Valkova-Valchanova M B, Saribas A S, Gibney B R, Dutton P L, Daldal F
Department of Biology, Plant Science Institute, University of Pennsylvania, Philadelphia 19104, USA.
Biochemistry. 1998 Nov 17;37(46):16242-51. doi: 10.1021/bi981651z.
The presence of a two-subunit cytochrome (cyt) b-c1 subcomplex in chromatophore membranes of Rhodobacter capsulatus mutants lacking the Rieske iron-sulfur (Fe-S) protein has been described previously [Davidson, E., Ohnishi, T., Tokito, M., and Daldal, F. (1992) Biochemistry 31, 3351-3358]. Here, this subcomplex was purified to homogeneity in large quantities, and its properties were characterized. As expected, it contained stoichiometric amounts of cyt b and cyt c1 subunits forming a stable entity devoid of the Fe-S protein subunit. The spectral and thermodynamic properties of its heme groups were largely similar to those of a wild-type bc1 complex, except that those of its cyt bL heme were modified as revealed by EPR spectroscopy. Dark potentiometric titrations indicated that the redox midpoint potential (Em7) values of cytochromes bH, bL, and c1 were very similar to those of a wild-type bc1 complex. The purified b-c1 subcomplex had a nonfunctional ubihydroquinone (UQH2) oxidation (Qo) site, but it contained an intact ubiquinone (UQ) reductase (Qi) site as judged by its ability to bind the Qi inhibitor antimycin A, and by the presence of antimycin A sensitive Qi semiquinone. Interestingly, its Qo site could be readily reconstituted by addition of purified Fe-S protein subunit. Reactivated complex exhibited myxothiazol, stigmatellin, and antimycin A sensitive cyt c reductase activity and an EPR gx signal comparable to that observed with a bc1 complex when the Qo site is partially occupied with UQ/UQH2. However, a mutant derivative of the Fe-S protein subunit lacking its first 43 amino acid residues was unable to reactivate the purified b-c1 subcomplex although it could bind to its Qo site in the presence of stigmatellin. These findings demonstrated for the first time that the amino-terminal membrane-anchoring domain of the Fe-S protein subunit is necessary for UQH2 oxidation even though its carboxyl-terminal domain is sufficient to provide wild-type-like interactions with stigmatellin at the Qo site of the bc1 complex.
先前已报道过，在缺乏 Rieske 铁硫（Fe-S）蛋白的荚膜红细菌突变体的载色体膜中存在一种双亚基细胞色素（cyt）b-c1 亚复合物[Davidson, E., Ohnishi, T., Tokito, M., and Daldal, F. (1992) Biochemistry 31, 3351 - 3358]。在此，该亚复合物被大量纯化至均一状态，并对其性质进行了表征。正如预期的那样，它含有化学计量的 cyt b 和 cyt c1 亚基，形成了一个不含 Fe-S 蛋白亚基的稳定实体。其血红素基团的光谱和热力学性质在很大程度上与野生型 bc1 复合物相似，不过通过电子顺磁共振光谱（EPR）显示，其 cyt bL 血红素的性质有所改变。暗电位滴定表明，细胞色素 bH、bL 和 c1 的氧化还原中点电位（Em7）值与野生型 bc1 复合物非常相似。纯化的 b-c1 亚复合物具有一个无功能的泛醇（UQH2）氧化（Qo）位点，但根据其结合 Qi 抑制剂抗霉素 A 的能力以及抗霉素 A 敏感的 Qi 半醌的存在情况判断，它含有一个完整的泛醌（UQ）还原酶（Qi）位点。有趣的是，通过添加纯化的 Fe-S 蛋白亚基，其 Qo 位点能够很容易地被重建。当 Qo 位点部分被 UQ/UQH2 占据时，重新激活的复合物表现出对粘噻唑、鱼藤酮和抗霉素 A 敏感的细胞色素 c 还原酶活性以及与 bc1 复合物观察到的相当的 EPR gx 信号。然而，缺乏其前 43 个氨基酸残基的 Fe-S 蛋白亚基的突变衍生物尽管在鱼藤酮存在的情况下能够结合到其 Qo 位点，但却无法重新激活纯化的 b-c1 亚复合物。这些发现首次证明，尽管 Fe-S 蛋白亚基的羧基末端结构域足以在 bc1 复合物的 Qo 位点提供与鱼藤酮类似野生型的相互作用，但 Fe-S 蛋白亚基的氨基末端膜锚定结构域对于 UQH2 氧化是必需的。
