Modified, large-scale purification of the cytochrome o complex (bo-type oxidase) of Escherichia coli yields a two heme/one copper terminal oxidase with high specific activity.
Modified, large-scale purification of the cytochrome o complex (bo-type oxidase) of Escherichia coli yields a two heme/one copper terminal oxidase with high specific activity.
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对大肠杆菌细胞色素 o 复合物（bo 型氧化酶）进行修饰、大规模纯化，产生具有高比活性的二血红素/一铜末端氧化酶。
DOI：
10.1021/bi00145a008
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发表时间：
1992
期刊：
影响因子：
2.9
通讯作者：
Gennis,RB
中科院分区：
文献类型：
--
作者：
Minghetti,KC;Goswitz,VC;Gabriel,NE;Hill,JJ;Barassi,CA;Georgiou,CD;Chan,SI;Gennis,RB
摘要：细胞色素o复合物是大肠杆菌有氧呼吸链中的一种6 o型泛醇氧化酶。该复合物与真核生物和原核生物的aa 3型细胞色素c氧化酶在结构和功能上有着密切的联系。纯化的细胞色素o复合物的比活性、亚基组成和金属含量对于文献中报道的不同制备方案是不一致的。本文介绍了一种相对简单的酶的制备方法，即从一株能过量生产氧化酶的大肠杆菌出发。经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分析，纯酶含有4个亚基。部分氨基酸序列数据证实了SDS-PAGE分析的亚基I、II和III分别为cyoB、cyoA和cyoC基因产物。对纯化方案稍作修改，得到含有可能的第五亚基的氧化酶纯化物，该亚基可能是cyoE基因产物。纯的四亚基酶含有2当量的铁，但只有1当量的铜。纯化的酶中没有电子顺磁共振检测到铜。因此，在该醌醇氧化酶中不存在aa 3型细胞色素c氧化酶的Cua的等价物。在纯化的醌醇氧化酶中也没有锌。最后，报道了与亚基II相互作用的单克隆抗体。这些单克隆抗体之一抑制洗涤剂溶解的纯化氧化酶的醌醇氧化酶活性。因此，尽管亚基II不含CuA，也不与细胞色素c相互作用，但它在thebo型泛醇氧化酶中仍具有重要功能。大肠杆菌的需氧呼吸链含有两种末端氧化酶，细胞色素o复合物（2> o型氧化酶）和细胞色素d复合物（M型氧化酶）-（Anraku，1988; Anraku & Gennis，1987）。这些酶中的每一种都起到醌醇氧化酶的作用，并将分子氧还原为水（Minghetti和Gennis，1988）。在生长培养基中氧张力高的条件下，细胞色素o复合物占主导地位，而细胞色素d复合物仅在氧变得有限或在厌氧生长条件下以高水平存在（Kranz & Gennis，1984; Rice & Hempfling，1978）。这些酶之所以令人感兴趣，不仅是因为它们是醌醇氧化酶，还因为通过任何一种酶的电子流都会导致跨膜质子动力的产生。这已经用在磷脂囊泡中重构的每种纯化酶的制备物证明（Carter & Gennis，1985; Kita等人，1982; Matsushita等人，1984;米勒和Gennis，1985）。Bo型泛醇氧化酶由于其结构和功能上与辅酶A的关系而受到特别的关注。
Revised Manuscript Received May 6, 1992 abstract: The cytochrome o complex is a 6o-type ubiquinol oxidase in the aerobic respiratory chain of Escherichia coli. This complex has a close structural and functional relationship with the eukaryotic and prokaryotic aa3-type cytochrome c oxidases. The specific activity, subunit composition, and metal content of the purified cytochrome o complex are not consistent for different preparative protocols reported in the literature. This paper presents a relatively simple preparation of theenzyme starting with a strain of Escherichia coli which overproduces theoxidase. The pure enzyme contains four subunits by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Partial amino acid sequence data confirm the identities of subunit I, II, and III from the SDS-PAGE analysis as the cyoB, cyoA, and cyoC gene products, respectively. A slight modification of the purification protocol yields an oxidase preparationthat contains a possible fifth subunit which may be the cyoE gene product. The pure four-subunit enzyme contains 2 equivs of iron but only 1 equiv of copper. There is no electron paramagnetic resonance detectable copper in the purified enzyme. Hence, the equivalent of Cua of the aa3-type cytochrome c oxidases is absent in this quinol oxidase. There is also no zinc in the purified quinol oxidase. Finally, monoclonal antibodies are reported that interact with subunit II. One of these monoclonals inhibits the quinol oxidase activity of the detergent-solubilized, purified oxidase. Hence, although subunit II does not contain CuA and does not interact with cytochrome c, it still must have an important function in thebo-type ubiquinol oxidase.The aerobic respiratory chain of Escherichia coli contains two terminal oxidases, the cytochrome o complex (2> o-type oxidase) and the cytochrome d complex (M-type oxidase)-(Anraku, 1988; Anraku & Gennis, 1987). Each of these enzymes functions as a quinol oxidase and reduces molecular oxygen to water (Minghetti & Gennis, 1988). Under conditions where the oxygen tension is high in the growth medium, the cytochrome o complex predominates, whereas the cytochrome d complex is present at high levels only when oxygen becomes limited or under anaerobic growth conditions (Kranz & Gennis, 1984; Rice & Hempfling, 1978). These enzymes are of interest not only because they are quinol ox-idases but also because electronflow through either enzyme results in the generation of a proton motive force across the membrane. This has been demonstrated with preparations of each of the purified enzymes reconstituted in phospholipid vesicles (Carter & Gennis, 1985; Kita et al., 1982; Matsushita et al., 1984; Miller & Gennis, 1985). The bo-type ubiquinol oxidase is of particular interest because of its structural and functional relationship to the