FUNCTION OF SUBUNIT ISOFORMS IN CYTOCHROME C OXIDASE

线粒体氧化磷酸化能够 提供超过95%的ATP总需求在呼吸 真核细胞 它是由呼吸链驱动的， 一系列多聚体膜蛋白， 影响电子从还原基质向氧的转移。 以前的研究强调了呼吸系统的重要性， 链本身调节氧化磷酸化， 确定了细胞色素c氧化酶，其末端成员，作为一个关键， 酶在细胞能量生产的整体调节。 目前，还不清楚真核细胞如何改变它们的 细胞色素c氧化酶活性水平响应于能量需求。 然而，最近发现的同种型的核编码 许多真核生物中的细胞色素c氧化酶亚基，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 我们将讨论这个假设。 首先，我们将使用 酵母细胞色素c氧化酶的两种亚基V亚型Va和Vb 做模特 以前的研究表明，这些异构体影响 全细胞色素c氧化酶在体内的某些催化性质 他们的基因COX 5a和COX 5 b的表达， 受氧气的不同调节。 在此，我们建议：1） 检查差异的结构功能基础 Va和Vb对电子传递活性的影响 全酶; 2）确定Va和Vb是否改变质子泵送 全酶的活性; 3）鉴定Va和Vb中的结构域 其调节全酶活性; 4）确定COX 5a和 COX 5 b是氧传感器，调节细胞色素的数量 c氧化酶分子在体内组装;和5）开发和 使用异源互补系统来确定人类 细胞色素c氧化酶具有亚单位同种型，其功能类似于酵母 Va和Vb。 这些研究应能增进我们对 细胞能量学和细胞色素c氧化酶结构-功能， 并可提供测定以及分子基础， 了解越来越多的人类疾病（即，组织 特定的肌病、心脏病和肝病）， 与细胞色素c氧化酶的缺陷有关。 此外他们 应该提供新的机会来检查，并可能修改， 细胞色素c氧化酶的催化机理。

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.