ORGANIZATION AND CONTROL OF ELECTRON TRANSFER CHAINS

这项提案的总体目标是确定 电子能量守恒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 提出了独特的调查问题，因为 涉及的多亚基蛋白质及其多肽阵列 和假肢小组。细胞色素c氧化酶是目前为止最好的。 以三大复合体为特征的，在序列方面 和结构信息，但它所依据的机制 转换能量仍然不被理解，许多其他的能量也不被理解 它的结构、功能和控制方面。相当可观 围绕几个基本问题存在争议，包括： 所需亚基的数量和作用；脂质的参与； 聚集态的作用；底物的数量和性质 相互作用；以及呼吸控制的机制。在这 我们将使用各种方案来解决这些问题 接近了。具体目标是：1)确定 通过分离单体和二聚体形成的聚集态 FPLC，选择针对该单体的单抗，以及 重组囊泡中分子形态的鉴定 电子显微镜；2)澄清准备，与和 不含亚基III，就动力学、光谱和物理而言 性质及其质子泵和呼吸控制， 能力；3)研究提出的几种质子机制 转位，以及核编码亚基的作用，通过利用 植物细胞色素的特殊光谱和结构特征 4)用FPLC研究心磷脂的作用 含脂和耗脂形式的分离和选择 心磷脂特异的单抗；5)确定 细胞色素c结合位点的性质和功能 探讨高亲和力单抗对小鼠免疫功能的影响 亚基II对细胞色素结合及动力学的影响；6)分析 亚基I和亚基II的性质，通过开发一个系统来 允许表达已克隆的线粒体基因 已被转换为“核”形式。我们预计这些研究 将提供对能量机制的新见解 细胞色素氧化酶的转导，导致更多 全面了解这一过程及其在 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.