Abstact

Mo/W-dependent formate dehydrogenases (Fdhs) catalyze the reversible reduction of CO(2) to formate and are key biocatalysts with high potential for CO(2) capture/conversion technologies. Although previous studies have suggested the presence of two substrate-access tunnels in Fdhs, experimental evidence for CO(2)-specific pathways has been lacking. Here, we present an integrated study of Nitratidesulfovibrio vulgaris FdhAB combining crystallography, molecular dynamics simulations, mutagenesis, and kinetic assays. NvFdhAB crystals pressurized with Kr, O(2), and CO(2) were used to map gas diffusion routes and uncovered a substrate-retention site consistently occupied by small molecules in multiple crystal structures. Our results indicate that both substrates mostly use the main tunnel to reach this retention site, but H(2)O and CO(2) can also enter through a novel side branch before following a shared route to the buried W active site. The retention site, located at the junction of both tunnels, plays a synergistic role in enhancing CO(2) reduction by increasing substrate concentration near the catalytic center, thereby improving catalytic efficiency. Notably, variants affecting this site showed a selective effect for CO(2) reduction, with no impact on formate oxidation. These findings provide experimental evidence of a CO(2)-specific pathway and identify structural determinants underpinning efficient CO(2) reduction in this enzyme family.