Redox potentials elucidate the electron transfer pathway of NAD < sup > + < /sup > -dependent formate dehydrogenases

J Inorg Biochem. 2024 Jan 20;253:112487. doi: 10.1016/j.jinorgbio.2024.112487. Online ahead of print.ABSTRACTMetal-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 (FeS) 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 [4Fe4S] clusters within electron transfer distance. To clarify this difference, the redox potentials of the bis-MGD and the FeS clusters were determined via redox titration by EPR spectroscopy. Redox potentials for the bis-MGD cofactor and five of the seven FeS 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 [4Fe4S] cluster. Finally, characterization of the monomeric FdsGBAD...

Source: Journal of Inorganic Biochemistry - February 2, 2024 Category: Biochemistry Authors: Benjamin R Duffus Marcel Gauglitz Christian Teutloff Silke Leimk ühler Source Type: research