PROTON AND ELECTRON TRANSFER AND ENERGY COUPLING

真核生物NADH-醌氧化还原酶（复合物I）是一种复杂的 由32个或更多的亚单位组成的系统，是能量的主要中心 在细胞中耦合。 在上一个资助期内， 在复合物1的研究领域已经取得了实质性进展， 有些是私家侦探做的（1）大部分的 已经在牛心脏复合物I中确定了一级序列， 粗糙脉孢菌复合体I和副球菌中的简单对应物 Escherichia coli（只有14个结构基因）。 比较研究为个体的功能角色提供了线索 多肽。(2)N. crassa复合物I 由两部分组成，并且每个部分中的氧化还原中心 部分已经通过基因转移技术单独修改。 (3)两种不同类型的原核复合体I已经部分地 提纯 它们是（i）P.acetificans和Rhodobacter capsulatus， 球状体携带同源的氧化还原系统，如真核生物所做的，但 仅由12 - 14个亚基组成，和（ii）嗜热栖热菌和 携带非同源氧化还原中心的大肠杆菌。(4)广泛 复合体I相关的肌病和神经病的谱已经被 发现，和特定的线粒体基因突变负责 其中一些疾病已经得到确认。 基于这些新的发现和我们以前的研究，我们计划采取 一种结合最先进的分子遗传学 技术和先进的生物物理技术，如EPR，ENDOR， 和ESEEM。 我们将重点关注以下两个方面 研究：（一）阐明了分子结构周围的 簇N-2和醌结合位点。 我们计划使用 生物工程系统，如新开发的N. crassa突变体， 在站点I区域中仅携带簇N-2。 细菌系统 具有独特的优点， 在他们身上。 使用这种技术， 将对现场I的能量耦合进行研究。(ii)研究的原因 相关疾病和确定复合物I的作用， 与这些疾病有关。 我们的多学科研究团队，包括 生物化学家-生物化学家（Ohnishi），分子生物学家（Gennis和 Yagi），在疾病方面有着深厚背景的分子生物学家（韦斯 和豪厄尔），以及物理化学家（霍夫曼和佩萨赫），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.