Comparative analysis of CO2 reduction by soluble Escherichia coli formate dehydrogenase H and its selenocysteine-to-cysteine substitution variant

Metal-dependent formate dehydrogenases (Me-FDHs) are highly active CO₂-reducing enzymes operating at low redox potentials and employ either molybdenum or tungsten to reduce the bound substrate. This makes them suitable for electrochemical applications such as fossil-free production of commodity chemicals utilizing renewable energy. Electrocatalytic CO₂ reduction by cathode-immobilized Me-FDHs has been recently demonstrated and rational protein engineering can be used to optimize Me-FDHs for various carbon reduction reactions. In the present study, CO₂ reduction by soluble monomeric Escherichia coli formate dehydrogenase H (EcFDH-H) was demonstrated and the function of its nucleophilic selenocysteine residue as a transient ligand of a centrally bound molybdenum atom was investigated. Kinetic analysis of the wildtype enzyme revealed maximum CO₂ reduction rates of 44 ± 6 s−1 at pH 5.8 that was decreased to 19% and 0% in the case of selenocysteine substitution with the structural homologues cysteine and serine, respectively. Further selenocysteine-to-cysteine substitution effects included an increased acid tolerance as well as stronger inhibition by nitrate and azide indicating a shift of the Mo oxidation state from IV to VI. Conversely, a destabilizing effect on the oxidized Mo(VI) center could be assigned to the native selenocysteine residue that may facilitate the observed efficient CO₂ reduction by rapid transition between Mo oxidation states. Taken together, the performed characterization of EcFDH-H as a catalyst for CO₂ reduction and the selenocysteine substitution analysis furthers the understanding of the active-site structure of Me-FDHs and thereby supports the development of more efficient biocatalysts for CO₂ reduction.