ORGANIZATION AND CONTROL OF ELECTRON TRANSFER CHAINS

电子转移与质子转移的耦合是基础 好氧生物的能量守恒机制。这项建议是 旨在阐明这一过程的分子基础。三大体系 将用于解决问题的不同方面。细菌， 球形红杆菌被认为是进化最近的细菌之一 真核生物线粒体的近亲。它产生一种细胞色素aa3型 一种在结构和功能上与哺乳动物同源的氧化酶 酵素。我们已经分离并鉴定了该酶，并克隆了 对其基因进行了测序。在协作工作中使用站点定向 突变技术，我们已经定义了四个中的三个的配体 金属中心，并制作了一个三维结构模型 催化点。这项建议的第一个目的是澄清 通过突变分析，筛选质子转运机制 在高度保守的基础上改变残留物， 它们与金属中心的预测关系，并提供了模型 细菌视紫红质。氧化物酶结构基础的其他方面 将解决的功能包括：CUA的连接，结合 细胞色素c的结构域，亚基I和II之间的相互作用，以及 双核金属中心的蛋白质环境。 真核细胞色素c氧化酶在结构上比 细菌酶，含有四到十个核编码肽。 核肽不仅在不同的生物体中存在差异，而且在 不同的组织。它们的功能意义是一个尚未解决的重大问题 问题。植物细胞色素c氧化酶具有独特的亚基组成。 我们有证据表明这是一种发展形式。我们建议对此进行探索 通过开发一种可能的发育肽抗体来实现的可能性 在小麦胚芽氧化酶中观察，并筛选其存在于 不同的成长阶段。核亚基的序列信息 也将获得这些多肽与其他多肽的关联 有机体。 我们的最新结果和其他调查人员的结果表明 线粒体基因向其通用密码子等价物的转化 可能是一种允许基因工具应用的有效方法 对于复杂的哺乳动物酶。我们建议进一步探讨这一点。 通过研究异源表达系统来指导 我们已经在卵母细胞中表达了COX II的哺乳动物基因 在试管中。球形红杆菌和酿酒酵母将成为 初步测试。 这些研究的结果预计将产生大量新的 关于电子和能量的机制和调节的信息 细胞色素c氧化酶的转移。

The coupling of electron transfer to proton translocation is the basic mechanism of energy conservation in aerobic organisms. This proposal is aimed at elucidating the molecular basis of this process. Three systems will be used to address different aspects of the problem. The bacterium, Rhodobacter sphaeroides, is considered one of the closest evolutionary relatives of eukaryotic mitochondria. It produces a cytochrome aa3-type oxidase that is structurally and functionally homologous to the mammalian enzyme. We have isolated and characterized the oxidase and cloned and sequenced its genes. In a collaborative effort using site-directed mutagenesis techniques, we have defined the ligands of three of the four metal centers and produced a model of the three dimensional structure of the catalytic site. The first aim of this proposal is to elucidate the mechanism of proton translocation by mutational analysis, selecting residues to be altered on the basis of a high degree of conservation, their predicted relationship to the metal centers, and the model provided by bacteriorhodopsin. Other aspects of the structural basis of oxidase function that will be addressed include: the ligation of CuA, the binding domain for cytochrome c, the interaction between subunits I and II, and the protein environment of the binuclear metal center. Eukaryotic cytochrome c oxidases are more complex in structure than bacterial enzymes, containing from four to ten nuclear encoded peptides. The nuclear peptides differ not only among various organisms but also in different tissues. Their functional significance is a major unsolved question. The plant cytochrome c oxidase has a unique subunit composition and we have evidence of a development form. We propose to explore this possibility by developing antibodies to a putative developmental peptide observed in wheat germ oxidase and to screen for its presence at different stages of growth. Sequence information on the nuclear subunits will also be obtained to relate these peptides to those of other organisms. Our recent results and those of other investigators indicate that conversion of mitochondrial genes into their universal codon equivalents may be an effective approach to allowing the application of genetic tools to the complex mammalian enzymes. We propose to further explore this direction by investigating heterologous expression systems for the mammalian gene for COX II which we have already expressed in oocytes and in vitro. Rhodobacter sphaeroides and Saccharomyces cereviseae will be tested initially. The results of these studies are expected to yield substantial new information regarding the mechanism and regulation of electron and energy transfer in cytochrome c oxidase.