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Metal-dependent, nicotine adenine dinucleotide (NAD+)-dependent formate dehydrogenases (FDHs) are complex metalloenzymes coupling biochemical transformations through intricate electron transfer pathways. Rhodobacter capsulatus FDH is a model enzyme for understanding coupled catalysis, in that reversible CO 2 reduction and formate oxidation are linked to a flavin mononuclotide (FMN)-bound diaphorase module via seven iron-sulfur (Fe S) clusters as a dimer of heterotetramers. Catalysis occurs at a bis-metal-binding pterin (Mo) binding two molybdopterin guanine dinucleotides (bis-MGD), a protein-based Cys residue and a participatory sulfido ligand. Insights regarding the proposed electron transfer mechanism between the bis-MGD and the FMN have been complicated by the discovery that an alternative pathway might occur via intersubunit electron transfer between two [4Fe 4S] clusters within electron transfer distance. To clarify this difference, the redox potentials of the bis-MGD and the Fe S clusters were determined via redox titration by EPR spectroscopy. Redox potentials for the bis-MGD cofactor and five of the seven Fe S clusters could be assigned. Furthermore, substitution of the active site residue Lys295 with Ala resulted in altered enzyme kinetics, primarily due to a more negative redox potential of the A1 [4Fe 4S] cluster. Finally, characterization of the monomeric FdsGBAD heterotetramer exhibited slightly decreased formate oxidation activity and similar iron-sulfur clusters reduced relative to the dimeric heterotetramer. Comparison of the measured redox potentials relative to structurally defined Fe S clusters support a mechanism by which electron transfer occurs within a heterotetrameric unit, with the interfacial [4Fe 4S] cluster serving as a structural component toward the integrity of the heterodimeric structure to drive efficient catalysis. The metal-dependent formate dehydrogenase from Rhodobacter capsulatus is a complex, molybdenum-containing iron-sulfur flavoenzyme. Herein, the redox potentials for five of the seven iron-sulfur clusters and the Mo(VI) → Mo(V) and Mo(V) → Mo(IV) reduction couples have been determined, and evidence for electron transfer through the monomeric heterotetramer unit is presented. [Display omitted] • Redox potentials of Mo active site and five of seven iron-sulfur clusters determined. • Use of Eu-based reductant improved discernment of cofactor redox potentials. • Alteration of an active site residue reveals identity of an iron-sulfur cluster. • Comparable monomer vs dimer activity and iron-sulfur cluster reduction observed. [ABSTRACT FROM AUTHOR]