STUDY ON FUNCTION OF CYTOCHROMEb-c_1 COMPLEX AND COENZYMEQ

由于球状红假单胞菌细胞色素B-C_1复合体(BC_1复合体)的亚基IV基因(FbcQ)已被克隆和测序，因此利用基因工程技术构建了球状红假单胞菌缺失部分fbcQ、具有卡那霉素抗性基因(Kn^R)的突变株。用含有Kn、R片段的多个探针进行Southern杂交，证实了野生型fbcQ被缺陷基因所取代。虽然球藻野生型菌株在有氧黑暗和厌氧光合作用条件下都能生长，但IV亚基缺陷突变株仅能有氧生长，而不能光合作用。突变株的有氧生长速度与野生型相同。缺乏IV亚基的菌株在72小时内没有观察到明显的光合作用生长，这是野生型菌株达到稳定生长所需的时间。突变株的光合作用生长的增殖曲线与野生型在抗霉素A存在下的生长情况相似。用纯化的亚基IV的多克隆抗体对突变株膜组分进行蛋白质印迹分析，证实了突变株膜组分中不存在亚基IV蛋白。用Triton X-100溶解突变株膜组分后，用羟基磷灰石柱层析法部分纯化了突变株BC1复合体。与野生型光合作用培养的细胞色素b和c1相比，部分纯化的复合体没有观察到光谱变化和细胞色素b和c1的含量。然而，该复合体中的泛醌-细胞色素c还原酶活性和对泛醌的表观Km分别降低和增加。因此，亚基IV可能在电子转移和质子转移中起辅助亚基的作用

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