Institut für Chemie, Max-Volmer-Laboratorium für Biophysikalische Chemie, PC14, Technische Universität Berlin, Strasse des 17. Juni 135, 10623, Berlin, Germany.
Institut für Biochemie und Biologie, Molekulare Enzymologie, Universität Potsdam, Karl-Liebknecht-Strasse 24-25, 14476, Potsdam, Germany.
Chemistry. 2022 Sep 27;28(54):e202201091. doi: 10.1002/chem.202201091. Epub 2022 Aug 3.
Biological carbon dioxide (CO ) 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 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 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.
生物二氧化碳（CO ）还原是生物体形成进一步代谢所需的有价值的能量更丰富的分子的重要步骤。来自荚膜红细菌的钼依赖型甲酸盐脱氢酶（FDH）在双钼喋呤鸟嘌呤二核苷酸（bis-MGD）辅因子的作用下催化甲酸盐的可逆氧化生成 CO 。为了阐明与机制相关的潜在底物结合位点，我们研究了与 CO 等电子且带电荷的叠氮化物和氰酸盐与抑制剂的相互作用。我们采用了红外（IR）光谱学与密度泛函理论（DFT）和抑制动力学相结合的方法。发现一个独特的抑制剂分子可以结合到活性位点的次级配位球中的非竞争性或竞争性结合位点。在 bis-MGD 辅因子附近的关键氨基酸残基的定点突变揭示了非竞争性和竞争性结合的变化，在后一种相互作用中，抑制剂可能在辅因子和相邻的 Arg587 之间结合。
