Immobilization of O₂-tolerant [NiFe] hydrogenase from Cupriavidus necator on Tin-rich Indium Oxide Alters the Catalytic Bias from H₂ Oxidation to Proton Reduction

The ability of hydrogenases to reversibly catalyze the production and oxidation of hydrogen with minimal overpotential makes them attractive electrocatalysts for hydrogen energy conversion devices. The oxygen tolerance demonstrated by the membrane-bound [NiFe] hydrogenase (MBH) from Cupriavidus necator (previously known as Ralstonia eutropha) provides a further advantage; however, this enzyme is well-known as being strongly biased toward hydrogen oxidation and shows little promise toward hydrogen production. Here, we have immobilized the MBH after genetically attaching two different affinity tags to the C terminus of the enzyme-a His-tag (MBHHis) and a Strep-tag (MBHStrep). The differences in adsorption and electrocatalytic behavior were investigated when wired to an amorphous, transparent, and planar tin-rich indium tin oxide (ITOTR) thin-film electrode with a Sn/In ratio of 1:1. As demonstrated by ATR-IR spectroelectrochemical studies, the affinity of the His-tag for the tin-rich ITO surface allows for quantitative immobilization of MBHHis in a direct electron transfer configuration. Remarkably, once immobilized on tin-rich ITO, hydrogen oxidation as well as an unusually high proton reduction current is observed especially under hydrogen. While this behavior is only observed for tin-rich ITO (as compared to classical crystalline ITO, with a lower tin content) and not fully understood so far, the conditions demonstrated herein promote catalytic bidirectionality in essentially unidirectional [NiFe] hydrogenases, and that is at least partially related to favorable, direct enzyme-semiconductor interactions.