Bratton M R, Hiser L, Antholine W E, Hoganson C, Hosler J P
Department of Biochemistry, University of Mississippi Medical Center, 2500 North State Street, Jackson, Mississippi 39216-4505, USA.
Biochemistry. 2000 Oct 24;39(42):12989-95. doi: 10.1021/bi0003083.
Genetic manipulation of the aa(3)-type cytochrome c oxidase of Rhodobacter sphaeroides was used to determine the minimal structural subunit associations required for the assembly of the heme A and copper centers of subunit I. In the absence of the genes for subunits II and III, expression of the gene for subunit I in Rb. sphaeroides allowed purification of a form of free subunit I (subunit I(a)()) that contained a single heme A. No copper was present in this protein, indicating that the heme a(3)-Cu(B) active site was not assembled. In cells expressing the genes for subunits I and II, but not subunit III, two oxidase forms were synthesized that were copurified by histidine affinity chromatography and separated by anion-exchange chromatography. One form was a highly active subunit I-II oxidase containing a full complement of structurally normal metal centers. This shows that association of subunit II with subunit I is required for stable formation of the active site in subunit I. In contrast, subunit III is not required for the formation of any of the metal centers or for the production of an oxidase with wild-type activity. The second product of the cells lacking subunit III was a large amount of a free form of subunit I that appeared identical to subunit I(a)(). Since significant amounts of subunit I(a)() were also isolated from wild-type cells, it is likely that subunit I(a)() will be present in any preparation of the aa(3)-type oxidase isolated via an affinity tag on subunit I.
利用球形红细菌aa(3)型细胞色素c氧化酶的基因操作来确定亚基I血红素A和铜中心组装所需的最小结构亚基关联。在缺乏亚基II和III基因的情况下，亚基I基因在球形红细菌中的表达使得能够纯化出一种游离亚基I形式（亚基I(a)()），其含有单个血红素A。该蛋白质中不存在铜，表明血红素a(3)-Cu(B)活性位点未组装。在表达亚基I和II基因但不表达亚基III基因的细胞中，合成了两种氧化酶形式，它们通过组氨酸亲和色谱共纯化，并通过阴离子交换色谱分离。一种形式是高度活跃的亚基I-II氧化酶，含有完整的结构正常的金属中心。这表明亚基II与亚基I的关联是亚基I中活性位点稳定形成所必需的。相比之下，亚基III对于任何金属中心的形成或具有野生型活性的氧化酶的产生都不是必需的。缺乏亚基III的细胞的第二种产物是大量与亚基I(a)()相同的游离亚基I形式。由于从野生型细胞中也分离出了大量的亚基I(a)()，因此在通过亚基I上的亲和标签分离的任何aa(3)型氧化酶制剂中都可能存在亚基I(a)()。
