Abstract
The molybdenum/tungsten—bis-pyranopterin guanine dinucleotide family of formate dehydrogenases (FDHs) plays roles in several metabolic pathways ranging from carbon fixation to energy harvesting because of their reaction with a wide variety of redox partners. Indeed, this metabolic plasticity results from the diverse structures, cofactor content, and substrates used by partner subunits interacting with the catalytic hub. Here, we unveiled two noncanonical FDHs in Bacillus subtilis, which are organized into two-subunit complexes with unique features, ForCE1 and ForCE2. We show that the formate oxidoreductase catalytic subunit interacts with an unprecedented partner subunit, formate oxidoreductase essential subunit, and that its amino acid sequence within the active site deviates from the consensus residues typically associated with FDH activity, as a histidine residue is naturally substituted with a glutamine. The formate oxidoreductase essential subunit mediates the utilization of menaquinone as an electron acceptor as shown by the formate:menadione oxidoreductase activity of both enzymes, their copurification with menaquinone, and the distinctive detection of a protein-bound neutral menasemiquinone radical by multifrequency electron paramagnetic resonance (EPR) experiments on the purified enzymes. Moreover, EPR characterization of both FDHs reveals the presence of several [Fe-S] clusters with distinct relaxation properties and a weakly anisotropic Mo(V) EPR signature, consistent with the characteristic molybdenum/bis-pyranopterin guanine dinucleotide cofactor of this enzyme family. Altogether, this work enlarges our knowledge of the FDH family by identifying a noncanonical FDH, which differs in terms of architecture, amino acid conservation around the molybdenum cofactor, and reactivity.
Highlights
Formate dehydrogenases catalyze the oxidation of formate into CO2 but have been shown to catalyze the reverse reaction in methanogenic and/or acetogenic microorganisms, namely the reduction of CO2 into formate [1]
The YjgC and YrhE sequences from B. subtilis encode putative metaldependent Formate Dehydrogenases (FDHs) Scrutiny of the B. subtilis 168 genome indicates that it encodes for yjgC and Journal Pre-proof yrhE, genes, whose ~110 kDa predicted proteins are paralogous with 61% of identity
While YjgC/YrhE are related to FdsA, as illustrated by the YjgC structural model based on the 3D-structure of R. capsulatus FdsA (Fig. 1C) and the phylogenic analysis (Fig. 1A and S1), YjgD and YrhD are distinct from FdsD or any of the other partner subunits previously characterized for a FDH, the complex I and [Ni-Fe] hydrogenases [6]
Summary
Formate dehydrogenases catalyze the oxidation of formate into CO2 but have been shown to catalyze the reverse reaction in methanogenic and/or acetogenic microorganisms, namely the reduction of CO2 into formate [1]. An histidine and an arginine residue located in proximity to the Mo atom [13] are strictly conserved in all FDHs described so far (Fig. 1A) and required for activity [14] These residues have been proposed to be involved in correct orientation or stabilization of the formate in the active site [15,16,17], but their role in activity is still debated. From a physiological point of view, FDHs are involved in a wide range of anaerobic metabolic pathways, such as energy harvesting and carbon fixation [6, 9] This is due to the interaction of the catalytic subunit which harbors the Mo/W-bisPGD cofactor with partner subunits with the greatest diversity amongst the Mo/W-bis PGD enzymes superfamily [9]. Both FDHs display a new architecture in which their catalytic subunit is associated with an unprecedented partner subunit that allows reactivity with menaquinone
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