STUDY ON FUNCTION OF CYTOCHROMEb-c_1 COMPLEX AND COENZYMEQ

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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