Hydrogenases are bidirectional redox enzymes that catalyze hydrogen turnover in archaea, bacteria, and algae. While all types of hydrogenase show H-2 oxidation activity, [FeFe]-hydrogenases are excellent H-2 evolution catalysts as well. Their active site cofactor comprises a [4Fe-4S] cluster covalently linked to a diiron site equipped with carbon monoxide and cyanide ligands. The active site niche is connected with the solvent by two distinct proton transfer pathways. To analyze the catalytic mechanism of [FeFe]-hydrogenase, we employ operando infrared spectroscopy and infrared spectro-electrochemistry. Titrating the pH under H-2 oxidation or H-2 evolution conditions reveals the influence of site-selective protonation on the equilibrium of reduced cofactor states. Governed by pK(a) differences across the active site niche and proton transfer pathways, we find that individual electrons are stabilized either at the [4Fe-4S] cluster (alkaline pH values) or at the diiron site (acidic pH values). This observation is discussed in the context of the complex interdependence of hydrogen turnover and bulk pH.
