PROTON AND ELECTRON TRANSFER AND ENERGY COUPLING

真核细胞中的NADH-Q氧化还原酶(复合体I)是一种复杂的 由32个或更多个亚单位组成的系统，是主要的能量中心 细胞内的耦合作用。在上一次授权期内，下列项目 在络合物1的研究领域取得了实质性进展， 其中一些是由P.I.S小组完成的：(1)大部分 在牛心复合体I中已经确定了初级序列， 粗糙脉孢菌复合体I和副球囊藻属中较简单的对应种 反硝化细菌(只有14个簇状结构基因)。 比较研究为个体的功能角色提供了线索 多肽。(2)粗毛拟青霉复合体的独特大体结构I 由两个部分组成，每个部分都有氧化还原中心 部分基因已经通过基因分散技术进行了个别修改。 (3)两种不同类型的原核复合体I已部分 纯净的。它们是(I)脱氮假单胞菌和胶囊红杆菌，以及 像真核生物一样携带同源氧化还原系统的球状生物，但 仅由12-14个亚基组成，以及(Ii)嗜热嗜热菌和 携带非同源氧化还原中心的大肠杆菌。(4)宽阔的 复杂的I相关肌病和中枢神经疾病的频谱已经被 和特定的线粒体基因突变负责 这些疾病中的一些已经被发现。 基于这些新发现和我们之前的研究，我们计划采取 一种结合最新分子遗传学的新方法 技术和复杂的生物物理技术，如EPR、Endor、 和ESEEM。我们将重点做好以下两个方面的工作 研究：(I)阐明分子周围的分子结构 N-2簇和苯二酚结合位点(S)。我们计划使用 生物工程系统，如新开发的粗毛拟青霉突变体 仅承载站点I区域中的群集N-2。细菌系统 有一个独特的优势，即可以进行定点突变 在他们身上。使用这项技术，最小的结构要求 将研究站点I的能量耦合。(二)研究事故原因 线粒体相关疾病与识别复合体I AS的作用 它与这些疾病有关。 我们的多学科研究团队，由一个 生物物理学家-生物化学家(Ohnishi)、分子生物学家(Gennis和 Yagi)，具有很强疾病背景的分子生物学家(Weiss 和豪厄尔)，以及物理化学家(霍夫曼和佩萨克)，将合作 在这个项目上。

Eukaryotic NADH-quinone oxidoreductase (Complex I) is a complicated system consisting of 32 or more subunits and is a major center for energy coupling in the cell. During the preceding grant period, the following substantial progress has been made in the research field of Complex 1, some of which was done by the P.I.'s group: (1) A major portion of the primary sequence has been determined in the bovine heart Complex I, Neurospora crassa Complex I, and in the simpler counterpart in Paracoccus denitrificans (which has only 14 clustered structural genes). Comparative studies provided clues for the functional roles of individual polypeptides. (2) A unique gross structure of N. crassa Complex I consisting of two parts has been determined, and redox centers in each part have been *individually modified by the gene distraction technique. (3) Two different types of prokaryotic Complex I have been partially purified. They are (i) P. denitrificans and Rhodobacter capsulatus and spheroides which carry homologous redox systems as eukaryotes do, but consist of only 12-14 subunits, and (ii) Thermus themophilus and Escherichia coli which carry non-homologous redox centers. (4) A broad spectrum of Complex I-related myopathies and meuropathies have been discovered, and specific mitochondrial gene mutations responsible for some of these diseases have been identified. Based upon these new findings and our previous studies, we plan to take a novel approach by combining state-of-the-art molecular genetic technology and sophisticated biophysical techniques, such as EPR, ENDOR, and ESEEM. We will focus on the following two aspects of Site I research: (i) The elucidation of the molecular structure around the cluster N-2 and the quinone binding site(s). We plan to use bio-engineered systems, such as a newly developed N. crassa mutant which carries only the cluster N-2 in the Site I region. Bacterial systems have the unique advantage that site-directed mutagenesis can be performed on them. Using this technique, the minimal structural requirements for Site I energy coupling will be investigated. (ii) Studying the cause of mitochondria-related diseases and identifying the role of complex I as it relates to these diseases. Our multi-disciplinary research team, consisting of a biophysicist-biochemist (Ohnishi), molecular biologists (Gennis and Yagi), molecular biologists with strong backgrounds in diseases (Weiss and Howell), and physicochemists (Hoffman and Peisach), will collaborate on this project.