Hacker B, Barquera B, Gennis R B, Crofts A R
School of Life Sciences, University of Illinois, Urbana 61801.
Biochemistry. 1994 Nov 8;33(44):13022-31. doi: 10.1021/bi00248a011.
The cytochrome b subunit of the ubiquinol:cytochrome c oxidoreductase (the bc1 complex) contains two heme prosthetic groups, cytochrome bL and cytochrome bH. In addition, this subunit also provides major elements of the quinol oxidation site (Qo) and a separate quinone reductase site (Qi), which are thought to be located on opposite sides of the membrane. Site-directed mutagenesis has been used to explore the role(s) of specific amino acid residues in this subunit from the photosynthetic bacterium Rhodobacter sphaeroides. Previous work identified five residues, Gly48 (Gly33), Ala52 (Gly37), His217 (His202), Lys251 (Lys228), and Asp252 (Asp229), as being either at or near the quinone reductase site (the residue numbers in parentheses designate the equivalent positions in the yeast mitochondrial enzyme). These residues are predicted to be near the cytoplasmic boundaries of transmembrane helices: helix A (G48, A52), helix D (H217), or helix E (K251, D252). In the current work, the importance of two additional highly conserved residues, which are also predicted to be near the cytoplasmic boundaries of transmembrane helices, is explored by site-directed mutagenesis. R114 (helix B) has been substituted with K, Q, and A, and W129 (helix C) has been changed to A and F. The results suggest that a positively charged residue at position 114 is important. The R114K mutation causes only subtle effects, which appear to be localized to cytochrome bH and the quinone reductase site. In contrast, R114Q is not assembled, and R114A, although partially assembled, is nonfunctional and appears to have a very low amount of cytochrome b associated with the complex. Both mutants at position 129 (W129A and W129F) are able to support the photosynthetic growth of the organism, but show abnormal characteristics. The defects associated with the W129A mutation appear to be primarily associated with the quinone reductase site and cytochrome bH, whereas the W129F mutation appears to result in more global defects that also perturb the cytochrome bL locus. The results are consistent with the placement of residues R114 and W129 near the cytoplasmic side of the membrane, but suggest that these residues are important for the assembly and overall stability of the complex.
泛醌-细胞色素c氧化还原酶（bc1复合物）的细胞色素b亚基包含两个血红素辅基，即细胞色素bL和细胞色素bH。此外，该亚基还构成了喹醇氧化位点（Qo）和一个独立的醌还原酶位点（Qi）的主要组成部分，这两个位点被认为位于膜的两侧。定点诱变已被用于探究光合细菌球形红杆菌中该亚基特定氨基酸残基的作用。先前的研究确定了五个残基，即甘氨酸48（甘氨酸33）、丙氨酸52（甘氨酸37）、组氨酸217（组氨酸202）、赖氨酸251（赖氨酸228）和天冬氨酸252（天冬氨酸229），它们位于醌还原酶位点或其附近（括号中的残基编号表示酵母线粒体酶中的等效位置）。预计这些残基靠近跨膜螺旋的胞质边界：螺旋A（G48、A52）、螺旋D（H217）或螺旋E（K251、D252）。在当前的研究中，通过定点诱变探究了另外两个高度保守的残基的重要性，预计它们也靠近跨膜螺旋的胞质边界。将R114（螺旋B）分别替换为K、Q和A，将W129（螺旋C）分别替换为A和F。结果表明，第114位带正电荷的残基很重要。R114K突变仅产生细微影响，似乎局限于细胞色素bH和醌还原酶位点。相比之下，R114Q不能组装，而R114A虽然部分组装，但无功能，并且似乎与该复合物相关的细胞色素b含量极低。129位的两个突变体（W129A和W129F）都能够支持该生物体的光合生长，但表现出异常特征。与W129A突变相关的缺陷似乎主要与醌还原酶位点和细胞色素bH有关，而W129F突变似乎导致更广泛的缺陷，也扰乱了细胞色素bL位点。这些结果与残基R114和W129位于膜胞质侧的位置一致，但表明这些残基对于该复合物的组装和整体稳定性很重要。
