Molecular Mechanisms of Copper Delivery to Mitochondrial Cytochrome c Oxidase

修改后的项目摘要/摘要部分 铜是一种必需的微量营养素，可作为多种酶的催化辅因子，包括细胞色素c氧化酶(CcO)，这是线粒体产生能量所必需的关键酶。铜向CcO的传递是一个复杂的过程，需要多种蛋白质，而编码这些蛋白质的几个基因的功能丧失突变会导致致命的婴儿疾病。目前，对于这些致命的疾病还没有有效的治疗方法。这在一定程度上是由于我们对铜是如何被贩运到线粒体并被输送到CcO的含铜亚单位的理解不完全。为了解决我们知识中的这一差距，我们使用涉及酵母遗传学和生物化学的互补方法来发现和表征促进铜向CcO输送的新型线粒体蛋白。利用这些方法，我们发现了进化上保守的线粒体蛋白Coa4和Coa6作为线粒体铜向CcO输送途径的新成员。现在，我们的目标是明确Coa4和Coa6在铜输送过程中的生化功能。我们的初步发现，包括人COA6的结构，使我们假设Coa4和Coa6在向CcO输送铜的过程中发挥着氧化还原作用。我们将通过确定Coa4和Coa6相互作用蛋白的体外和体内氧化还原状态，以及通过基因和蛋白质相互作用研究确定相互作用的物理基础来检验这一假说。因此，拟议的项目解决了向CCOO提供铜的机制中的基本问题。

Modified Project Summary/Abstract Section Copper (Cu) is an essential micronutrient that acts as a catalytic cofactor for a number of enzymes including cytochrome c oxidase (CcO), a critical enzyme required for mitochondrial energy production. Cu delivery to CcO is a complex process requiring multiple proteins, and loss-of-function mutations in several of the genes encoding these proteins cause fatal infantile disorders. Currently, no effective therapy exists for these fatal disorders. This is due, in part, to our incomplete understanding of how Cu is trafficked to the mitochondria and is delivered to the Cu-containing subunits of CcO. To address this gap in our knowledge, we use complementary approaches involving yeast genetics and biochemistry to discover and characterize the novel mitochondrial proteins that facilitate Cu delivery to CcO. Using these approaches, we have found the evolutionarily conserved mitochondrial proteins Coa4 and Coa6 as new members of the mitochondrial Cu delivery pathways to CcO. Now, we aim to define the biochemical functions of Coa4 and Coa6 in the Cu delivery process. Our preliminary findings, including the structure of human COA6, have led us to hypothesize that Coa4 and Coa6 play redox roles in Cu delivery to CcO. We will test this hypothesis by determining the in vitro and in vivo redox states of Coa4- and Coa6- interacting proteins and by defining the physical basis of interaction through genetic and protein:protein interaction studies. Thus, the proposed project addresses fundamental questions in Cu delivery mechanisms to CcO.