[4Fe-4S] cluster. Bottom. In presence of fumarate (0.2mM) with electrode rotating at 3000 rpm. Note the boost that is observed at a potential corresponding to the [4Fe-4S] cluster. (Adapted with permission from J. Am. Chem. Soc. 1997, 119, 11628–11638. Copyright 1997 American Chemical Society.)

The NiFe hydrogenase contains a buried NiFe center (which is the active site for H2 oxidation) and a ‘wire’ of Fe-S clusters leading to the protein surface [94]. In order of their distance from the active site, these are [4Fe-4S] (proximal), [3Fe-4S] (medial), and [4Fe-4S] (distal). However, the reduction potential of the medial cluster, [3Fe-4S], is more than 0.2 volts higher than its neighbors in the chain. The enzyme adsorbs at a rotating disc PGE electrode to give a film that is extremely active in oxidizing H2 [92]. Under 0.1 atm H2, sigmoidal voltammograms are obtained (Figure 25) for which the current shows an almost linear increase with (rotation rate)1/2, as predicted by the Levich equation for a reaction that is controlled by mass transport of substrate to the electrode. Based on there being up to a monolayer coverage of electrocatalytically active enzyme, the turnover number lies in the range 1500-9000 s, which is much higher than values reported for studies using small-molecule electron donors. The enzyme is inhibited by CO, and experiments carried out under a stream of CO reveal, quite clearly, the presence

of two reversible (non-turnover) signals-one at −30 mV and one (integrating to double amplitude) at −300 mV-which can be assigned respectively to the [3Fe-4S] and two [4Fe-4S] clusters. These reduction potentials are in quite close agreement with values determined by EPR potentiometry, and their positions with respect to

the catalytic H2-oxidation wave are indicated in the figure. It is immediately evident that H2 oxidation is diffusion controlled even in the potential range where the [3Fe-4S] cluster must remain almost entirely in the reduced form. Evidently, this uphill electron transfer does not seriously compromise activity.