Mechanism of Hydrogen Sulfide-Dependent Inhibition of FeFe Hydrogenase

The so-called FeFe hydrogenases catalyze H 2 production and oxidation at a dinuclear inorganic active site. Some of them can be natively purified in an overoxidized, O 2 -resistant "H inact " state, recently identified by Rodríguez-Maciá et al . as the product of the reaction of the enzyme with sulfide (J. Am. Chem. Soc. 140, 9346 (2018)). We used a combination of direct electrochemistry experiments with the FeFe hydrogenase from C. reinhardtii , site-directed mutagenesis, and molecular dynamics and DFT calculations to describe the mechanism of inhibition: the diffusion of the inhibitor in the enzyme and its subsequent reaction at the active site. We conclude that hydrogen sulfide (H 2 S) inhibits the enzyme non-competitively, in a first step by replacing a conserved water molecule that is involved in proton transfer to/from the active site, and then binding to the active site as a hydrosulfide ligand (HS − ). DFT calculations with the PBE0-D3 functional successfully describe the redox state of the cubane subcluster in the resulting "H trans " state. In contrast with the proposal of Rodríguez-Maciá et al. , our experimental and theoretical results are consistent with the reactivation involving the reduction of H trans , followed by the potentiometric or catalytic re-oxidation of the enzyme. This mechanism reconciles all experimental observations and we suggest that it is common to all FeFe hydrogenases. In addition, we observe that the hydrogenases from M. elsdenii , C. acetobutylicum (CaI) and C. pasteurianum (CpI) are also inhibited by sulfide, but with very slow kinetics. Whereas sulfide inhibition is fully reversible, we observed an irreversible inactivation by polysulfide contaminants, which should be avoided if the hydrogenase is exposed to sulfide to prepare samples that are protected from air, e.g. for transport or storage.