Probing the Active Site of an O2‐Tolerant NAD+‐Reducing [NiFe]‐Hydrogenase from Ralstonia eutropha H16 by In Situ EPR and FTIR Spectroscopy

A clear picture: In situ EPR and FTIR spectroscopic studies on the soluble, NAD+-reducing [NiFe]-hydrogenase of Ralstonia eutropha reveal that the catalytic site resides predominantly in the intermediate Nia-C state within whole cells. This state, can either be reversibly oxidized to a “Nir-B”-like state or further reduced to various Nia-SR species. The data suggest that the iron center in the active site contains a standard set (one CO and two CN−) of inorganic ligands.