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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]

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]

The organometallic H-cluster of the [FeFe]-hydrogenase consists of a [4Fe–4S] cubane bridged via a cysteinyl thiolate to a 2Fe subcluster ([2Fe]H) containing CO, CN−, and dithiomethylamine (DTMA) ligands. The H-cluster is synthesized by three dedicated maturation proteins: the radical SAM enzymes HydE and HydG synthesize the non-protein ligands, while the GTPase HydF serves as a scaffold for assembly of [2Fe]H prior to its delivery to the [FeFe]-hydrogenase containing the [4Fe–4S] cubane. HydG uses L -tyrosine as a substrate, cleaving it to produce p-cresol as well as the CO and CN− ligands to the H-cluster, although there is some question as to whether these are formed as free diatomics or as part of a [Fe(CO)2(CN)] synthon. Here we show that Clostridium acetobutylicum (C.a.) HydG catalyzes formation of multiple equivalents of free CO at rates comparable to those for CN− formation. Free CN− is also formed in excess molar equivalents over protein. A g = 8.9 EPR signal is observed for C.a. HydG reconstituted to load the 5th "dangler" iron of the auxiliary [4Fe–4S][FeCys] cluster and is assigned to this "dangler-loaded" cluster state. Free CO and CN− formation and the degree of activation of [FeFe]-hydrogenase all occur regardless of dangler loading, but are increased 10–35% in the dangler-loaded HydG; this indicates the dangler iron is not essential to this process but may affect relevant catalysis. During HydG turnover in the presence of myoglobin, the g = 8.9 signal remains unchanged, indicating that a [Fe(CO)2(CN)(Cys)] synthon is not formed at the dangler iron. Mutation of the only protein ligand to the dangler iron, H272, to alanine nearly completely abolishes both free CO formation and hydrogenase activation, however results show this is not due solely to the loss of the dangler iron. In experiments with wild type and H272A HydG, and with different degrees of dangler loading, we observe a consistent correlation between free CO/CN− formation and hydrogenase activation. Taken in full, our results point to free CO/CN−, but not an [Fe(CO)2(CN)(Cys)] synthon, as essential species in hydrogenase maturation. [ABSTRACT FROM AUTHOR]

Biological carbon dioxide (CO 2 ) reduction is an important step by which organisms form valuable energy-richer molecules required for further metabolic processes. The Mo-dependent formate dehydrogenase (FDH) from Rhodobacter capsulatus catalyzes reversible formate oxidation to CO 2 at a bis-molybdopterin guanine dinucleotide (bis-MGD) cofactor. To elucidate potential substrate binding sites relevant for the mechanism, we studied herein the interaction with the inhibitory molecules azide and cyanate, which are isoelectronic to CO 2 and charged as formate. We employed infrared (IR) spectroscopy in combination with density functional theory (DFT) and inhibition kinetics. One distinct inhibitory molecule was found to bind to either a non-competitive or a competitive binding site in the secondary coordination sphere of the active site. Site-directed mutagenesis of key amino acid residues in the vicinity of the bis-MGD cofactor revealed changes in both non-competitive and competitive binding, whereby the inhibitor is in case of the latter interaction presumably bound between the cofactor and the adjacent Arg587., (© 2022 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

Formate dehydrogenase (FDH) enzymes are versatile catalysts for CO 2 conversion. The FDH from Rhodobacter capsulatus contains a molybdenum cofactor with the dithiolene functions of two pyranopterin guanine dinucleotide molecules, a conserved cysteine, and a sulfido group bound at Mo(VI). In this study, we focused on metal oxidation state and coordination changes in response to exposure to O 2 , inhibitory anions, and redox agents using X-ray absorption spectroscopy (XAS) at the Mo K-edge. Differences in the oxidative modification of the bis-molybdopterin guanine dinucleotide (bis-MGD) cofactor relative to samples prepared aerobically without inhibitor, such as variations in the relative numbers of sulfido (Mo═S) and oxo (Mo═O) bonds, were observed in the presence of azide (N 3 - ) or cyanate (OCN - ). Azide provided best protection against O 2 , resulting in a quantitatively sulfurated cofactor with a displaced cysteine ligand and optimized formate oxidation activity. Replacement of the cysteine ligand by a formate (HCO 2 - ) ligand at the molybdenum in active enzyme is compatible with our XAS data. Cyanide (CN - ) inactivated the enzyme by replacing the sulfido ligand at Mo(VI) with an oxo ligand. Evidence that the sulfido group may become protonated upon molybdenum reduction was obtained. Our results emphasize the role of coordination flexibility at the molybdenum center during inhibitory and catalytic processes of FDH enzymes.