Assembly of Redox Metal Centers in Cytochrome Oxidase

描述（由申请人提供）： 该建议的重点是使用酵母作为实验系统的金属中心细胞色素c氧化酶（CcO）的组装。CcO缺乏症是婴儿呼吸链紊乱的重要原因，可表现为Leigh综合征、心肌病、脑心肌病或肝功能衰竭。孟德尔遗传性CcO缺乏症代表CcO组装因子的突变，其在CcO的铜或血红素氧化还原辅因子的合成或插入中起作用。这些因子在真核生物中是保守的，对它们功能的了解首先是从酵母的研究中收集的。因此，酵母研究的组装CcO的氧化还原辅因子中心有直接的关系，人类生物学和CcO缺乏症。我们将阐明两个血红素和两个铜中心在Cox1和Cox2的装配途径。我们假设，血红素插入到Cox1是一个连续的过程与血红素a网站形成的早期Cox1成熟，随后血红素a3插入涉及Shy1。人类Shy1同源基因SURF 1是Leigh综合征患者中最常见的突变基因。我们的数据表明，同时血红素a3插入，Cox11依赖铜代谢的CuB网站发生。我们将定义Cox11在Cox1的CuB位点形成中的机械作用。我们假设，辅酶插入到Cox1完成之前，加入Cox2含有CuA的辅因子网站。我们将定义的作用，Sco1在CuA网站形成Cox2和Cox17作为铜供体的Sco1和Cox11。在血红素a3插入的下游步骤处CcO组装的停滞产生了促氧化剂中间体，其导致用过氧化氢应激的细胞中的生长受损。我们使用H2 O2敏感性测定来确定Shy 1在血红素a3插入中的作用，并表征两个新的CcO组装因子Coa 1和Coa 2的功能。在CcO缺乏症患者中存在促氧化血红素a3：Cox1停滞中间体可能会增加所观察到的病理学的氧化应激。铜转移步骤发生在对氧化应激敏感的线粒体的膜间隙中。因此，CcO组装可能对诱导氧化应激的环境因子敏感。这项资助的统一主题是，在酵母中定义CcO组装途径将开辟解剖人类细胞中的途径的策略，并可能识别人类特定的组装因子。公共卫生相关性：细胞色素c氧化酶中金属辅因子中心的组装是酵母和人类细胞中的保守过程。在铜和血红素氧化还原辅因子的合成或插入中起作用的细胞色素氧化酶组装因子中的突变导致细胞色素氧化酶缺陷，其可表现为Leigh综合征、心肌病、脑心肌病或肝功能衰竭。Leigh综合征患者中最常见的突变基因是SURF 1，其编码一种蛋白质，该蛋白质在细胞色素氧化酶中的血红素插入中起作用。

DESCRIPTION (provided by applicant): This proposal focuses on the assembly of metal centers cytochrome c oxidase (CcO) using yeast as an experimental system. CcO deficiency is an important cause of respiratory chain disorders in infants that can present as Leigh Syndrome, cardiomyopathy, encephalocardiomyopathy or hepatic failure. The Mendelian- inherited CcO deficiency disorders represent mutations in CcO assembly factors that act either in the synthesis or insertion of copper or heme redox cofactors for CcO. These factors are conserved in eukaryotes and insights into their function were first gleaned from studies in yeast. Thus, yeast studies on the assembly of redox cofactor centers of CcO have direct relevance to human biology and CcO deficiencies disorders. We will elucidate the assembly pathway of the two heme a and two copper centers in Cox1 and Cox2. We postulate that heme insertion into Cox1 is a sequential process with the heme a site forming early in Cox1 maturation followed by heme a3 insertion involving Shy1. The human Shy1 ortholog SURF1 is the most frequently mutated gene in Leigh Syndrome patients. Our data indicate that concurrent to heme a3 insertion, Cox11-dependent copper metallation of the CuB site occurs. We will define the mechanistic roles of Cox11 in CuB site formation in Cox1. We hypothesize that cofactor insertion into Cox1 is completed prior to the addition of Cox2 containing the CuA cofactor site. We will define the role of Sco1 in CuA site formation in Cox2 and Cox17 as the copper donor to both Sco1 and Cox11. Arrest of CcO assembly at steps downstream of heme a3 insertion generates a pro- oxidant intermediate that leads to growth impairment in cells stressed with hydrogen peroxide. We used the H2O2 sensitivity assay to establish the role of Shy1 in heme a3 insertion and to characterize the function of two new CcO assembly factors Coa1 and Coa2. The presence of the pro-oxidant heme a3:Cox1 stalled intermediate in patients with CcO deficiencies may add oxidative stress to the observed pathology. The Cu- transfer steps occur in the intermembrane space of mitochondria that is susceptible to oxidative stress. Thus, CcO assembly will be likely sensitive to environmental agents that induce oxidative stress. The unifying theme in this grant is that defining the CcO assembly pathway in yeast will open up strategies to dissect the pathway in human cells and perhaps identify human specific assembly factors. PUBLIC HEALTH RELEVANCE: Assembly of the metal cofactor centers in cytochrome c oxidase is a conserved process in yeast and human cells. Mutations in cytochrome oxidase assembly factors that act in the synthesis or insertion of copper and heme redox cofactors leads to cytochrome oxidase deficiencies that can present as Leigh Syndrome, cardiomyopathy, encephalocardiomyopathy or hepatic failure. The most frequently mutated gene in Leigh Syndrome patients is SURF1 that encodes a protein that acts in heme a insertion in cytochrome oxidase.