Cytochrome bo is a member of the heme-copper terminal oxidase superfamily and serves as a four-subunit ubiquinol oxidase in the aerobic respiratory chain of Escherichia coli. To probe the location and structural properties of the ubiquinol oxidation site, we isolated and characterized five or 10 spontaneous mutants resistant to either 2,6-dimethyl-1,4-benzoquinone, 2,6-dichloro-4-nitrophenol, or 2,6-dichloro-4-dicyanovinylphenol, the potent competitive inhibitors for the oxidation of ubiquinol-1 [Sato-Watanabe, M., Mogi, T., Miyoshi, H., Iwamura, H., Matsushita, K., Adachi, O., and Anraku, Y. (1994) J. Biol. Chem. 269, 28899-28907]. Analyses of the growth yields and the ubiquinol-1 oxidase activities of the mutant membranes showed that the mutations increased the degree of the resistance to the selecting compounds. Notably, several mutants showed the cross-resistance. These data indicate that the binding sites for substrate and the competitive inhibitors are partially overlapped in the ubiquinol oxidation site. All the mutations were linked to the expression vector, and 23 mutations examined were all present in the C-terminal hydrophilic domain (Pro96-His315) of subunit II. Sequencing analysis revealed that seven mutations examined are localized near both ends of the cupredoxin fold. Met248Ile, Ser258Asn, Phe281Ser, and His284Pro are present in a quinol oxidase-specific (Qox) domain and proximal to low-spin heme b in subunit I and the lost CuA site in subunit II, whereas Ile129Thr, Asn198Thr, and Gln233His are rather scattered in a three-dimensional structure and closer to transmembrane helices of subunit II. Our data suggest that the Qox domain and the CuA end of the cupredoxin fold provide the quinol oxidation site and are involved in electron transfer to the metal centers in subunit I.

译文

细胞色素bo是血红素铜末端氧化酶超家族的成员,在大肠杆菌的有氧呼吸链中充当四亚基泛醇氧化酶。为了探究泛醇氧化位点的位置和结构性质,我们分离并鉴定了5个或10个对2,6-二甲基-1,4-苯醌,2,6-二氯-4-硝基苯酚或2,6-二氯-4-二氰基苯酚具有抗性的自发突变体,ubiquinol-1的有效竞争抑制剂 [Sato-Watanabe,M.,Mogi,T.,三好,H.,岩村,H.,松下,K.,足立,O.,和安乐,Y. (1994) J. Biol. Chem. 269,28899-28907]。对突变膜的生长产量和ubiquinol-1氧化酶活性的分析表明,突变增加了对选择化合物的抗性程度。值得注意的是,几个突变体显示出交叉抗性。这些数据表明,底物和竞争性抑制剂的结合位点在泛醇氧化位点部分重叠。所有突变均与表达载体连接,并且所检查的23个突变均存在于亚基II的C末端亲水结构域 (Pro96-His315) 中。测序分析显示,所检查的七个突变位于cupredoxin折叠的两端附近。Met248Ile,Ser258Asn,Phe281Ser和His284Pro存在于喹啉氧化酶特异性 (Qox) 结构域中,并且靠近亚基I中的低自旋血红素b和亚基II中丢失的CuA位点,而Ile129Thr,Asn198Thr,gln233His相当分散在三维结构中,更接近亚基II的跨膜螺旋。我们的数据表明,cupredoxin折叠的Qox结构域和CuA末端提供了quinol氧化位点,并参与了向亚基I中的金属中心的电子转移。

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