We have examined the rapid reaction kinetics and spectroscopicpropertiesofthemolybdenum-containing,NAD ؉ -dependent FdsABG formate dehydrogenase from Ralstonia eutropha. We confirm previous steady-state studies of the enzyme and extend its characterization to a rapid kinetic study of the reductive half-reaction (the reaction of formate with oxidized enzyme). We have also characterized the electron paramagnetic resonance signal of the molybdenum center in its Mo V state and demonstrated the direct transfer of the substrate C␣ hydrogen to the molybdenum center in the course of the reaction. Varying temperature, microwave power, and level of enzyme reduction, we are able to clearly identify the electron paramagnetic resonance signals for four of the iron/sulfur clusters of the enzyme and find suggestive evidence for two others; we observe a magnetic interaction between the molybdenum center and one of the iron/sulfur centers, permitting assignment of this signal to a specific iron/sulfur cluster in the enzyme. In light of recent advances in our understanding of the structure of the molybdenum center, we propose a reaction mechanism involving direct hydride transfer from formate to a molybdenum-sulfur group of the molybdenum center.The molybdenum-containing, NAD ϩ -dependent formate dehydrogenases from bacteria such as Ralstonia eutropha catalyze the oxidation of formate to CO 2 , reducing NAD ϩ to NADH, and are members of the NADH dehydrogenase superfamily of enzymes. These cytosolic formate dehydrogenases are expressed under aerobic conditions and are distinct from the predominant bacterial formate dehydrogenases expressed under anaerobic conditions, which are typically membrane-associated and extremely O 2 -sensitive; they are also distinct from the cofactorless formate dehydrogenases from many eukaryotes, including humans, that catalyze the direct hydride transfer from formate to NAD ϩ (1). In R. eutropha, the trimeric FdsABG (soluble, NAD ϩ -dependent, molybdenum-containing formate dehydrogenase, product of the fdsABG gene cluster) enzyme is encoded by the fdsGBACD operon and is predicted to contain seven iron/sulfur clusters, FMN, and a molybdenum center; each subunit bears significant sequence similarity (ϳ21% identity) and expected strong structural homology to a corresponding subunit of the matrix-or cytosol-exposed portion of NADH dehydrogenase, and the spatial layout of the several redox-active centers is particularly highly conserved. The 105-kDa FdsA is cognate to subunit Nqo3 in the crystallographically characterized Thermus thermophilus NADH dehydrogenase (2, 3) and is predicted to have four [4Fe-4S] clusters and one [2Fe-2S] cluster (4). The close structural homology predicted between FdsA and Nqo3 extends to the presence of a histidine ligand to one of the [4Fe-4S] clusters near the N terminus of the FdsA/Nqo3 subunit. The C terminus of FdsA contains a molybdenum center and has ϳ60% sequence similarity to the structurally characterized molybdenum-containing formate dehydrogenase FdhF of Escherichia ...