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.

钼/钨-双-吡喃蝶呤鸟嘌呤二核苷酸家族的甲酸脱氢酶 (FDH) 在从碳固定到能量收集的多种代谢途径中发挥作用，因为它们与多种氧化还原伙伴发生反应。事实上，这种代谢可塑性是由与催化中心相互作用的伙伴亚基使用的多种结构、辅因子含量和底物造成的。在这里，我们揭示了枯草芽孢杆菌中的两种非规范 FDH，它们被组织成具有独特功能的两个亚基复合物 ForCE1 和 ForCE2。我们表明甲酸氧化还原酶催化亚基与前所未有的伙伴亚基甲酸氧化还原酶必需亚基相互作用，并且其活性位点内的氨基酸序列偏离通常与 FDH 活性相关的共有残基，因为组氨酸残基天然取代为谷氨酰胺。甲酸氧化还原酶必需亚基介导甲基萘醌作为电子受体的利用，如甲酸：甲萘醌氧化还原酶活性、它们与甲基萘醌的共纯化以及通过多频电子顺磁共振对蛋白质结合的中性甲基萘醌自由基的独特检测所示。 EPR) 对纯化酶的实验。而且，两种 FDH 的 EPR 表征揭示了几个具有不同弛豫特性和弱各向异性 Mo(V) EPR 特征的 [Fe-S] 簇的存在，这与该酶家族的特征性钼/双吡喃蝶呤鸟嘌呤二核苷酸辅因子一致。总而言之，这项工作通过识别非规范 FDH 扩大了我们对 FDH 家族的了解，该 FDH 在结构、钼辅因子周围的氨基酸保守性和反应性方面有所不同。