Electrochemical characterization of C. pasteurianum hydrogenase II and three 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 iron-sulfur clusters (up to four), and the composition of the so-called P1 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. 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 the FeFe-hydrogenase family, we also examined the effects of replacing three CpII residues with the corresponding residues from CpI: S99A, 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 (S99A), but not on the irreversibility of the catalytic response 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.