OUP accepted manuscript

While formate dehydrogenases (FDHs) have been used for cofactor recycling in chemoenzymatic synthesis, the ability of FDH to reduce CO₂ could also be utilized in the conversion of CO₂ to useful products via formate (HCOO^-). In this study, we investigated the reduction of CO₂ in the form of hydrogen carbonate (HCO₃^-) to formate by FDHs from Candida methylica (CmFDH) and Chaetomium thermophilum (CtFDH) in a NADH-dependent reaction. The catalytic performance with HCO₃^- as a substrate was evaluated by measuring the kinetic rates and conducting productivity assays. CtFDH showed a higher efficiency in converting HCO₃^- to formate than CmFDH, whereas CmFDH was better in the oxidation of formate. The pH optimum of the reduction was at pH 7-8. However, the high concentrations of HCO₃^- reduced the reaction rate. CtFDH was modeled in the presence of HCO₃^- showing that it fits to the active site. The active site setting for hydride transfer in CO₂ reduction was modeled. The hydride donated by NADH would form a favorable contact to the carbon atom of HCO₃^-, resulting in a surplus of electrons within the molecule. This would cause the complex formed by hydrogen carbonate and the hydride to break into formate and hydroxide ions.