Site-selective protonation controls hydrogen turnover in [FeFe]-hydrogenase

Hydrogenases are metalloenzymes that catalyze H2 oxidation and H2 evolution. In particular, [FeFe]-hydrogenases have been shown to be excellent H2 evolution catalysts. Their active site cofactor comprises a [4Fe-4S] cluster covalently linked to a diiron site modified with CO and CN- ligands (H-cluster). Distinct proton transfer pathways connect the H-cluster on opposing sites facilitating hydrogen turnover at low overptential. In this study, we employ in situ ATR FTIR spectroscopy and spectro-electrochemistry to analyze H2 oxidation and H2 evolution of [FeFe]-hydrogenase at a wide range of proton concentrations. Our data reveals the influence of site-selective proton transfer on the equilibrium of redox states. Under reducing conditions, protonation facilitates electron uptake at the [4Fe-4S] cluster and impedes premature reduction of the diiron site, the later which dominates at acidic pH values. This observation is discussed in the context of the pH dependence of H2 evolution as catalyzed by [FeFe]-hydrogenase.