Replacing a cysteine ligand by selenocysteine in a [NiFe]-hydrogenase unlocks hydrogen production activity and addresses the role of concerted proton-coupled electron transfer in electrocatalytic reversibility

Hydrogenases catalyze hydrogen/proton interconversion that is normally electrochemically reversible (having minimal overpotential requirement), a special property otherwise almost exclusive to platinum metals. The mechanism of [NiFe]-hydrogenases includes a long-range proton-coupled electron-transfer process involving a specific Ni-coordinated cysteine and the carboxylate of a nearby glutamate. A variant in which this cysteine has been exchanged for selenocysteine displays two distinct changes in electrocatalytic properties, as determined by protein film voltammetry. First, proton reduction, even in the presence of H₂ (a strong product inhibitor), is greatly enhanced relative to H₂ oxidation: this result parallels a characteristic of natural [NiFeSe]-hydrogenases which are superior H₂ production catalysts. Second, an inflection (an <em>S</em>-shaped “twist” in the trace) appears around the formal potential, the small overpotentials introduced in each direction (oxidation and reduction) signaling a departure from electrocatalytic reversibility. Concerted proton–electron transfer offers a lower energy pathway compared to stepwise transfers. Given the much lower proton affinity of Se compared to that of S, the inflection provides compelling evidence that concerted proton–electron transfer is important in determining why [NiFe]-hydrogenases are reversible electrocatalysts.