SUBUNIT III AND CYTOCHROME OXIDASE FUNCTION

类球红细菌（Rhodobacter sphaeroides）的aa3型细胞色素c氧化酶是一种 哺乳动物线粒体氧化酶的优秀模型，和一个系统， 制备用于快速定点诱变和过表达 Rb中突变氧化酶的研究。sphaeroides 最近的高分辨率 细胞色素氧化酶的结构提供了 从分子水平探讨亚基III在氧化酶功能中的作用， 水平 亚基III是三个高度保守的核心亚基之一 细胞色素氧化酶，但它的作用，氧化酶的功能是远远没有 清楚 亚基III具有独特的结构，包括3个紧密结合的 与亚基III和I的残基相互作用的磷脂。 我们的初步数据表明，亚基III是必要的组装 的血红素a3-CuB活性位点的亚基I，并阻止组装 氧化酶在催化过程中迅速失活。 自杀- 失活涉及一种结构变化， 以前的报道和新的结构表明， 亚基III也通过调节质子泵的效率， 细胞色素氧化酶，结合ADP/ATP在一个网站，可以直接 影响泵的活性，并通过脂质提供路径， 池，用于02进入掩埋的血红素a3-CuB位点。 探讨 氧化酶组装的分子机制，自杀失活，泵送 效率、核苷酸结合和O2递送、定点突变体 的关键残基和氧化酶缺乏亚基III已制备。 此外，发现亚基III中的四个突变导致线粒体 已经将人的疾病引入类球Rb氧化酶。 将测定突变氧化酶的表达改变， 活性，氧化还原活性金属中心的结构，内部 电子转移，CO，细胞色素c和核苷酸结合，以及质子 泵送效率 目标是：1）确定亚单位III的作用 在氧化酶功能中，2）定义自杀失活的机制， 3)定义细胞色素氧化酶的功能缺陷， 线粒体疾病，以及4）使用亚基III与 进一步探讨血红素a3-CuB活性位点的供能机制 通过细胞色素氧化酶偶联。

The aa3-type cytochrome c oxidase of Rhodobacter sphaeroides is an excellent model of the mammalian mitochondrial oxidase, and a system has been prepared for rapid site-directed mutagenesis and over-expression of mutant oxidases in Rb. sphaeroides. Recent high resolution structures of cytochrome oxidase provide the information needed to explore the roles of subunit III in oxidase function at the molecular level. Subunit III is one of the three highly conserved core subunits of cytochrome oxidase, but its role in oxidase function is far from clear. Subunit III has a unique structure that includes 3 tightly bound phospholipids that interact with residues of both subunits III and I. Our preliminary data show that subunit III is necessary for the assembly of the heme a3-CuB active site in subunit I, and prevents the assembled oxidase from rapidly inactivating during catalysis. Suicide- inactivation involves a structural change that eliminates the ability of heme a3 to bind CO. Previous reports and the new structures suggest that subunit III also modulates the efficiency of proton pumping by cytochrome oxidase, binds ADP/ATP at a site and that could directly influence the activity of the pump, and provides a path, via the lipid pool, for 02 to access the buried heme a3-CuB site. To explore the molecular mechanisms of oxidase assembly, suicide inactivation, pumping efficiency, nucleotide binding, and 02 delivery, site directed mutants of key residues and oxidases lacking subunit III have been prepared. In addition, four mutations in subunit III found to cause mitochondrial diseases in humans have been introduced into the Rb sphaeroides oxidase. The mutant oxidases will be assayed for alterations in expression, activity, the structure of the redox-active metal centers, internal electron transfer, CO, cytochrome c and nucleotide binding, and proton pumping efficiency. The goals are to 1) define the roles of subunit III in oxidase function, 2) define the mechanism of suicide-inactivation, 3) define the functional defects in cytochrome oxidase that cause the mitochondrial diseases, and 4) use the interaction of subunit III with the heme a3-CuB active site to further explore the mechanisms of energy coupling by cytochrome oxidase.