Elucidating the Biosynthesis and Transport of Heme A

阐明血红素 A 的生物合成和运输

• 批准号: 6927814
• 负责人: Eric L. Hegg
• 金额: $ 20.66万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-08-01 至 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6927814
• 关键词: active sites; cytochrome oxidase; enzyme activity; enzyme biosynthesis; heme; hydro lyase; porphyrin biosynthesis; prostaglandin endoperoxide synthase; protein protein interaction; protein purification; protein transport; stable isotope double label

DESCRIPTION (provided by applicant): Cytochrome c oxidase is the terminal oxidase in all plants, animals, aerobic yeasts, and some bacteria. Catalyzing the reduction of molecular oxygen to water concomitant with the pumping of protons across the inner mitochondrial membrane, cytochrome c oxidase generates a proton gradient responsible for approximately 50 percent of the ATP formed during eukaryotic aerobic metabolism. Heme A is an obligatory cofactor in eukaryotic cytochrome c oxidase, present at both an electron transfer site and the site of oxygen reduction. Heme A differs from heme B (protoheme) in that a farnesyl moiety has been added to one of the vinyl groups, and a methyl substituent has been oxidized to an aldehyde. These conversions (both of which are required to obtain active cytochrome c oxidase) are catalyzed by heme 0 synthase and heme A synthase, respectively. Surprisingly, despite the obvious importance of heme A to both cytochrome c oxidase and the energy transduction pathway, very little is known about how heme A is synthesized, how it is transported, or how it is inserted into cytochrome c oxidase.The focus of this research is to elucidate the biosynthesis of heme A and its method of transport, including the formation of associated multi-component complexes. Preliminary data obtained in this lab demonstrates that heme A synthase alters the activity of heme 0 synthase, suggesting that the two enzymes are part of a larger protein complex. We have also obtained the first direct evidence indicating that heme A synthase represents a novel heme-containing oxygenase. To confirm these tantalizing results and to enhance our understanding of heme A biosynthesis and transport, an interdisciplinary research plan has been developed. Biochemical and molecular approaches will be used to define specific macromolecular complexes and protein-protein interactions involving heme A synthase. The proteins hypothesized to interact with heme A synthase are 1) heme 0 synthase, 2) two COX1 translation activators of the mitochondrial ribosome complex, and 3) subunit I of cytochrome c oxidase. In addition, a variety of chemical and spectroscopic techniques will be employed to 1) fully characterize the active site of heme A synthase, 2) determine the nature of the active oxidant, and 3) elucidate the mechanism of heme A biosynthesis. Combined, the proposed experiments represent a comprehensive attack on heme A synthase, thus providing fundamental information about the assembly of cytochrome c oxidase and further insight into biological 02 activation.

描述（由申请人提供）：细胞色素c氧化酶是所有植物、动物、好氧酵母和某些细菌的终末氧化酶。细胞色素c氧化酶催化分子氧还原为水，同时通过线粒体内膜泵送质子，产生质子梯度，负责真核生物有氧代谢过程中形成的大约50%的ATP。血红素A是真核细胞色素c氧化酶的一种必需辅因子，存在于电子转移位点和氧还原位点。血红素A与血红素B（原血红素）的不同之处在于，其中一个乙烯基上加入了一个法尼基片段，一个甲基取代基被氧化成醛。这些转化（两者都是获得活性细胞色素c氧化酶所必需的）分别由血红素0合成酶和血红素A合成酶催化。令人惊讶的是，尽管血红素A对细胞色素c氧化酶和能量转导途径都具有明显的重要性，但人们对血红素A如何合成、如何运输或如何插入细胞色素c氧化酶知之甚少。本研究的重点是阐明血红素A的生物合成及其转运方法，包括形成相关的多组分配合物。本实验室获得的初步数据表明，血红素A合成酶会改变血红素0合成酶的活性，这表明这两种酶是一个更大的蛋白质复合物的一部分。我们还获得了第一个直接证据，表明血红素A合酶是一种新的含血红素加氧酶。为了证实这些诱人的结果，并加强我们对血红素A生物合成和运输的理解，一项跨学科的研究计划已经制定。生化和分子方法将用于定义涉及血红素A合成酶的特定大分子复合物和蛋白质-蛋白质相互作用。假设与血红素A合成酶相互作用的蛋白质是1)血红素0合成酶，2)线粒体核糖体复合体的两个COX1翻译激活因子，以及3)细胞色素c氧化酶的亚基I。此外，各种化学和光谱学技术将用于1)充分表征血红素a合成酶的活性位点，2)确定活性氧化剂的性质，以及3)阐明血红素a生物合成的机制。综上所述，这些实验代表了对血红素a合成酶的全面攻击，从而为细胞色素c氧化酶的组装提供了基础信息，并进一步了解了生物02活化。