Saribaş A S, Valkova-Valchanova M, Tokito M K, Zhang Z, Berry E A, Daldal F
Department of Biology, Plant Science Institute, University of Pennsylvania, Philadelphia 19104, USA.
Biochemistry. 1998 Jun 2;37(22):8105-14. doi: 10.1021/bi973146s.
Ubihydroquinone:cytochrome (cyt) c oxidoreductase (bc1 complex and its plant counterpart b6f complex) is a vital component of energy-transducing systems in most organisms from bacteria to eukaryotes. In the facultative phototrophic (Ps) bacterium Rhodobacter capsulatus, it is constituted by the cyt b, cyt c1, and Rieske Fe-S protein subunits and is essential for Ps growth. Of these subunits, cyt b has two nontransmembrane helices, cd1 and cd2, which are critical for its structure and function. In particular, substitution of threonine (T) at position 163 on cd1 with phenylalanine (F) or proline (P) leads to the absence of the bc1 complex. Here, Ps+ revertants of B:T163F were obtained, and their detailed characterizations indicated that position 163 is important for the assembly of the bc1 complex by mediating subunit interactions at the Qo site. The loss of the hydroxyl group at position 163 of cyt b was compensated for by the gain of either a hydroxyl group at position 182 of cyt b or 46 of the Fe-S protein or a sulfhydryl group at position 46 of cyt c1. Examination of the mitochondrial bc1 complex crystal structure [Zhang, Z., Huang, L., Shulmeister, V. M., Chi, Y.-I., Kim, K. K., Hung, L.-W., Crofts, A. R., Berry, E. A., and Kim, S.-H. (1998) Nature 392, 677-684] revealed that the counterparts of B:G182 (i.e., G167) and F:A46 (i.e. , A70) are located close to B:T163 (i.e., T148), whereas the C:R46 (i.e., R28) is remarkably far from it. The revertants contained substoichiometric amounts of the Fe-S protein subunit and exhibited steady-state and single-turnover, electron transfer activities lower than that of a wild-type bc1 complex. Interestingly, their membrane supernatants contained a smaller form of this subunit with physicochemical properties identical to those of its membrane-bound form. Determination of the amino-terminal amino acid sequence of this soluble Fe-S protein revealed that it was derived from the wild-type protein by proteolytic cleavage at V44. This work revealed for the first time that position 163 of cyt b is important both for proper subunit interactions at the Qo site and for inactivation of the bc1 complex by proteolytic cleavage of its Fe-S protein subunit at a region apparently responsible for its mobility during Qo site catalysis.
细胞色素c氧化还原酶（bc1复合物及其植物对应物b6f复合物）是从细菌到真核生物的大多数生物体中能量转换系统的重要组成部分。在兼性光养细菌荚膜红细菌中，它由细胞色素b、细胞色素c1和 Rieske铁硫蛋白亚基组成，对光养生长至关重要。在这些亚基中，细胞色素b有两个非跨膜螺旋，cd1和cd2，对其结构和功能至关重要。特别是，将cd1上第163位的苏氨酸（T）替换为苯丙氨酸（F）或脯氨酸（P）会导致bc1复合物缺失。在这里，获得了B:T163F的光合阳性回复突变体，其详细表征表明第163位对于通过介导Qo位点的亚基相互作用来组装bc1复合物很重要。细胞色素b第163位羟基的缺失通过细胞色素b第182位、铁硫蛋白第46位的羟基或细胞色素c1第46位的巯基的获得得到补偿。对线粒体bc1复合物晶体结构的研究[Zhang, Z., Huang, L., Shulmeister, V. M., Chi, Y.-I., Kim, K. K., Hung, L.-W., Crofts, A. R., Berry, E. A., and Kim, S.-H. (1998) Nature 392, 677 - 684]表明，B:G182（即G167）和F:A46（即A70）的对应位置靠近B:T163（即T148），而C:R46（即R28）则与之相距甚远。回复突变体中Fe-S蛋白亚基的含量低于化学计量，并且表现出稳态和单周转电子传递活性低于野生型bc1复合物。有趣的是，它们的膜上清液中含有这种亚基的一种较小形式，其物理化学性质与其膜结合形式相同。对这种可溶性Fe-S蛋白的氨基末端氨基酸序列的测定表明，它是通过在V44处的蛋白水解切割从野生型蛋白衍生而来的。这项工作首次揭示，细胞色素b的第163位对于Qo位点的正确亚基相互作用以及通过在其Fe-S蛋白亚基的一个显然负责其在Qo位点催化过程中移动性的区域进行蛋白水解切割来使bc1复合物失活都很重要。
