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: 7106450
• 负责人: Eric L. Hegg
• 金额: $ 3.46万
• 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 2006-08-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7106450
• 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、需氧酵母和一些细菌中的末端氧化酶。细胞色素c氧化酶催化分子氧还原为水，同时泵送质子穿过线粒体内膜，细胞色素c氧化酶产生质子梯度，负责真核生物有氧代谢期间形成的约50%的ATP。血红素A是真核细胞色素c氧化酶中的一种必需辅因子，存在于电子传递位点和氧还原位点。血红素A与血红素B（原血红素）的不同之处在于，法呢基部分已被添加到乙烯基之一，并且甲基取代基已被氧化成醛。这些转化（两者都是获得活性细胞色素c氧化酶所需的）分别由血红素0合酶和血红素A合酶催化。令人惊讶的是，尽管血红素A的细胞色素c氧化酶和能量转导途径的明显的重要性，很少有人知道血红素A是如何合成的，它是如何运输，或它是如何插入到细胞色素c oxidase.The本研究的重点是阐明血红素A的生物合成及其运输方法，包括相关的多组分复合物的形成。本实验室获得的初步数据表明，血红素A合酶改变血红素0合酶的活性，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的证据表明，血红素A合酶代表一种新的含血红素加氧酶。为了证实这些诱人的结果，并提高我们的理解血红素A的生物合成和运输，一个跨学科的研究计划已经制定。生物化学和分子生物学的方法将被用来定义特定的大分子复合物和蛋白质-蛋白质相互作用，涉及血红素A合酶。假设与血红素A合酶相互作用的蛋白质是1）血红素0合酶，2）线粒体核糖体复合物的两个COX 1翻译激活剂，和3）细胞色素c氧化酶的亚基I。此外，各种化学和光谱技术将被用来1）充分表征血红素A合酶的活性位点，2）确定活性氧化剂的性质，和3）阐明血红素A生物合成的机制。结合起来，所提出的实验代表了对血红素A合酶的全面攻击，从而提供了关于细胞色素c氧化酶组装的基本信息和对生物O2活化的进一步了解。