Biosynthesis and Insertion of Heme A during Cytochrome c Oxidase Biogenesis

说明(申请人提供)：血红素A是真核细胞色素C氧化酶(CcO)的必需辅因子，但人们对血红素A如何插入CcO，或血红素通过生物合成途径的流动如何与CcO组装协调知之甚少。由于CcO间接负责有氧代谢过程中形成的~50%的ATP，因此细胞保持适当的血红素A水平是至关重要的，而血红素A动态平衡的缺陷会导致几种临床和致命的早期线粒体疾病。然而，与此同时，过量的血红素a是有毒的，因此对细胞来说，调节其生产以确保 有足够的但不是过剩的血红素A可用。我们对影响血红素生物合成和将其插入CcO的过程缺乏了解，这是有氧代谢的一个基本方面的重大知识缺口。这项提议的目的是揭示通过血红素生物合成途径的血红素流动是如何耦合到一氧化碳生物合成的，以及帮助和帮助调节这些过程的关键蛋白质-蛋白质相互作用。至少有三种不同的蛋白质对血红素A的生物合成及其随后插入CcO的亚基1(Cox1)至关重要：血红素O合成酶(HOS)、血红素A合成酶(HAS)和Surf1/Shy1。我们假设HOS和HAS形成不同的、大的蛋白质复合体，改变酶的活性。在我们的模型中，HAS复合体将血红素A输送到Cox1，而Surf1/Shy1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帮助这一过程。我们进一步假设HOS和HAS的活性与Cox1的翻译有关。这些假说将通过追求两个特定的目标来验证：1)表征血红素A生物合成和CcO组装之间的关系，2)确定HOS和HAS蛋白质复合体的意义。为了实现这些目标，我们将使用球形红杆菌和酿酒酵母作为模型进行体内和体外研究。这项拟议的研究意义重大，因为它涉及CcO生物发生的两个最核心但也最不为人所知的方面。(1)细胞如何调节重要但有毒的血红素(一种辅因子)的产生？(2)血红素A是如何插入到一氧化碳中的？我们的方法是创新的，因为我们同时使用球形葡萄球菌和酿酒酵母来利用细菌氧化酶的稳定性和相对简单性，同时在基因易驯化的真核生物中验证我们的结果。使用这两个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。 与公共健康相关：血红素A是细胞色素C氧化酶的必备辅因子，该酶负责将O2转化为水，并在呼吸过程中以电化学梯度的形式储存能量。拟议的关于血红素A动态平衡的研究与公共健康相关，因为对血红素a水平的不当调节会导致多种线粒体疾病，包括贫血、Leigh综合征和婴儿心肌病。我们将揭示血红素a的生物合成是如何调节的，以及这个关键的辅因子是如何在细胞色素c氧化酶的生物发生过程中插入的。

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. PUBLIC HEALTH RELEVANCE: Heme A is an obligatory cofactor in cytochrome c oxidase, the enzyme responsible for converting O2 to water and storing the energy as an electrochemical gradient during respiration. The proposed research on heme A homeostasis is relevant to public health because improper regulation of heme a levels leads to a variety of mitochondrial diseases including anemia, Leigh Syndrome, and infantile cardiomyopathy. We will reveal how heme a biosynthesis is regulated and how this critical cofactor is inserted during cytochrome c oxidase biogenesis.