Metal-dependent formate dehydrogenases are important enzymes due to their activity of CO 2 reduction to formate. The tungstencontaining FdhAB formate dehydrogenase from Desulfovibrio vulgaris Hildenborough is a good example displaying high activity, simple composition, and a notable structural and catalytic robustness. Here, we report the first spectroscopic redox characterization of FdhAB metal centers by EPR. Titration with dithionite or formate leads to reduction of three [4Fe−4S] 1+ clusters, and full reduction requires Ti(III)−citrate. The redox potentials of the four [4Fe−4S] 1+ centers range between −250 and −530 mV. Two distinct W V signals were detected, W D V and W F V, which differ in only the g 2 -value. This difference can be explained by small variations in the twist angle of the two pyranopterins, as determined through DFT calculations of model compounds. The redox potential of W VI/V was determined to be −370 mV when reduced by dithionite and −340 mV when reduced by formate. The crystal structure of dithionitereduced FdhAB was determined at high resolution (1.5 Å), revealing the same structural alterations as reported for the formatereduced structure. These results corroborate a stable six-ligand W coordination in the catalytic intermediate W V state of FdhAB.
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