PROTON-TRANSFER IN CYTOCHROME BO(3) UBIQUINOL OXIDASE OF ESCHERICHIA-COLI - 2ND-SITE MUTATIONS IN SUBUNIT-I THAT RESTORE PROTON-PUMPING IN THE MUTANT ASP135-]ASN
PROTON-TRANSFER IN CYTOCHROME BO(3) UBIQUINOL OXIDASE OF ESCHERICHIA-COLI - 2ND-SITE MUTATIONS IN SUBUNIT-I THAT RESTORE PROTON-PUMPING IN THE MUTANT ASP135-]ASN
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DOI：
10.1021/bi00013a035
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发表时间：
1995-04-04
期刊：
影响因子：
2.9
通讯作者：
WIKSTROM, M
中科院分区：
文献类型：
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作者：
GARCIAHORSMAN, JA;PUUSTINEN, A;WIKSTROM, M
大肠埃希菌的泛喹酚氧化酶，细胞色素BO(3)，是呼吸性血红素-铜氧化酶家族的成员，它通过质子跨细菌膜转移将氧气还原到水中来保存能量。先前发现该酶亚基I中天冬氨酸残基(Asp135)突变为天冬酰胺会损害质子转运[Thomas等人。(1993年)生物化学32,10923-10928]。该残基位于跨膜螺旋II和III之间的螺旋间“环”中，包含6个保守的残基(Asn124、Pro128、G1y132、Asp135、Pro139和Asn142)。用定点突变的方法研究了整个结构域的功能。Asn124和Asn142的非保守突变也会导致质子转位的丧失，而它们对谷氨酰胺的保守替换没有影响。这个结构域中其他八个位置的突变不会影响质子的转移。在139或142位引入酸性基团，但不在其他8个测试位引入，恢复了Asp135-->；ASN突变蛋白中的质子泵。这些结果表明，结构域的C-末端部分可能是α螺旋的，整个‘环’作为质子转运机制的输入通道的一部分，在结构和功能上起着重要的作用。
The ubiquinol oxidase, cytochrome bo(3), of Escherichia coli is a member of the respiratory heme-copper oxidase family and conserves energy from the reduction of dioxygen to water by translocation of protons across the bacterial membrane. Mutation of an aspartic acid residue (Asp135) to asparagine in subunit I of this enzyme was previously found to impair proton translocation [Thomas et al. (1993) Biochemistry 32, 10923-10928]. This residue is located in an interhelical ''loop'' between transmembranous helices II and III, which contains six well-conserved residues (Asn124, Pro128, G1y132, Asp135, Pro139, and Asn142). Site-directed mutagenesis was performed to study the function of this entire domain. Nonconservative mutations of Asn124 and Asn142 also resulted in a loss of proton translocation, whereas their conservative substitution to glutamine had no effect. Mutations in eight other positions within this domain did not affect proton translocation. Introduction of an acidic group at positions 139 or 142, but not at eight other tested positions, restored proton pumping in the Asp135--> Asn mutated protein. These results suggest that the C-terminal part of the domain may be alpha-helical and that the entire ''loop'' plays an important structural and functional role as part of an input channel of the proton translocation machinery.