ORGANIZATION AND CONTROL OF ELECTRON TRANSFER CHAINS

本提案的总体目标是确定 能量守恒电子和质子转移的分子基础 在线粒体中。 这个复杂的，膜相关的过程 由于大的， 涉及的多亚基蛋白质及其不同的肽阵列 和假体组。 细胞色素c氧化酶是目前最好的 以三个主要的复合体为特征， 和结构信息，但它的机制， 能量转换仍然不为人所知，也没有许多其他的 它的结构、功能和控制。 相当大 争议围绕着几个基本问题，包括： 所需亚单位的数量和作用;脂质的参与; 聚集态的作用;底物的数量和性质 相互作用;和呼吸控制的机制。 在这 我们将通过各种方式解决这些问题， 接近。 具体目标是：1）确定 通过分离单体和二聚体形式， FPLC，选择单体的单克隆抗体，和 通过以下方式鉴定重构囊泡中的分子形式： 电子显微镜; 2）澄清制剂， 不含亚基III，在动力学、光谱和物理方面 性质及其质子泵送和呼吸控制， 能力; 3）检查几个建议的机制，质子 易位，和核编码亚基的作用，通过利用 植物细胞色素特殊光谱和结构特征 用FPLC研究心磷脂的作用 分离含脂和贫脂形式， 心磷脂特异性单克隆抗体; 5）定义 细胞色素c结合位点的性质和功能 研究高亲和力单克隆抗体对 亚基II对细胞色素c比宁和动力学的影响;以及6）分析 亚基I和II的性质，通过开发一个系统， 允许克隆的线粒体基因的表达， 变成了“核”的形式。 我们希望这些研究 将提供新的洞察力的机制，能源 转导细胞色素氧化酶，导致更多的 全面了解过程及其调节， 整个线粒体呼吸链

The overall objective of this proposal is to determine the molecular basis of energyconserving electron and proton transfer in mitochondria. This complex, membraneassociated process presents unique problems for investigation because of the large, multisubunit proteins involved and their diverse array of peptides and prosthetic groups. Cytochrome c oxidase is by far the best characterized of the three major complexes, in terms of sequence and sturctural information, but the mechanism by which it transforms energy is still not understood, nor are many other aspects of its structure, function and control. Considerable controversy surrounds several fundamental issues, including: the number and role of required subunits; the involvement of lipid; the role of aggregation state; the number and nature of substrate interactions; and the mechanism of respiratory control. In this proposal we will address these issues using a variety of approaches. The specific aims are: 1) to determine the role of aggregation state by separating monomer and dimer forms by FPLC, selecting for monoclonal antibodies to the monomer, and identifying the molecular form in reconstituted vesicles by electron microscopy; 2) to clarify the preparations, with and without subunit III, with respect to kinetic, spectral and physical properties and their proton pumping and respiratory control, capacities; 3) to examine several proposed mechanism of proton translocation, and the roles of nuclearcoded subunits, by utilizing the unusual spectral and structural features of plant cytochrome oxidase; 4) to investigate the role of cardiolipin using FPLC separation of lipidcontaining and depleted forms, and selection of cardiolipinspecific monoclonal antibodies; 5) to define the nature and functionality of cytochrome c binding sites by investigating the effects of high affinity monoclonal antibodies to subunit II on cytochrome c bining and kinetics; and 6) to analyze the properties of subunits I and II, by developing a system to permit expression of the cloned mitochondrial genes that have been converted to 'nuclear' forms. We expect that these studies will provide new insight into the mechanism of energy transduction in cytochrome oxidase, resulting in a more comprehensive understanding of the process and its regulation in the whole mitochondrial respiratory chain.