Structural Insights into the Active-Ready Form of [FeFe]-Hydrogenase and Mechanistic Details of Its Inhibition by Carbon Monoxide

[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.