STUDY ON FUNCTION OF CYTOCHROMEb-c_1 COMPLEX AND COENZYMEQ

细胞色素b-c_1复合物与辅酶MEQ的功能研究

• 批准号: 06672190
• 负责人: USUI Shigeyuki
• 金额: $ 0.9万
• 依托单位: GIFU PHARMACEUTICAL UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1994
• 资助国家: 日本
• 起止时间: 1994 至 1995
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-06672190/
• 关键词: Cytochrome b-c1 complex; Rhodobactor Sphaeroides; Coenzyme Q; Electron Transfer Chain; Q Binding Protein; シトクロムb-C_1複合体; Rhodobactor sphaereides

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

由于已克隆并测序了球形红杆菌最小亚基细胞色素b-c_1复合体（bc_1复合体）的IV亚基基因（fbcQ），利用基因工程技术构建了球形红杆菌缺失部分细胞色素b-c_1复合体的突变株，该突变株含有卡那霉素耐药基因（Kn^R）代替fbcQ。利用含有Kn^R片段的多个探针进行Southern杂交，证实了该缺陷基因取代了野生型fbcQ。尽管野生型菌株在好氧黑暗和厌氧光合条件下都能生长，但缺乏亚基IV的突变体只进行好氧而不进行光合作用。突变体的有氧生长速率与野生型相同。在野生型菌株达到稳定生长所需的72小时内，没有观察到IV亚基缺陷菌株的明显光合生长。在抗霉素a存在的情况下，突变株的光合生长增殖特征与野生型相似，利用针对纯化的IV亚基产生的多克隆抗体，对膜部分的总蛋白进行了Western blot分析，证实突变株中没有IV亚基蛋白Triton X-100的分数。部分纯化的配合物与光合作用野生型相比，光谱无变化，细胞色素b和c_1含量无变化。复合物中泛醇-细胞色素c还原酶活性降低，泛醇-细胞色素c还原酶表观Km增加。因此，亚基IV可以在电子转移和质子易位中起辅助亚基的作用