In vitro sulfuration of Rhodobacter capsulatus formate dehydrogenase

Metal-dependent formate dehydrogenases (FDHs) are of considerable interest as a bioinspired metalloenzyme target to efficiently reduce the greenhouse gas CO₂ into the portable energy carrier formate under physiological conditions. These enzymes were shown to harbor an active site sulfido ligand that is essential for the formate oxidation and CO₂ reduction activity and contributes to the oxygen sensitivity of the enzyme, since the ligand is rapidly lost in the presence of O₂. Inhibitors like azide or nitrate are routinely employed to protect the active site from oxidative damage. The demonstrated unitary in vitro sulfido ligand incorporation to the active site bis metal-binding pterin guanine dinucleotide (bis-MGD) cofactor in FDH from Rhodobacter capsulatus of this study also completely reactivates the enzyme. Reductive treatment with either sulfide or bisulfite, or with sodium dithionite under weakly acidic conditions in the strict absence of O₂ resulted in comparable enzymatic activity to FDH purified after heterologous expression in Escherichia coli. Confirmation of the inserted sulfido ligand was afforded by EPR spectroscopy of a Mo^V intermediate species associated with MoS₆ coordination. Specific insertion of a ³³S sulfido ligand to the bis-MGD Mo evidenced the chemical insertion of the sulfido ligand and confirmed its role to serve in defining the electronic character of the sulfurated bis-MGD Mo^V-SH state. The relevance of these results, in relation to known in vitro sulfuration assays described for other molybdoenzymes, is discussed.