Inactivation of Formate Dehydrogenase from Methanobacterium formicicum by Cyanide

Formate dehydrogenase from Methanobacterium formicicum contains Mo, FAD, and Fe/S clusters as prosthetic groups. Partial reduction of the native enzyme with either formate or dithionite yielded a Mo(V) electron paramagnetic resonance (EPR) signal, g₁ = 2.020, g₂ = 2.006, and g₃ = 1.997, exhibiting nearly isotropic superhyperfine interaction to two, equivalent, strongly coupled protons (A₁ = 0.45, A₂= 0.55, and A₃ = 0.5 mT), identified by ²H substitution. Incubation of oxidized formate dehydrogenase with cyanide resulted in an irreversible loss of enzyme activity, which could not be restored by treatment with sulfide. Equimolar amounts of thiocyanate were released from cyanide-treated formate dehydrogenase, indicating the loss of one terminal sulfur ligand to molybdenum. Cyanide inactivation of the oxidized native enzyme resulted in a shift of the Mo(V) EPR signal to higher field (g₁= 2.005, g₂ = 1.998, and g₃ = 1.989) and a reduction in the complexity of the superhyperfine splitting pattern due to loss of a strongly coupled, exchangeable proton, the remaining coupled proton yielding coupling constants of A₁ = 1.05, A₂ = 0.85, and A₃= 0.88 mT, respectively. In addition, cyanide treatment altered the behavior of the Mo(V) EPR signal during potentiometric titration of the enzyme, yielding midpoint potentials for the Mo(VI)/Mo(V) and Mo(V)/Mo(IV) redox couples of-319 and -321 mV, respectively. Comparison of the spectroscopic and thermodynamic properties of the native and cyanide-treated forms of formate dehydrogenase indicates the mode of inactivation to be analogous to that established for the related enzyme, xanthine oxidase, resulting in the loss of a sulfur residue yielding the “desulfo” form of formate dehydrogenase.