Unifying Activity, Structure, and Spectroscopy of [NiFe] Hydrogenases: Combining Techniques To Clarify Mechanistic Understanding

ConspectusAchieving a unified understanding of the mechanism of a multicenter redox enzyme such as [NiFe] hydrogenase is complicated by difficulties in reconciling information obtained by using different techniques and on samples in different physical forms. Measurements of the activity of the enzyme, and of factors which perturb activity, are generally carried out using biochemical assays in solution or with electrode-immobilized enzymes using protein film electrochemistry (PFE). Conversely, spectroscopy aimed at reporting on features of the metalloclusters in the enzyme, such as electron paramagnetic resonance (EPR) or X-ray absorption spectroscopy (XAS), is often conducted on frozen samples and is thus difficult to relate to catalytically relevant states as information about turnover and activity has been lost. To complicate matters further, most of our knowledge of the atomic-level structure of metalloenzymes comes from X-ray diffraction studies in the solid, crystalline state, which are again difficu...