Site-directed mutations of conserved residues of the Rieske iron-sulfur subunit of the cytochrome bc1 complex of Rhodobacter sphaeroides blocking or impairing quinol oxidation.
Site-directed mutations of conserved residues of the Rieske iron-sulfur subunit of the cytochrome bc1 complex of Rhodobacter sphaeroides blocking or impairing quinol oxidation.
复制标题
球形红杆菌细胞色素 bc1 复合物的 Rieske 铁硫亚基保守残基的定点突变可阻断或损害对苯二酚氧化。
DOI：
10.1021/bi00083a005
复制
发表时间：
1993
期刊：
影响因子：
2.9
通讯作者：
Crofts,AR
中科院分区：
文献类型：
--
作者：
VanDoren,SR;Gennis,RB;Barquera,B;Crofts,AR
修订稿于 1993 年 3 月 16 日收到摘要：已构建了与球形红杆菌泛醇木糖色素 C2 氧化还原酶（bc\复合物）的 Rieske 亚基的 2Fe-2S 簇结合的结构域中保守残基的定点突变。 Rb 中 133 位的天冬氨酸取代甘氨酸。球状序列（突变体 FG133D），它模仿了 Gatti 等人之前在酵母中分离和表征的突变。[Gatti, D. L., Meinhardt, S. W., Ohnishi, T., & Tzagoloff, A.(1989) J. Mol.生物。 205,421-435]，允许更详细地研究泛醇木糖色素 ci 氧化还原酶对光合链瞬时激活的热力学行为和动力学。该突变体复合物中受损的催化作用位于对苯二酚氧化位点。通过对苯二酚氧化位点还原细胞色素bn的表观二阶速率常数比野生型低约20倍，并且与其相对于野生型增加的表观激活势垒相关。 Rieske 亚基的假定 2Fe-2S 结合结构域中保守的半胱氨酸和组氨酸的取代选择性地敲除 2Fe-2S 簇和对苯二酚氧化活性，同时保留细胞色素和其他催化位点基本完整。这些菌株的回复特性与必需的突变残基一致。细胞色素 ci 突变体 CQ228stop 的膜 [Konishi, K.、Van Doren, S. R.、Kramer, D. M.、Crofts, AR 和 Gennis, R. B.(1991) J. Biol.。化学。 266, 14270-14276]，细胞色素 ci 的可溶结构域从细胞质膜释放到周质，保留了一个残缺的复合物，其中包含相对不受干扰的 2Fe-2S 中心和细胞色素 b 滴定范围与细胞色素 bn 相同，但具有更宽的 a 带和峰移至红色（\m 在 563 nm）。该复合物在不存在膜结合细胞色素 ci 和任何具有低电位细胞色素 b 血红素特性的中心的情况下结合抗霉素和豆菌素。因此，根据 EPR 光谱和豆毒素结合报告，2Fe-2S 簇的基本结构独立于细胞色素 Ci 亚基。泛醇木糖色素 c 氧化还原酶家族（cyt1 bc\复合物或 b（叶绿体中的 f 复合物））是许多呼吸和光合电子传递链中普遍存在的成分。通过高电位铁硫簇和ci 血红素并通过这些复合物的两个较低电位的 b 血红素通过 Q 循环机制驱动质子泵送（Mitchell，
Revised Manuscript Received March 16, 1993 abstract: Site-directed mutations of conserved residues in the domain binding the 2Fe-2S cluster of the Rieske subunit of the ubiquinolxytochrome C2 oxidoreductase {bc\complex) of Rhodobacter sphaeroides have been constructed. The substitution of aspartate for glycine at position 133 in the Rb. sphaeroides sequence (mutant FG133D), which mimicked a mutation previously isolatedand characterized in yeast by Gatti et al.[Gatti, D. L., Meinhardt, S. W., Ohnishi, T., & Tzagoloff, A.(1989) J. Mol. Biol. 205,421-435], allowed more detailed studies of thermodynamic behavior and the kinetics of the ubiquinolxytochrome ci oxidoreductase on flash activation of the photosynthetic chain. The impaired catalysis in this mutant complex is localized to the quinol oxidizing site. The apparent second-order rate constant for reduction of cytochrome bn via the quinol oxidizing site is about 20-fold lower than that of the wild-type and correlates with itsapparent activation barrier being increased relative to that of the wild-type. Substitutions for the cysteines and a histidine which are conserved in the putative 2Fe-2S binding domain of the Rieske subunit selectively knock out the 2Fe-2S cluster and quinol oxidizingactivity, while leavingthe cytochromes and other catalytic sites essentially intact. Reversion properties of these strains are consistent with the mutated residues being essential. Membranes of the cytochrome ci mutant CQ228stop [Konishi, K., Van Doren, S. R., Kramer, D. M., Crofts, AR, & Gennis, R. B.(1991) J. Biol. Chem. 266, 14270-14276], with the soluble domain of cytochrome ci released from the cytoplasmic membraneto the periplasm, retain a crippled complex which contains a relatively unperturbed 2Fe-2S center and cytochrome b titrating in the same range as cytochrome bn, but with a broader a band and a peak shifted to the red (\m at 563 nm). The complex binds antimycin and stigmatellin in the absence of both membrane-bound cytochrome ci and any center with the properties of the low-potential cytochrome b heme. Hence, the essential architecture of the 2Fe-2S cluster, as reported by EPR spectroscopy and by stigmatellin binding is independent of the cytochrome Ci subunit.Enzymes of the ubiquinolxytochrome c oxidoreductase family (cyt1 bc\complex or b (f complex in chloroplasts) are ubiquitous components of many respiratory and photosynthetic electron transport chains. The electron-transfer reactions through the high-potential iron-sulfur cluster and ci heme and through the two lower potential b hemes of these complexes drive proton pumping by a Q-cycle mechanisms (Mitchell,