While formate dehydrogenases (FDHs) have been used for cofactor recycling in chemoenzymatic synthesis, the ability of FDH to reduce CO could also be utilized in the conversion of CO to useful products via formate (HCOO). In this study, we investigated the reduction of CO in the form of hydrogen carbonate (HCO) to formate by FDHs from Candida methylica (CmFDH) and Chaetomium thermophilum (CtFDH) in a NADH-dependent reaction. The catalytic performance with HCO as a substrate was evaluated by measuring the kinetic rates and conducting productivity assays. CtFDH showed a higher efficiency in converting HCO 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 HCO reduced the reaction rate. CtFDH was modeled in the presence of HCO showing that it fits to the active site. The active site setting for hydride transfer in CO reduction was modeled. The hydride donated by NADH would form a favorable contact to the carbon atom of HCO, 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.
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