FUNCTION OF SUBUNIT ISOFORMS IN CYTOCHROME C OXIDASE

细胞色素 C 氧化酶中亚基异构体的功能

• 批准号: 3296192
• 负责人: ROBERT Oliver POYTON
• 金额: $ 21.12万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-04-01 至 1995-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3296192
• 关键词: Saccharomyces cerevisiae; cytochrome c; cytochrome oxidase; enzyme mechanism; enzyme structure; gene expression; high performance liquid chromatography; isozymes; microorganism culture; mitochondrial membrane; oxidative phosphorylation; oxygen; protein purification; protein structure function

Mitochondrial oxidative phosphorylation is capable of supplying more than 95% of the total ATP requirement in respiring eucaryotic cells. It is driven by a respiratory chain composed of a number of multimeric membrane proteins that act in series to affect the transfer of electrons from reduced substrates to oxygen. Previous studies have emphasized the importance of the respiratory chain itself in regulating oxidative phosphorylation and have identified cytochrome c oxidase, its terminal member, as a key enzyme in the overall regulation of cellular energy production. At present, it is unclear how eucaryotic cells alter their cytochrome c oxidase activity levels in response to energy demand. However, the recent discovery of isoforms to the nuclearcoded subunits of cytochrome c oxidase in many eucaryotes, including humans, has led to the hypothesis that these polypeptides play a role in the modulation of cytochrome c oxidase activity. In this grant we will address this hypothesis. Initially, we will use the two subunit V isoforms, Va and Vb, of yeast cytochrome c oxidase as a model. Previous studies have shown that these isoforms affect some catalytic properties of holocytochrome c oxidase in vivo and that the expression of their genes, COX5a and COX5b, is differentially regulated by oxygen. Here, we propose to: 1) examine the structural-functional basis for the differential effects of Va and Vb on the electron transport activities of the holoenzyme; 2) determine if Va and Vb alter the proton pumping activity of the holoenzyme; 3) identify the domain(s) in Va and Vb that modulate holoenzyme activities; 4) determine if COX5a and COX5b are oxygen sensors that regulate the number of holocytochrome c oxidase molecules that are assembled in vivo; and 5) develop and use a heterologous complementation system to determine if human cytochrome c oxidase has subunit isoforms that function like yeast Va and Vb. These studies should enhance our understanding of cellular energetics and cytochrome c oxidase structure-function, and may provide an assay as well as a molecular basis for understanding the growing number of human diseases (i.e., tissue specific myopathies, cardiopathies, and hepatopathies) that are being linked to defects in cytochrome c oxidase. In addition, they should provide new opportunities to examine, and possibly modify, the mechanism of cytochrome c oxidase catalysis.

线粒体氧化磷酸化能够 在呼吸中提供总ATP总需求的95％以上 卵巢细胞。 它是由由 许多多聚体膜蛋白串联起作 影响电子从还原底物到氧的转移。 先前的研究强调了呼吸的重要性 链本身在调节氧化磷酸化方面，并具有 确定的细胞色素C氧化酶，其末端成员，作为钥匙 细胞能量产生总体调节中的酶。 目前，尚不清楚桉树细胞如何改变其 细胞色素C氧化酶活性响应能量需求。 但是，最近发现的核编码的同工型 许多桉树中的细胞色素C氧化酶的亚基，包括 人类导致了这样的假设，即这些多肽发挥了 在调节细胞色素C氧化酶活性中的作用。 在这个 授予我们将解决这一假设。 最初，我们将使用 酵母菌C氧化酶的两个亚基V同工型VA和VB 作为模型。 先前的研究表明，这些同工型会影响 全体内全囊色素C氧化酶的某些催化特性和 他们的基因Cox5a和Cox5b的表达是 由氧气差异调节。 在这里，我们建议：1） 检查差异的结构功能基础 VA和VB对电子传输活动的影响 Holoenzyme; 2）确定VA和VB是否改变了质子泵送 全酶的活性； 3）在VA和VB中识别域 调节全酶活性； 4）确定Cox5a和 COX5B是调节全胃色素数量的氧气传感器 c在体内组装的氧化酶分子； 5）发展和 使用异源补充系统来确定是否人 细胞色素C氧化酶的亚基同工型与酵母一样 VA和VB。 这些研究应该增强我们对 细胞能量和细胞色素C氧化酶结构功能， 并可能提供一个分子和分子基础 了解人类疾病数量不断增长（即组织 特定的肌病，心脏病和肝病） 与细胞色素C氧化酶缺陷有关。 另外，他们 应该提供新的机会来检查并可能修改， 细胞色素C氧化酶催化的机制。