Electrochemical characterization of C. pasteurianum hydrogenase II and four site- directed variants: catalysis and inhibition

Certain Clostridium pasteurianum strains encode up to four FeFe-hydrogenases, which are homologous (identity > 20%) but can be readily distinguished by their number of residues (450–550), the nature and number of accessory clusters (up to four), and the composition of the so-called L1 motif (TSCCP in CpI and CpII, TSCCCP in CpIII, or NSCCP in CpIV). CpI is one of the most extensively characterized FeFe-hydrogenases ; here we compare it with CpII using protein film electrochemistry, the technique invented and popularised by Fraser Armstrong in Oxford, to whom this paper is dedicated. In this approach the enzyme is wired to an electrode and then interrogated to obtain information on their catalytic responses and reactions with the inhibitors. With the aim of elucidating the relation between structure and function within this family of enzymes, we also examined the effects of replacing four CpII residues with the corresponding residues from CpI : S99A and T223M, near the active-site dinuclear cluster; T377A, between the cubane and the proximal cluster; and S73A, near the distal cluster. Small effects are observed on the catalytic bias (S99A) and the Michaelis constant for H₂ (T223M), but not on the irreversibility of the catalytic voltammetry of CpII. The most significant changes concern the reaction with O₂: unlike CpI, CpII reacts fully irreversibly with O₂; the T377A and S99A mutations significantly slow this reaction, whereas the S73A mutation makes it more reversible. These findings have far-reaching implications for ongoing research aimed at understanding why homologous hydrogenases exhibit distinct catalytic properties, by suggesting that these differences likely arise from a combination of small changes rather than a single underlying cause.