Hiser L, Hosler J P
Department of Biochemistry, The University of Mississippi Medical Center, Jackson, Mississippi 39216, USA.
J Biol Chem. 2001 Nov 30;276(48):45403-7. doi: 10.1074/jbc.M107016200. Epub 2001 Sep 26.
The aa(3)-type cytochrome c oxidase of Rhodobacter sphaeroides, a proteobacterium of the alpha subgroup, is structurally similar to the core subunits of the terminal oxidase in the mitochondrial electron transport chain. Subunit I, the product of the coxI gene, normally binds two heme A molecules. A deletion of cox10, the gene for the farnesyltransferase required for heme A synthesis, did not prevent high level accumulation of subunit I in the cytoplasmic membrane. Thus, subunit I can be expressed and stably inserted into the cytoplasmic membrane in the absence of heme A. Aposubunit I was purified via affinity chromatography to a polyhistidine tag. Copurification of subunits II and III with aposubunit I indicated that assembly of the core oxidase complex occurred without the binding of heme A. In addition to formation of the apooxidase containing all three large structural proteins, CoxI-II and CoxI-III heterodimers were isolated from cox10 deletion strains harboring expression plasmids with coxI and coxII or with coxI and coxIII, respectively. This demonstrated that subunit assembly of the apoenzyme was not an inherently ordered or sequential process. Thus, multiple paths must be considered for understanding the assembly of this integral membrane metalloprotein complex.
球形红杆菌（一种α亚群的变形菌）的aa(3)型细胞色素c氧化酶在结构上与线粒体电子传递链中末端氧化酶的核心亚基相似。亚基I是coxI基因的产物，通常结合两个血红素A分子。血红素A合成所需的法尼基转移酶基因cox10的缺失，并不妨碍亚基I在细胞质膜中的高水平积累。因此，在没有血红素A的情况下，亚基I可以表达并稳定插入细胞质膜。通过亲和色谱法将脱辅基亚基I纯化至带有多组氨酸标签。脱辅基亚基I与亚基II和III的共纯化表明，核心氧化酶复合物的组装在没有血红素A结合的情况下发生。除了形成包含所有三种大型结构蛋白的脱辅基氧化酶外，还分别从携带含有coxI和coxII或coxI和coxIII的表达质粒的cox10缺失菌株中分离出CoxI-II和CoxI-III异二聚体。这表明脱辅基酶的亚基组装不是一个固有有序或连续的过程。因此，在理解这种整合膜金属蛋白复合物的组装时，必须考虑多种途径。
