Redox potentials elucidate the electron transfer pathway of NAD+-dependent formate dehydrogenases

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 CO2 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 reduction potentials of the bis-MGD and the Fe-S clusters were determined via redox titration by EPR spectroscopy. Reduction 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 reduction 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 reduction 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.