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

Metal-dependent formate dehydrogenases are important enzymes due to their activity of CO reduction to formate. The tungsten-containing FdhAB formate dehydrogenase from 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] clusters, and full reduction requires Ti(III)-citrate. The redox potentials of the four [4Fe-4S] centers range between -250 and -530 mV. Two distinct W signals were detected, W and W, which differ in only the -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 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 state of FdhAB.