Site-selective Protonation of the Catalytic Cofactor in [FeFe]-Hydrogenase

Hydrogenases are microbial redox enzymes that catalyze H2 oxidation and proton reduction (H2 evolution). While all hydrogenases show high H2 oxidation activities, [FeFe]-hydrogenases are excellent H2 evolution catalysts as well. Their hydrogen-forming active site cofactor (“H-cluster”) comprises a [4Fe-4S] cluster covalently linked to a diiron site modified with CO and CN– ligands that tune the redox potential and anchor the cofactor within the protein. Two distinct proton transfer pathways connect the H-cluster and facilitate hydrogen turnover at low overpotential. In this study, we employ in situ ATR FTIR spectroscopy and spectro-electrochemistry to analyze the mechanics of hydrogen turnover in [FeFe]-hydrogenase. Titrating the proton concentration from pH 10 to pH 5 under H2 oxidation or H2 evolution conditions reveals the influence of site-selective protonation on the equilibrium of H-cluster redox states. Under H2 evolution 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 natural pH dependence of H2 evolution as catalyzed by [FeFe]-hydrogenase.