Elucidating the Biosynthesis and Transport of Heme A

说明(申请人提供)：细胞色素c氧化酶是所有植物、动物、需氧酵母菌和某些细菌的末端氧化酶。细胞色素c氧化酶催化分子氧还原为水，同时伴随着质子通过线粒体内膜的泵送，产生的质子梯度约占真核生物有氧代谢过程中形成的ATP的50%。血红素A是真核细胞色素C氧化酶的必需辅因子，既存在于电子转移部位，也存在于氧还原部位。亚铁血红素A与亚铁血红素B(原亚铁血红素)的不同之处在于，在其中一个乙烯基团上添加了法尼基，甲基取代基被氧化成醛。这些转化(这两种转化都是获得有活性的细胞色素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有明显的重要性，但人们对血红素A是如何合成的，它是如何运输的，或者它是如何插入细胞色素c氧化物酶的知之甚少。本研究的重点是阐明血红素A的生物合成及其运输方式，包括相关多组分复合体的形成。本实验室获得的初步数据表明，血红素A合成酶改变了血红素0合成酶的活性，这表明这两种酶是更大的蛋白质复合体的一部分。我们还获得了第一个直接证据，表明血红素A合成酶代表了一种新的含血红素的加氧酶。为了证实这些诱人的结果，并加强我们对血红素A生物合成和运输的了解，已经制定了一个跨学科的研究计划。将使用生物化学和分子方法来定义涉及血红素A合成酶的特定大分子复合体和蛋白质-蛋白质相互作用。推测与血红素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翻译激活剂，以及3)细胞色素C氧化酶I亚单位。此外，还将使用各种化学和光谱技术来1)充分表征血红素A合成酶的活性部位，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，从而提供了关于细胞色素C氧化酶组装的基本信息，并进一步深入了解了生物02的激活。

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.