PROTON AND ELECTRON TRANSFER AND ENERGY COUPLING

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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.