Mechanism of Hydrogen Sulfide-Dependent Inhibition of FeFe Hydrogenase

The
so-called FeFe hydrogenases catalyze H2 production
and oxidation at a dinuclear inorganic active site. Some of them can
be natively purified in an overoxidized, O2-resistant “Hinact” state, recently identified by Rodríguez-Maciá
et al. as the product of the reaction of the enzyme with sulfide [Rodríguez-Maciá, P.; 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 “Htrans” state. Our experimental and theoretical results
are consistent with the reactivation involving the reduction of the
H-cluster in the Htrans 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.