Formate dehydrogenase from methylosinus trichosporium Ob3b: Purification and spectroscopic characterization of the cofactors

NAD⁺-coupled formate dehydrogenase has been purified to near-homogeneity from the obligate methanotroph Methylosinus trichosporium OB3b. The inclusion of stabilizing reagents in the purification buffers has resulted in a 3-fold increase in specific activity (98 μM/min/mg; turnover number 600 s^-1) and as much as a 25-fold increase in yield over previously reported purification protocols. The enzyme, (molecular weight 400,000 ± 20,000) is composed of four subunit types (α, 98,000; β, 56,000; γ, 20,000; δ, 11,500) apparently associated as 2 αβγδ protomers. The holoenzyme contains flavin (1.8 ± 0.2), iron (46 ± 6), inorganic sulfide (38 ± 4), and molybdenum (1.5 ± 0.1). The flavin is optically similar to the common flavin cofactors, but it is chromatographically distinct. Anaerobic incubation of the enzyme with formate, NADH, or sodium dithionite, resulted in ∼50% reduction of the iron and elicited an electron paramagnetic resonance (EPR) spectrum (∼2.5 spins/protomer) from which the spectra of five distinct EPR-active centers could be resolved in the g = 1.94 region. Four of these spectra were characteristic of [Fe-S]_x clusters. The fifth (g_ave = 1.99; ∼0.1 spins/protomer) was similar to that observed for the molybdenum cofactor of xanthine oxidase, and it exhibited the expected hyperfine splitting when the enzyme was enriched with ⁹⁵Mo (I = 5/2). Mössbauer spectroscopy showed that all of the iron in the enzyme became reduced upon the addition of a redox mediator, proflavin, to the dithionite reduced enzyme at pH 8.0. Nevertheless, a decrease in the EPR-active spin concentration in the g = 1.94 region of the spectrum occurred and was attributed to the reduction of the molybdenum center to the EPR-silent Mo(IV) state (S = 1). The fully reduced enzyme also exhibited a new species with an S = 3/2 ground state (1-2 spins/protomer). Addition of 50% ethylene glycol to the fully reduced enzyme revealed no new species, but caused an increase in the EPR-detectable spin quantitation to 5-6 spins/protomer. This suggests that cluster spin-spin interactions may occur in both the partially and fully reduced native enzyme.