SUBUNIT III AND CYTOCHROME OXIDASE FUNCTION

球形红杆菌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，并且有一个系统 为快速定点突变和过度表达做好了准备 Rb中突变的氧化酶。球形藻。最近的高分辨率 细胞色素氧化酶的结构提供了需要的信息 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，但它在氧化酶功能中的作用远不是 安全。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。 我们的初步数据显示，亚基III是组装所必需的 亚基I中的血红素A3-幼崽活性部位，并防止组装 酶在催化过程中不会迅速失活。自杀- 失活涉及到一种结构变化，该变化会消除 血红素A3结合CO.之前的报告和新的结构表明 该亚基III还通过以下方式调节质子泵的效率 细胞色素氧化酶，在一个位点与ADP/ATP结合，这可以直接 通过脂质影响泵的活性，并提供一条途径 水池，供02人进入埋藏的血红素A3幼崽遗址。要探索 氧化酶组装、自杀性失活、泵送的分子机制 效率、核苷酸结合和02递送、定点突变体 缺少亚基III的关键残基和氧化酶的制备。 此外，亚基III中的四个突变被发现会导致线粒体 人类中的疾病已被引入RB球状氧化物酶。 突变的氧化酶将被检测其表达的变化， 活性，氧化还原活性金属中心的结构，内部 电子转移，一氧化碳，细胞色素c和核苷酸结合，以及质子 抽水效率。目标是1)确定亚基III的角色 在氧化酶功能中，2)定义自杀失活的机制， 3)确定导致细胞色素氧化酶功能缺陷的原因 线粒体疾病，以及4)利用亚基III与 进一步探讨血红素A3-幼体活性部位的能量机制 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.