Structural Insights into the Active-Ready Form of [FeFe]-Hydrogenase and Mechanistic Details of Its Inhibition by Carbon Monoxide Claudio Greco Maurizio Bruschi Jimmy Heimdal Piercarlo Fantucci Luca De Gioia Ulf Ryde 10.1021/ic701051h.s002 https://acs.figshare.com/articles/journal_contribution/Structural_Insights_into_the_Active_Ready_Form_of_FeFe_Hydrogenase_and_Mechanistic_Details_of_Its_Inhibition_by_Carbon_Monoxide/2987989 [FeFe]-Hydrogenases harbor a {2Fe3S} assembly bearing two CO and two CN<sup>-</sup> groups, a μ-CO ligand, and a vacant coordination site trans to the μ-CO group. Recent theoretical results obtained studying the <i>isolated</i> {2Fe3S} subsite indicated that one of the CN<sup>-</sup> ligands can easily move from the crystallographic position to the coordination site trans to the μ-CO group; such an isomerization would have a major impact on substrates and inhibitors binding regiochemistry and, consequently, on the catalytic mechanism. To shed light on this crucial issue, we have carried out hybrid QM/MM and free energy perturbation calculations on the whole enzyme, which demonstrate that the protein environment plays a crucial role and maintains the CN<sup>-</sup> group fixed in the position observed in the crystal structure; these results strongly support the hypothesis that the vacant coordination site trans to the μ-CO group has a crucial functional relevance both in the context of CO-mediated inhibition of the enzyme and in dihydrogen oxidation/evolution catalysis. 2007-09-03 00:00:00 crystal structure 3S inhibitors binding regiochemistry energy perturbation calculations CO ligand QM enzyme Fe coordination site trans CN Structural Insights Mechanistic Details protein environment