Brandt U, Haase U, Schägger H, von Jagow G
Universität Frankfurt, Zentrum der Biologischen Chemie, Frankfurt am Main, Federal Republic of Germany.
J Biol Chem. 1991 Oct 25;266(30):19958-64.
The binding of specific inhibitors to the ubiquinol oxidation pocket ("QP center") of cytochrome c reductase was analyzed before and after removal of bound phospholipid and the "Rieske" iron-sulfur protein using optical spectroscopy and fluorescence quench binding assays. The enzyme lacking iron-sulfur protein showed almost unchanged, tight binding of the E-beta-methoxyacrylate inhibitors oudemansin A and MOA-stilbene, whereas binding of the chromone inhibitor stigmatellin was almost completely abolished. The affinity of the weak inhibitor 3-undecyl-2-hydroxy-naphthoquinone was decreased. Oudemansin A binding to the defective pocket of the iron-sulfur protein-depleted enzyme was lowered by added phospholipid. It was deduced from these results that the QP center is a spacious pocket formed by domains of cytochrome b, bearing the E-beta-methoxcyacrylate binding site, and the iron-sulfur protein, bearing the stigmatellin binding site. Moreover, removal of the iron-sulfur protein leaves this pocket defective but essentially unchanged in its remaining binding capability. The affinity of three preparations of cytochrome c reductase, the complete, the delipidated, and the iron-sulfur depleted enzyme for E-beta-methoxyacrylate-stilbene, was analyzed for different redox states of the catalytic centers of cytochrome c reductase. The apparent Kd values for the different redox states were interpreted in terms of two conformational states. It is suggested that these changes reflect the two states of the "catalytic switch" proposed recently for the QP pocket of cytochrome c reductase (Brandt, U., and von Jagow, G. (1991) Eur. J. Biochem. 195, 163-170). According to the refined model presented in this work, changeover to the "b" state is triggered by reduction of the iron-sulfur cluster, and changeover back to the "FeS" state is triggered by electron transfer from the low potential onto the high potential heme b center. Our interpretation implies that the stability of the two states is affected by the redox states of the enzyme, but that additionally changing the redox states of the two centers is required for "switching" on a catalytic time scale.
利用光谱学和荧光猝灭结合分析方法，在去除结合的磷脂和“里氏”铁硫蛋白之前和之后，分析了特异性抑制剂与细胞色素c还原酶的泛醇氧化口袋（“QP中心”）的结合情况。缺乏铁硫蛋白的酶对E-β-甲氧基丙烯酸酯抑制剂奥德曼辛A和MOA-芪表现出几乎不变的紧密结合，而色酮抑制剂杀粉蝶菌素的结合几乎完全被消除。弱抑制剂3-十一烷基-2-羟基萘醌的亲和力降低。添加磷脂会降低奥德曼辛A与铁硫蛋白缺失酶的缺陷口袋的结合。从这些结果推断，QP中心是一个宽敞的口袋，由带有E-β-甲氧基丙烯酸酯结合位点的细胞色素b结构域和带有杀粉蝶菌素结合位点的铁硫蛋白形成。此外，去除铁硫蛋白会使这个口袋有缺陷，但其余结合能力基本不变。分析了细胞色素c还原酶的三种制剂，即完整的、脱脂的和铁硫缺失的酶，在细胞色素c还原酶催化中心的不同氧化还原状态下对E-β-甲氧基丙烯酸酯-芪的亲和力。根据两种构象状态解释了不同氧化还原状态下的表观解离常数（Kd）值。有人提出，这些变化反映了最近为细胞色素c还原酶的QP口袋提出的“催化开关”的两种状态（布兰特，U.，和冯·雅戈，G.（1991年）《欧洲生物化学杂志》195，163 - 170）。根据本工作提出的精细模型，向“b”状态的转变是由铁硫簇的还原触发的，而回到“FeS”状态的转变是由电子从低电位转移到高电位血红素b中心触发的。我们的解释意味着这两种状态的稳定性受酶的氧化还原状态影响，但在催化时间尺度上“切换”还需要另外改变两个中心的氧化还原状态。
