- hydrogenase from E. coli
was bound. Current densities higher than -0.5 mA Â cm
−2 for electrocatalytic H 2 -
production at only −0.2 V of overpotential [38]. Electroenzymatic H 2− production
has also been studied by co-immobilization of a NiFe-hydrogenase and a viologen
polymer on a high-surface-area carbon electrode. High current densities of
−100 mA/cm
−3 were reported in this system [39].
An important issue for biocatalytic H 2 -production is the effect of ambient O 2 on
the activity of hydrogenases. Moreover, if H 2 is obtained from the water splitting
reaction O 2 is also formed as by-product [40]. As already pointed out above, the O 2 -
tolerant NiFe-hydrogenases are poor catalysts for H
+ reduction to H 2 . On the other
hand, NiFeSe-hydrogenases, which are quickly inactivated under low concentrations of O 2 in the H 2 -oxidation and H/D exchange assays, have shown to be able to
produce H 2 in the presence of 1% O 2 when adsorbed on graphite electrodes [23] or
covalently bound to a SAM thiol-modified gold electrode poised at low redox
potentials [41]. Even if this amount of O 2 quickly oxidizes the active site of the
NiFeSe-hydrogenases to the inactive state, DET from the electrode to the adequately oriented enzyme molecules allows their immediate reactivation. Moreover,
it has been recently shown that the D. desulphuricans FeFe-hydrogenase, which
belongs to the type of hydrogenases more sensitive to irreversible inactivation by
either O 2 or light in spite of displaying high H 2 -production activity, becomes more
resistant when covalently bound to a graphite electrode poised at potentials lower
than −0.46 V versus NHE. The half-live time for electrocatalytic H 2 -production
under at 1% O 2 increased 4 times. This effect was explained by the lower reactivity
of the super reduced state of the hydrogenase active state towards the O 2 inhibitor
[42]. Therefore, due to its very high turnover, this hydrogenase has potential
applications in H 2 -production if an excess overpotential is applied on the electrode
in order to decrease the rate of O 2 inactivation.
Genetic engineering tools are also very useful for increasing biotechnological
applications of hydrogenases on electrodes. A truncated mutant of the
FeFe-hydrogenase from Megasphaera elsdeini showed the following improved
properties for electroenzymatic H 2 -production: (i) a smaller size that allows higher
biocatalyst coverage on the electrode; (ii) an enhanced catalytic bias towards H 2
production; (iii) a less sensitivity towards O 2 inhibition compared to other
FeFe-hydrogenases [43].
Biological Production of Hydrogen
253
used to confirm structural integrity of the adsorbed hydrogenase. However, the
quantification of the total amount of immobilized hydrogenase suggested that a high
percentage of enzyme molecules were not participating on the electrocatalytic
process [37]. This result could be due to incorrect orientation of hydrogenase
molecules for DET with the electrode, or to protein inactivation during adsorption.
Another strategy developed has been the coating of a TiO 2 with electrospun polyacrylonate fibres, to which the membrane-bound NiFe- hydrogenase from E. coli
was bound. Current densities higher than -0.5 mA Â cm
−2 for electrocatalytic H 2 -
production at only −0.2 V of overpotential [38]. Electroenzymatic H 2− production
has also been studied by co-immobilization of a NiFe-hydrogenase and a viologen
polymer on a high-surface-area carbon electrode. High current densities of
−100 mA/cm
−3 were reported in this system [39].
An important issue for biocatalytic H 2 -production is the effect of ambient O 2 on
the activity of hydrogenases. Moreover, if H 2 is obtained from the water splitting
reaction O 2 is also formed as by-product [40]. As already pointed out above, the O 2 -
tolerant NiFe-hydrogenases are poor catalysts for H
+ reduction to H 2 . On the other
hand, NiFeSe-hydrogenases, which are quickly inactivated under low concentrations of O 2 in the H 2 -oxidation and H/D exchange assays, have shown to be able to
produce H 2 in the presence of 1% O 2 when adsorbed on graphite electrodes [23] or
covalently bound to a SAM thiol-modified gold electrode poised at low redox
potentials [41]. Even if this amount of O 2 quickly oxidizes the active site of the
NiFeSe-hydrogenases to the inactive state, DET from the electrode to the adequately oriented enzyme molecules allows their immediate reactivation. Moreover,
it has been recently shown that the D. desulphuricans FeFe-hydrogenase, which
belongs to the type of hydrogenases more sensitive to irreversible inactivation by
either O 2 or light in spite of displaying high H 2 -production activity, becomes more
resistant when covalently bound to a graphite electrode poised at potentials lower
than −0.46 V versus NHE. The half-live time for electrocatalytic H 2 -production
under at 1% O 2 increased 4 times. This effect was explained by the lower reactivity
of the super reduced state of the hydrogenase active state towards the O 2 inhibitor
[42]. Therefore, due to its very high turnover, this hydrogenase has potential
applications in H 2 -production if an excess overpotential is applied on the electrode
in order to decrease the rate of O 2 inactivation.
Genetic engineering tools are also very useful for increasing biotechnological
applications of hydrogenases on electrodes. A truncated mutant of the
FeFe-hydrogenase from Megasphaera elsdeini showed the following improved
properties for electroenzymatic H 2 -production: (i) a smaller size that allows higher
biocatalyst coverage on the electrode; (ii) an enhanced catalytic bias towards H 2
production; (iii) a less sensitivity towards O 2 inhibition compared to other
FeFe-hydrogenases [43].
Biological Production of Hydrogen
253