Structural Insights Into CO ₂ Transport Pathways in a W‐Formate Dehydrogenase: Structural Basis for CO ₂ Reduction

Mo/W-dependent formate dehydrogenases (Fdhs) catalyze the reversible reduction of CO₂ to formate and are key biocatalysts with high potential for CO₂ capture/conversion technologies. Although previous studies have suggested the presence of two substrate-access tunnels in Fdhs, experimental evidence for CO₂-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, O₂, and CO₂ 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 H₂O and CO₂ 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 CO₂ reduction by increasing substrate concentration near the catalytic center, thereby improving catalytic efficiency. Notably, variants affecting this site showed a selective effect for CO₂ reduction, with no impact on formate oxidation. These findings provide experimental evidence of a CO₂-specific pathway and identify structural determinants underpinning efficient CO₂ reduction in this enzyme family.