Wolfe B M, Lui S M, Cowan J A
Evans Laboratory of Chemistry, Ohio State University, Columbus 43210.
Eur J Biochem. 1994 Jul 1;223(1):79-89. doi: 10.1111/j.1432-1033.1994.tb18968.x.
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.
已确定了从普通脱硫弧菌（希登伯勒）中严格纯化异化亚硫酸盐还原酶脱硫绿素的条件。通过快速蛋白质液相色谱进行的最终纯化产生了至少三条不同的条带，每条条带都显示出脱硫绿素的特征吸收光谱。其中两条已通过氨基酸分析、等电聚焦、聚丙烯酰胺凝胶电泳和辅基中心的组成进行了广泛表征。每条都包含两对[Fe4S4]和丝氨酸亚铁血红素单元。这些结果与最近声称丝氨酸亚铁血红素大量脱金属、铁含量过高以及存在Fe6S6簇的工作形成了鲜明对比。根据我们的结果和其他已发表的工作对这些提议进行了严格评估。已确定了稳态动力学参数：kcat(SO3(2-)=0.31 mol SO3(2-).s-1.mol血红素-1，Km = 0.06 mM；kcat(NO2-)=0.038 mol NO2-.s-1.mol血红素-1，Km = 0.028 mM；kcat(NH2OH)=29 mol NH2OH.s-1.mol血红素-1，Km = 48 mM。与大肠杆菌和菠菜同化亚硫酸盐还原酶以及菠菜亚硝酸还原酶进行了详细比较。高度纯化的异化亚硫酸盐还原酶样品显示出菱形高自旋铁血红素中心特有的电子顺磁共振光谱，而完全还原的酶则显示出[Fe4S4]簇特有的EPR特征。通过变温实验和自旋定量进一步表征了辅基中心的磁性。
