Biosynthesis and Insertion of Heme A during Cytochrome c Oxidase Biogenesis

描述（由申请人提供）：血红素A是真核细胞色素c氧化酶（CcO）中的一种强制性辅因子，但关于血红素A如何插入CcO中，或血红素通过血红素a生物合成途径的通量如何与CcO组装协调的知之甚少。由于CcO间接负责有氧代谢过程中形成的约50%的ATP，因此细胞维持适当水平的血红素A至关重要，血红素A稳态的不足导致几种临床和致命的早发性线粒体疾病。然而，与此同时，过量的血红素a是有毒的，因此细胞调节其生产以确保 有足够的血红素A，但不是过量的我们缺乏了解的过程中，影响血红素的生物合成和它的插入到CcO代表了一个主要的知识差距在有氧代谢的一个基本方面。该提案的目的是揭示如何通过血红素的生物合成途径的血红素通量耦合到CcO的生物合成和关键的蛋白质-蛋白质相互作用，协助和帮助调节这些过程。至少有三种不同的蛋白质对于血红素A的生物合成及其随后插入CcO的亚基1（Cox 1）中是关键的：血红素O合酶（HOS）、血红素A合酶（HAS）和Surf 1/Shy 1。我们假设，HOS和HAS形成不同的，大的蛋白质复合物，改变酶活性。在我们的模型中，HAS复合物将血红素A传递给Cox 1，而Surf 1/Shy 1通过稳定新插入的血红素A并防止其在组装过程中丢失来辅助这一过程。我们进一步假设HOS和HAS的活性与Cox 1的翻译有关。这些假设将通过追求两个具体目标进行测试：1）表征血红素A生物合成和CcO组装之间的关系，以及2）确定HOS和HAS蛋白复合物的意义。为了实现这些目标，我们将采用在体内和体外研究使用球形红细菌和酿酒酵母作为模型。这项研究意义重大，因为它解决了CcO生物发生中最核心但也是最不了解的两个方面。(1)细胞如何调节重要但有毒的血红素辅因子的产生？(2)血红素A是如何插入CcO的？我们的方法是创新的，因为我们同时使用R。sphaeroides和S.酿酒酵母利用稳定性和相对简单的细菌氧化酶，同时验证我们的结果在遗传上易处理的真核生物。采用这两种系统提供的协同作用使我们能够在解决CcO生物发生的这些基本问题方面取得快速进展。

DESCRIPTION (provided by applicant): Heme A is an obligatory cofactor in eukaryotic cytochrome c oxidase (CcO), but little is known about how heme A is inserted into CcO, or how the flux of heme through the heme a biosynthetic pathway is coordinated to CcO assembly. Because CcO is indirectly responsible for ~50% of the ATP formed during aerobic metabolism, it is crucial that cells maintain a proper level of heme A, and deficiencies in heme a homeostasis lead to several clinical and fatal early-onset mitochondrial disorders. At the same time, however, excess heme a is toxic, and it is therefore critical for cells to regulate its production to ensure that there is sufficient, but not excess, heme A available. Our lack of understanding about the processes that affect heme a biosynthesis and its insertion into CcO represents a major knowledge gap in a fundamental aspect of aerobic metabolism. The objectives of this proposal are to reveal how the flux of heme through the heme a biosynthetic pathway is coupled to CcO biosynthesis and the key protein-protein interactions that assist and help regulate these processes. At least three different proteins are critical for the biosynthesis of heme A and its subsequent insertion into subunit 1 (Cox1) of CcO: heme O synthase (HOS), heme A synthase (HAS) and Surf1/Shy1. We hypothesize that HOS and HAS form distinct, large protein complexes that alter enzymatic activity. In our model, the HAS complex delivers heme A to Cox1 while Surf1/Shy1 aids in this process by stabilizing the newly inserted heme A and preventing its loss during assembly. We further hypothesize that the activity of HOS and HAS is coupled to Cox1 translation. These hypotheses will be tested by pursuing two specific aims: 1) characterize the relationship between heme A biosynthesis and CcO assembly, and 2) ascertain the significance of the HOS and HAS protein complexes. To accomplish these goals, we will employ both in vivo and in vitro studies using Rhodobacter sphaeroides and Saccharomyces cerevisiae as models. The proposed research is significant because it addresses two of the most central but also least understood aspects of CcO biogenesis. (1) How does the cell regulate the production of the vital yet toxic heme a cofactor? (2) How is heme A inserted into CcO? Our approach is innovative because we are simultaneously employing both R. sphaeroides and S. cerevisiae to capitalize on the stability and relative simplicity of bacterial oxidases while validating our results in a genetically tractable eukaryote. The synergy provided by employing both systems positions us to make rapid progress addressing these essential questions of CcO biogenesis.