Electrocatalysis for Fuel Cells at Enzyme-Modified Electrodes - Fuel Cell Catalysis: A Surface Science Approach - page 610

orientations of the enzyme on the electrode that lead to a distribution of interfacial
electron transfer rates such that some molecules require a high driving force to undergo
catalysis [L´ger et al., 2002]. At lower temperatures, electron transfer keeps up
with catalysis, but as the enzyme turns over faster at higher temperatures, interfacial
electron transfer becomes the rate-limiting step. This effect is also evident in two
reported comparisons of H 2 oxidation catalyzed by hydrogenase-modified and plati-
nized electrodes operating in aqueous solution: the precious metal catalyst is more
active at potentials close to the value of E(H þ /H 2 ), but, at higher overpotentials,
both the platinized and hydrogenase-modified electrodes show a similar and
diffusion-controlled level of H 2 oxidation [Jones et al., 2002; Karyakin et al.,
2005]. This effect suggests that improved coupling of hydrogenase to the electrode
would enhance electrocatalysis at lower applied potentials.
Non-turnover signals arising from electron transfer to the FeS relay clusters in
the enzyme are just visible in voltammetry for high-coverage films of A. vinosum
hydrogenase, suggesting that an upper limit for the electroactive coverage is about
3 pmol cm 22 (for a review, see Vincent et al. [2007]). The response in Fig. 17.14
therefore results from a low density of extremely active catalytic sites, and there is con-
siderable scope for optimizing the current by increasing the effective electrode area.
A method was recently reported for covalently attaching hydrogenase to graphite or
multiwalled carbon nanotubes via a carbodiimide/N-hydroxysuccinamide coupling reac-
tion to aminophenyl functionalities introduced onto the nanotubes [Alonso-Lomillo et al.,
2007]. This strategy relies upon a surface patch that is rich in glutamate residues close to
the surface FeS cluster of Desulfovibrio gigas [NiFe]-hydrogenase to favor attachment
close to the electron entry point, but probably still results in a distribution of orientations.
The resulting hydrogenase film exhibits diffusion-controlled H 2 oxidation currents that
persist for over 30 days. The authors also show that hydrogenase can be adsorbed onto
unmodified carbon nanotubes, but this procedure results in a less active and less stable
protein film. The negatively charged surface patch probably also explains the stabilization
of directly adsorbed hydrogenase films in the presence of positively charged co-
adsorbates such as polymyxin and polylysine (for a review, see Vincent et al. [2007]).
Further advances are still needed for obtaining stable, high coverage films of hydrogenase
that are orientated correctly for efficient interfacial electron transfer.
17.3.2.2 Tolerance of Hydrogenases to O 2 , CO, and Sulfide Figure 17.15
summarizes the reactions of [NiFe]-hydrogenases with small molecules that can
poison Pt sites (for a review, see Vincent et al. [2007]). A. vinosum, the source of
the [NiFe]-hydrogenase addressed in Fig. 17.14, lives in semi-aerobic soils or water-
ways, and therefore infrequently encounters high levels of O 2 . The H 2 oxidation
activity of this enzyme is fully inhibited by O 2 , with oxygenic bridging ligands
becoming trapped in the active site (Fig. 17.15). Under reducing potentials, it is poss-
ible to recover the activity almost fully, but the reaction, presumably requiring reduc-
tive removal of the bridging ligand as H 2 O, is very slow (the half life is approximately
1 hour at 20 8C for re-activation of the hydroperoxide form) [Vincent et al., 2007]. For
the purposes of operation of the enzyme within a fuel cell, or indeed probably a
biological cell, the activity of A. vinosum hydrogenase is essentially destroyed by O 2 .