STUDY ON FUNCTION OF CYTOCHROMEb-c_1 COMPLEX AND COENZYMEQ

由于编码球形红杆菌（Rhodobactorsphaeroides，R. sphaeroides）细胞色素b-c_1复合物（bc_1 complex）最小亚基的IV亚基基因（fbcQ）已被克隆并测序，因此利用基因工程技术构建了缺失fbcQ部分基因并含有卡那霉素抗性基因（Kn^R）的球形红杆菌突变株。用含有Kn^R片段的多个探针进行Southern杂交，证实野生型fbcQ被缺陷基因所取代。虽然野生型球形红球藻既能在有氧黑暗条件下生长，也能在厌氧光合条件下生长，但亚基IV缺陷型突变体只能在有氧条件下生长，而不能在光合条件下生长。突变株的好氧生长速率与野生型相同。没有观察到明显的光合生长的亚基IV缺陷型菌株长达72小时，野生型菌株达到稳定生长所需的时间。在抗霉素A存在下，突变株的光合生长的增殖曲线与野生型的光合生长的增殖曲线相似。通过使用针对纯化的亚基IV产生的多克隆抗体对膜级分中的总蛋白进行Western印迹分析，证实突变株中不存在亚基IV蛋白。1复合物从有氧生长的突变体部分纯化后，用Triton X-100溶解的膜馏分通过羟基磷灰石柱层析。与野生型光合生长的细胞色素B和c_1相比，未观察到部分纯化的复合物中细胞色素b和c_1的光谱变化和含量。然而，泛喹啉-细胞色素c还原酶的活性和泛醌在此复合物的表观Km被发现，分别减少和增加。因此，亚基IV可能在电子转移和质子转运中充当辅助亚基

Since the subunit IV gene (fbcQ) encoding the smallest subunit cytochrome b-c_1 complex (bc_1 complex) from Rhodobactor sphaeroides (R.spheroides) has been cloned and sequenced, the mutant strain of R.sphaeroides which lacked a part of fbcQ and had a kanamycin resistant gene (Kn^R)instead of fbcQ was constructed using genetic engineering techniques. The replacement of wild-type fbcQ by the defective gene was confirmed by Southern hybridization using several probes containing Kn^R fragment. Although the wild-type strain of R.sphaeroides grew under both aerobic with dark and anaerobic with photosynthetic conditions, subunit IV deficient mutant did aerobically only but not photosynthetically. The rate of aerobic growth of mutant was the same as that of wild-type strain. No apparent photosynthetic growth was observed for the subunit IV deficient strain up to 72 hours, a time required for the wild-type strain to reach stationary growth. The proliferation profile of photosynthetic growth of mutant strain was similar to that of photosynthetic growth of wild-type in the presence of antimycin A.The absence of subunit IV protein in mutant was confirmed by Western blot analysis of total protein in the membrane fraction using polyclonal antibodies generated against purified subunit IV.The bc_1 complex from the mutant grown aerobically was partially purified by hydroxylapatite column chromatography after solubilization of the membrane fraction with Triton X-100. No spectral changes and contents of cytochrome b and c_1 in the partially purified complex was observed, compared with those from wild-type grown photosynthetically. However, the ubiquinol-cytochrome c reductase activity and the apparent Km for ubiquinone in this complex was found to decrease and increase, respectively. Thus subunit IV may function as a helper subunit in the electron transfer and the proton translocation