Anion Binding and Oxidative Modification at the Molybdenum Cofactor of Formate Dehydrogenase from Rhodobacter capsulatus Studied by X‑ray Absorption Spectroscopy

Formate dehydrogenase (FDH) enzymes are versatile catalysts for CO₂ 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 O₂, 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 (N₃^–) or cyanate (OCN^–). Azide provided best protection against O₂, resulting in a quantitatively sulfurated cofactor with a displaced cysteine ligand and optimized formate oxidation activity. Replacement of the cysteine ligand by a formate (HCO₂^–) 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.