UC Riverside

Formate oxidation to carbon dioxide is a key reaction in one-carbon compound metabolism, and its reverse reaction represents the first step in carbon assimilation in the acetogenic and methanogenic branches of many anaerobic organisms. The molybdenum-containing dehydrogenase FdsDABG is a soluble NAD-dependent formate dehydrogenase and a member of the DMSO reductase family of molybdoenzymes and NADH dehydrogenase superfamily. Here, we present the first structure of the FdsBG subcomplex from the cytosolic FdsDABG formate dehydrogenase from the hydrogen-oxidizing bacterium Cupriavidus necator H16 – both with and without bound NADH. The structures revealed that the two iron-sulfur clusters, Fe4S4 cluster B6 in FdsB and Fe2S2 cluster G7 in FdsG, are closer to the FMN than they are in other NADH dehydrogenases. Rapid kinetic studies and EPR measurements of rapid freeze-quenched samples of the NADH reduction of FdsBG identified a neutral flavin semiquinone, not previously observed to participate in NADH-mediated reduction of the FdsDABG holoenzyme. We found that this semiquinone forms through the transfer of one electron from the fully reduced FMNH-, initially formed via NADH-mediated reduction, to the G7 Fe2S2 cluster. This Fe2S2 cluster is not part of the on-path electron transferring chain of iron-sulfur clusters connecting the FMN in the FdsB subunit with the active-site molybdenum center of FdsA. According to the NADH-bound structure, the nicotinamide ring stacks onto the re-face of the FMN; the structure of FdsDABG in Rhodobacter capsulatus shows a much further binding of the nicotinamide ring relative to the FMN. Regardless, NADH binding significantly reduced the electron density for the isoalloxazine ring of FMN and induced a conformational change in residues of the FMN-binding pocket that display peptide-bond flipping upon NAD+ binding in proper NADH dehydrogenases.