How does methylviologen cation radical supply two electrons to the formate dehydrogenase in the catalytic reduction process of CO to formate?

Formate dehydrogenase from Candida boidinii (EC.1.2.1.2; CbFDH) is a commercially available enzyme and can be easily handled as a catalyst for the CO reduction to formate in the presence of NADH, single-electron reduced methylviologen (MV˙) and so on. It was found that the formate oxidation to CO with CbFDH was suppressed using the oxidized MV as a co-enzyme and the single-electron reduced MV (MV˙) was effective for the catalytic activity of CbFDH for the CO reduction to formate compared with that using the natural co-enzyme of NADH [Y. Amao, Chem. Lett., 2017, 46, 780-788]. The CO reduction to formate catalyzed by CbFDH requires two molecules of the MV˙. In order to clarify the two-electron reduction process using MV˙ in the CO reduction to formate catalyzed with CbFDH, we attempted enzyme reaction kinetics, electrochemical and quantum chemical analyses. Kinetic parameters obtained from the enzymatic kinetic analysis metric revealed an index of affinity of MV˙ for CbFDH in the CO reduction to formate. From the results of the electrochemical analysis, it was predicted that only one molecule of MV˙ was bound to CbFDH, and the MV bound to CbFDH was to be necessarily re-reduced by the electron source outside of CbFDH to supply the second electron in the CO reduction to formate. From the results of docking simulation and density functional theory (DFT) calculations, it was indicated that one molecule of MV bound to the position close to CO in the inner part of the substrate binding pocket of CbFDH contributed to the two-electron CO reduction to formate.