Electrochemical and Infrared Spectroscopic Studies Provide Insight into Reactions of the NiFe Regulatory Hydrogenase from Ralstonia eutropha with O₂ and CO

The regulatory hydrogenase (RH) from Ralstonia eutropha acts as the H₂-sensing unit of a two-component system that regulates biosynthesis of the energy conserving hydrogenases of the organism according to the availability of H₂. The H₂ oxidation activity, which was so far determined in vitro with artificial electron acceptors, has been considered to be insensitive to O₂ and CO. It is assumed that bulky isoleucine and phenylalanine amino acid residues close to the NiFe active site gate gas access, preventing molecules larger than H₂ interacting with the active site. We have carried out sensitive electrochemical measurements to demonstrate that O₂ is in fact an inhibitor of H₂ oxidation by the RH, and that both H⁺ reduction and H₂ oxidation are inhibited by CO. Furthermore, we have demonstrated that the inhibitory effect of O₂ arises due to interaction of O₂ with the active site. Using protein film infrared electrochemistry (PFIRE) under H₂ oxidation conditions, in conjunction with solution infrared measurements, we have identified previously unreported oxidized inactive and catalytically active reduced states of the RH active site. These findings suggest that the RH has a rich active site chemistry similar to that of other NiFe hydrogenases.