anaerobic NiFe-hydrogenases, such as the one from D. gigas and others, because
they require a long activation process (several hours at room temperature) of the
oxidized enzyme in order to recover optimal activity [2]. The faster reactivation of
NiFeSe-hydrogenases is explained by the fact that in the presence of O 2 the Se-Cys
reacts fast with it by forming a selenoate, which can be quickly reduced back to the
Se-Cys in presence of a low potential electron donor. Cys are slower to react with
O 2 but sulphenates are also reduced slowly, thus if one or more of the active site
Cys of NiFe-hydrogenases become oxidized it reactivation under reducing conditions will have a large kinetic barrier [25]. However, an irreversibly oxidized state
has also been observed for the NiFeSe-hydrogenase from Desulphovibrio vulgaris
Hildenborough, which is formed upon prolonged oxygen exposure during purification and crystallization [26, 27]. This oxidized species contains a terminal cysteine ligand to nickel doubly oxidized to the sulphinate state, a modification that can
be prevented by blocking access of O 2 to the active site through a hydrophilic
channel [28].
The powerful methodology of directed evolution for improving or obtaining new
enzymatic activities has also been used with the aim of optimizing H 2 -production
catalyzed by hydrogenases. This strategy has been used to evolve the Fe–Fe
hydrogenase genes of two species of Clostridia bacteria. Chimeric genes were
expressed in E. coli and the H 2 -production activity of the cell cultures with reduced
MV was measured by gas chromatography. However, the best chimera mutant had
only 4% of the activity of the positive control C. acetobutylicum [29].
High-throughput screening has also been used for simultaneous evaluation of
NADPH-driven H 2 production activity by a library of more than 10,000 randomly
mutated C. pasteurianum FeFe-hydrogenases [30].
2.2 Electroenzymatic Production of H 2
Electrodes can work directly as electron donor or acceptor of hydrogenases,
replacing the natural redox proteins, co-factors or artificial redox compounds as
co-substrates. In consequence, strategies for electroenzymatic H 2 production are
possible [31]. A requisite for direct electron transfer (DET) of enzymes is that the
redox centre located on the protein surface is at a short distance (less than 20 Å) of
the electrode surface. In the case of hydrogenases, a redox relay formed by an
alignment of iron-sulphur clusters connects the protein surface with the bi-metallic
active site, allowing for fast intramolecular electron transfer in both directions [3].
Therefore, the most exposed iron-sulphur cluster, normally known as the distal
cluster, must be facing the electrode surface in order to allow measurement of
electroenzymatic H 2 -production (Fig. 3a). Most of the published work in this topic
is based on direct adsorption of a small amount of enzyme on rough carbon surfaces, such as carbon black or pyrolytic edge graphite [32]. These electrode
materials facilitate direct electron transfer of hydrogenase, even if the orientation of
the adsorbed enzyme molecules is random, because the roughness of the surface at
the nanometric scale allows that a significant hydrogenase population has its distal
Biological Production of Hydrogen
251
they require a long activation process (several hours at room temperature) of the
oxidized enzyme in order to recover optimal activity [2]. The faster reactivation of
NiFeSe-hydrogenases is explained by the fact that in the presence of O 2 the Se-Cys
reacts fast with it by forming a selenoate, which can be quickly reduced back to the
Se-Cys in presence of a low potential electron donor. Cys are slower to react with
O 2 but sulphenates are also reduced slowly, thus if one or more of the active site
Cys of NiFe-hydrogenases become oxidized it reactivation under reducing conditions will have a large kinetic barrier [25]. However, an irreversibly oxidized state
has also been observed for the NiFeSe-hydrogenase from Desulphovibrio vulgaris
Hildenborough, which is formed upon prolonged oxygen exposure during purification and crystallization [26, 27]. This oxidized species contains a terminal cysteine ligand to nickel doubly oxidized to the sulphinate state, a modification that can
be prevented by blocking access of O 2 to the active site through a hydrophilic
channel [28].
The powerful methodology of directed evolution for improving or obtaining new
enzymatic activities has also been used with the aim of optimizing H 2 -production
catalyzed by hydrogenases. This strategy has been used to evolve the Fe–Fe
hydrogenase genes of two species of Clostridia bacteria. Chimeric genes were
expressed in E. coli and the H 2 -production activity of the cell cultures with reduced
MV was measured by gas chromatography. However, the best chimera mutant had
only 4% of the activity of the positive control C. acetobutylicum [29].
High-throughput screening has also been used for simultaneous evaluation of
NADPH-driven H 2 production activity by a library of more than 10,000 randomly
mutated C. pasteurianum FeFe-hydrogenases [30].
2.2 Electroenzymatic Production of H 2
Electrodes can work directly as electron donor or acceptor of hydrogenases,
replacing the natural redox proteins, co-factors or artificial redox compounds as
co-substrates. In consequence, strategies for electroenzymatic H 2 production are
possible [31]. A requisite for direct electron transfer (DET) of enzymes is that the
redox centre located on the protein surface is at a short distance (less than 20 Å) of
the electrode surface. In the case of hydrogenases, a redox relay formed by an
alignment of iron-sulphur clusters connects the protein surface with the bi-metallic
active site, allowing for fast intramolecular electron transfer in both directions [3].
Therefore, the most exposed iron-sulphur cluster, normally known as the distal
cluster, must be facing the electrode surface in order to allow measurement of
electroenzymatic H 2 -production (Fig. 3a). Most of the published work in this topic
is based on direct adsorption of a small amount of enzyme on rough carbon surfaces, such as carbon black or pyrolytic edge graphite [32]. These electrode
materials facilitate direct electron transfer of hydrogenase, even if the orientation of
the adsorbed enzyme molecules is random, because the roughness of the surface at
the nanometric scale allows that a significant hydrogenase population has its distal
Biological Production of Hydrogen
251