Assembly of Redox Metal Centers in Cytochrome Oxidase

描述(由申请人提供)： 本研究以酵母菌为实验体系，研究金属中心细胞色素c氧化酶(CcO)的组装。CCO缺乏是婴儿呼吸链紊乱的重要原因，可表现为Leigh综合征、心肌病、脑型心肌病或肝功能衰竭。孟德尔遗传的CcO缺乏症代表CcO组装因子的突变，这些突变作用于CcO的铜或血红素氧化还原辅因子的合成或插入。这些因子在真核生物中是保守的，对它们功能的洞察首先是从对酵母的研究中收集到的。因此，酵母菌对CcO氧化还原辅因子中心组装的研究与人类生物学和CcO缺乏症有直接关系。我们将阐明两个血红素a和两个铜中心在Cox1和Cox2中的组装途径。我们假设，血红素插入Cox1是一个连续的过程，血红素是在Cox1成熟早期形成的位置，随后是涉及Shy1的血红素A3插入。人类Shy1基因SURF1是Leigh综合征患者中最常见的突变基因。我们的数据表明，在血红素A3插入的同时，发生了依赖于Cox11的CuB位置的铜金属化。我们将定义Cox11在Cox1幼崽位置形成中的机械作用。我们假设Cox1中的辅因子插入是在添加包含CUA辅因子位点的COX2之前完成的。我们将确定Sco1在COX2和Cox17中CUA位置形成中的作用，作为Sco1和Cox11的铜供体。在血红素A3插入的下游步骤阻止CCOO组装会产生一种促氧化中间体，导致过氧化氢胁迫下细胞的生长障碍。我们使用过氧化氢敏感性实验来确定Shy1在血红素A3插入中的作用，并表征两个新的CcO组装因子Coa1和Coa2的功能。在CcO缺乏的患者中，促氧化剂血红素A3：Cox1停滞中间体的存在可能会增加观察到的病理学中的氧化应激。铜的转移步骤发生在线粒体的膜间隙，而线粒体对氧化应激很敏感。因此，CCOO组装可能对诱导氧化应激的环境因素敏感。这项资助的统一主题是，定义酵母中的CcO组装途径将开启在人类细胞中剖析该途径的策略，并可能识别人类特有的组装因子。

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.