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 microM/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 (alpha, 98,000; beta, 56,000; gamma, 20,000; delta, 11,500) apparently associated as 2 alpha beta gamma delta 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 approximately 50% reduction of the iron and elicited an electron paramagnetic resonance (EPR) spectrum (approximately 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 (gave = 1.99; approximately 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.
Jollie
D R
DR
Department of Biochemistry, Medical School, University of Minnesota, Minneapolis 55455.
Lipscomb
J D
JD
D000596:Amino Acids / Q000032:analysis; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003574:Cytochrome c Group / Q000302:isolation & purification / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D005415:Flavins / Q000032:analysis; D005560:Formate Dehydrogenases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D007501:Iron / Q000032:analysis; D007700:Kinetics; D008772:Methylococcaceae; D008970:Molecular Weight; D008982:Molybdenum / Q000032:analysis; D013053:Spectrophotometry; D015204:Spectroscopy, Mossbauer; D013440:Sulfides / Q000032:analysis
D000596:Amino Acids; D003574:Cytochrome c Group; D005415:Flavins; D013440:Sulfides; D008982:Molybdenum; D007501:Iron; D005560:Formate Dehydrogenases