Desulfoviridin, a multimeric-dissimilatory sulfite reductase from Desulfovibrio vulgaris (Hildenborough). Purification, characterization, kinetics and EPR studies.
Conditions for the rigorous purification of desulfoviridin, the dissimilatory sulfite reductase from the sulfate-reducing bacterium Desulfovibrio vulgaris (Hildenborough) have been established. A final purification by fast protein liquid chromatography yields at least three distinct bands that each exhibit the characteristic absorption spectrum of desulfoviridin. Two of these have been extensively characterized by amino acid analysis, isoelectric focusing, polyacrylamide gel electrophoresis, and formulation of the prosthetic centers. Each contains two pairs of [Fe4S4] and siroheme units. These results stand in marked contrast to recent work claiming significant demetallation of siroheme, excess iron content, and the presence of Fe6S6 clusters. These proposals are critically assessed in light of our results and other published work. Steady-state kinetic parameters have been determined: kcat(SO3(2-) = 0.31 mol SO3(2-).s-1.mol heme-1, Km = 0.06 mM; kcat(NO2-) = 0.038 mol NO2-.s-1.mol heme-1, Km = 0.028 mM; kcat(NH2OH) = 29 mol NH2OH.s-1.mol heme-1, Km = 48 mM. A detailed comparison is made with the Escherichia coli and spinach assimilatory sulfite reductase enzymes and spinach nitrite reductase. Highly purified samples of dissimilatory sulfite reductase display an electron paramagnetic resonance spectrum characteristic of rhombic high spin ferric heme centers, while the fully reduced enzyme shows EPR features typical of [Fe4S4] clusters. The magnetic properties of the prosthetic centers are further characterized by variable temperature experiments and spin quantitation.
Evans Laboratory of Chemistry, Ohio State University, Columbus 43210.
B M
BM
Wolfe
S M
SM
Lui
J A
JA
Cowan
D015153:Blotting, Western; D002853:Chromatography, Liquid; D003545:Cysteine / Q000032:analysis; D003553:Cystine / Q000032:analysis; D016969:Desulfovibrio vulgaris / Q000201:enzymology*; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D050862:Oxidoreductases Acting on Sulfur Group Donors / Q000737:chemistry / Q000302:isolation & purification*
D003553:Cystine; D050862:Oxidoreductases Acting on Sulfur Group Donors; D003545:Cysteine