Spectroscopic and Structural Characterization of Reduced Desulfovibrio vulgaris Hildenborough W-FdhAB Reveals Stable Metal Coordination during Catalysis

[Image: see text] Metal-dependent formate dehydrogenases are important enzymes due to their activity of CO(2) reduction to formate. The tungsten-containing 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 dithionite-reduced FdhAB was determined at high resolution (1.5 Å), revealing the same structural alterations as reported for the formate-reduced structure. These results corroborate a stable six-ligand W coordination in the catalytic intermediate W(V) state of FdhAB.