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 Hinact state, recently identified by Rodrig ' uez-Macia et al. as the product of the reaction of the enzyme with sulfide [Rodriguez-Macia, P.; et al. J. Am. Chem. Soc. 2018, 140, 9346]. We used a combination of direct electrochemistry experiments with the FeFe hydrogenase from Chlamydomonas reinhardtii, site-directed mutagenesis, molecular dynamics and density functional theory (DFT) calculations to describe the mechanism of inhibition: the diffusion of the inhibitor in the enzyme and its subsequent reaction at the active site H-cluster. We conclude that hydrogen sulfide (H2S) inhibits the enzyme noncompetitively, in a first step by replacing a conserved water molecule that is involved in proton transfer, 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 fragment of the H-cluster in the resulting H-trans state. Our experimental and theoretical results are consistent with the reactivation involving the reduction of the H-cluster in the H-trans state, followed by the potentiometric or catalytic reoxidation 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 Megasphaera elsdenii, Clostridium acetobutylicum (CaI), and Clostridium pasteurianum (CpI) are also inhibited by sulfide, but with very slow kinetics. Although 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.

Automated summary

The study investigates the inhibitory mechanism of hydrogen sulfide on FeFe hydrogenase through a combination of various experimental and theoretical methods, revealing the underlying mechanisms and providing insights into the oxidation and reduction of the enzyme. Additionally, it was observed that the inhibition by sulfide may lead to irreversible inactivation of the hydrogenase.