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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]

All kingdoms of life have more than 150 different forms of RNA alterations, with tRNA accounting for around 80% of them. These chemical alterations include, among others, methylation, sulfuration, hydroxylation, and acetylation. These changes are necessary for the proper codon recognition and stability of tRNA. In Escherichia coli, sulfur modification at the wobble uridine (34) of lysine, glutamic acid, and glutamine is essential for codon and anticodon binding and prevents frameshifting during translation. Two important proteins that are involved in this thiolation modification are the L-cysteine desulfurase IscS, the initial sulfur donor, and tRNA-specific 2-thiouridylase MnmA, which adenylates and finally transfers the sulfur from IscS to the tRNA. tRNA-specific 2-thiouridylases are iron–sulfur clusters (Fe-S), either dependent or independent depending on the organism. Here, we dissect the controversy of whether the E. coli MnmA protein is an Fe-S cluster-dependent or independent protein. We show that when Fe-S clusters are bound to MnmA, tRNA thiolation is inhibited, making MnmA an Fe-S cluster-independent protein. We further show that 2-thiouridylase only binds to tRNA from its own organism. [ABSTRACT FROM AUTHOR]