The Geometry of the Catalytic Active Site in [FeFe]-Hydrogenases is Determined by Hydrogen Bonding and Proton Transfer

The H2 conversion and CO inhibition reactivity of nine [FeFe]-hydrogenase constructs with semi-artificial cofactors was studied by in situ and time-resolved infrared spectroscopy, X-ray crystallography, and theoretical methods. Impaired hydrogen turnover and proton transfer as well as characteristic CO inhibition/ reactivation kinetics are assigned to varying degrees of hydrogen-bonding interactions at the active site. We show that the probability to adopt catalytic intermediates is modulated by intramolecular and protein-cofactor interactions that govern structural dynamics at the active site of [FeFe]-hydrogenases.