SUBUNIT III AND CYTOCHROME OXIDASE FUNCTION

亚基 III 和细胞色素氧化酶功能

• 批准号: 6519847
• 负责人: JONATHAN P HOSLER
• 金额: $ 17.09万
• 依托单位: UNIVERSITY OF MISSISSIPPI MED CTR
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-06-01 至 2003-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6519847
• 关键词: Rhodospirillales; active sites; bioenergetics; carbon monoxide; cytochrome oxidase; enzyme activity; enzyme biosynthesis; enzyme deficiency; enzyme mechanism; enzyme structure; gene mutation; hydrogen transporting ATP synthase; metalloenzyme; mitochondria; site directed mutagenesis

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.

Rhodobacter sphaeroides的AA3型细胞色素C氧化酶是 哺乳动物线粒体氧化酶的优秀模型，并且系统具有 准备用于快速定向的诱变和过表达 Rb中突变氧化酶的sphaeroides。 最近的高分辨率 细胞色素氧化酶的结构提供了所需的信息 探索亚基III在氧化酶功能上的作用 等级。 亚基III是三个高度保守的核心亚基之一 细胞色素氧化酶的作用，但其在氧化酶功能中的作用远非 清除。 亚基III具有独特的结构，其中包括3个紧密结合 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对于组装是必需的 亚基I中的血红素A3-cub活性位点的 氧化酶在催化过程中迅速灭活。 自杀- 灭活涉及结构性变化，以消除能力 Heme A3 bind Co。先前的报告和新结构建议 该亚基III还调节了质子泵送的效率 细胞色素氧化酶，结合位点上的ADP/ATP，可以直接 影响泵的活性，并通过脂质提供路径 游泳池，用于02访问埋入的血红素A3-cub站点。 探索 氧化酶组装的分子机制，自杀灭活，抽水 效率，核苷酸结合和02递送，位点定向突变体 已经制备了缺乏亚基III的关键残基和氧化酶。 此外，在亚基III中的四个突变发现引起线粒体 人类中的疾病已被引入RB SPHAEREIDES氧化酶。 将分析突变氧化酶以改变表达的改变， 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 抽水效率。 目标是1）定义亚基III的角色 在氧化酶功能中，2）定义自杀灭活机制， 3）定义引起的细胞色素氧化酶的功能缺陷 线粒体疾病和4）使用亚基III的相互作用 血红素A3-cub活性位点，以进一步探索能量机理 通过细胞色素氧化酶偶联。