Metal-dependent formate dehydrogenases (FDHs) are of considerable interest as a bioinspired metalloenzyme target to efficiently reduce the greenhouse gas CO2 into the simplest carboxylic acid formic acid and its conjugate base formate as a portable energy carrier under physiological conditions. These enzymes harbor an active site sulfido ligand bound to the molybdenum or tungsten metal 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. We have 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, that completely reactivated 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 MoV 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 MoV-SH state. These results provide an important benchmark by which targeted in vitro sulfuration can be applied in other enzymatic systems without an apparent association of maturation enzymes identified to biosynthetically install a sulfido ligand.