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A minimal light‐driven approach was established for studying enzymatic CO2 conversion spectroscopically. The system consists of a photosensitizer Eosin Y, EDTA as a sacrificial electron donor and substrate source, and formate dehydrogenase from Rhodobacter capsulatus (RcFDH) as a biocatalyst. This simplified three‐component system provides a photo‐triggered control for in situ characterization of the entire catalytic reaction. Direct reduction of RcFDH by the photosensitizer without additional electron carriers was confirmed via UV‐Vis spectroscopy, while GC‐MS and IR spectroscopy were used to follow photoinduced CO2 generation from EDTA and its subsequent enzymatic reduction, yielding the product formate. Photo‐driven and in vitro, dye‐based CO2 reduction was inhibited by azide under a mixed (competitive‐non‐competitive) inhibition mode. IR spectroscopy reveals displacement of the competitively‐bound azide by CO2, reflecting an interaction of both with the active site cofactor. This work comprises a proof‐of‐concept for a new approach to employ light for regulating the reaction of formate dehydrogenases and other CO2 reductases. [ABSTRACT FROM AUTHOR]

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]

Metal-containing formate dehydrogenases (FDH) catalyse the reversible oxidation of formate to carbon dioxide at their molybdenum or tungsten active site. They display a diverse subunit and cofactor composition, but structural information on these enzymes is limited. Here we report the cryo-electron microscopic structures of the soluble Rhodobacter capsulatus FDH (RcFDH) as isolated and in the presence of reduced nicotinamide adenine dinucleotide (NADH). RcFDH assembles into a 360 kDa dimer of heterotetramers revealing a putative interconnection of electron pathway chains. In the presence of NADH, the RcFDH structure shows charging of cofactors, indicative of an increased electron load. Rhodobacter capsulatus NAD+ dependent formate dehydrogenase (RcFDH) is a molybdoenzyme that catalyses the reversible oxidation of formate to carbon dioxide, and is of interest for biotechnological applications. Here the authors present the cryo-EM structures of RcFDH as isolated from R. capsulatus and in the reduced state with bound NADH, and discuss the enzyme mechanism. [ABSTRACT FROM AUTHOR]