The ultimate goal of the project is to elucidate the structure- function relationship of the Site I and Site III energy coupling. We have three major long term objectives, namely, (A) To test the hypothesis that the mechanism of electron transfer in the Site I is of a cyclic type. This hypothesis enables us to explain (a) the extreme complexity of redox components and protein subunit structure, (b) a high (four to five) stoichiometry of H+/2e- (c) the extremely low midpotential of the cluster N-la. For this goal, we will do the following: (i) study the physico- chemical properties of the H+-carriers, (ii) investigate new inhibitors which are different from conventional rotenone-type compounds, (iii) measure the spatial organization of the redox components in the membrane using continuous wave and pulsed EPR techniques at cryogenic temperature, and (iv) study simpler bacterial systems, such as Paracoccus denitrificans and Thermus thermophilus HB-8. (B) To apply our experience in the study of Site I to clinical problems. Certain diseases are caused by a genetic defect in the iron sulfur proteins in the Site I. We have demonstrated that this defect can be clearly identified by EPR measurement on pathological specimens. We will continue this line of study. (C) To elucidate the structure function relationship of Site III respiration. Since we pioneered the measurement of spin-spin interaction between cyt.a3-NO and other paramagnetic species in bovine heart cytochrome oxidase, we will complete the study by conducting the following experiments: (i) definitive measurement of inter-cluster distance in bovine heart cytochrome oxidase, (ii) direct measurement of T1 and T2 by pulsed EPR with careful resolution of redox poise of cyt.a and CuA, (iii) use of yeast strain whose cyt. c oxidase protein and chromophore structure are perturbed by double mutation. Taking full advantage of studying microorganism, we will study spectral and thermodynamic characteristics of individual redox components and analysis of the redox and spin-spin interactions among redox centers. These studies are expected to open a new horizon in our understanding of the electron and proton transfer process in Site I and III on the molecular level.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM030736-10
Application #
3278582
Study Section
Physical Biochemistry Study Section (PB)
Project Start
1983-02-01
Project End
1993-01-31
Budget Start
1992-02-01
Budget End
1993-01-31
Support Year
10
Fiscal Year
1992
Total Cost
Indirect Cost
Name
University of Pennsylvania
Department
Type
Schools of Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Sinha, Prem Kumar; Nakamaru-Ogiso, Eiko; Torres-Bacete, Jesus et al. (2012) Electron transfer in subunit NuoI (TYKY) of Escherichia coli NADH:quinone oxidoreductase (NDH-1). J Biol Chem 287:17363-73
Ohnishi, Tomoko; Nakamaru-Ogiso, Eiko; Ohnishi, S Tsuyoshi (2010) A new hypothesis on the simultaneous direct and indirect proton pump mechanisms in NADH-quinone oxidoreductase (complex I). FEBS Lett 584:4131-7
Nakamaru-Ogiso, Eiko; Kao, Mou-Chieh; Chen, Han et al. (2010) The membrane subunit NuoL(ND5) is involved in the indirect proton pumping mechanism of Escherichia coli complex I. J Biol Chem 285:39070-8
Ohnishi, S Tsuyoshi; Ohnishi, Tomoko (2010) An adder-mixer for adding a few microliters of reagent into an electron paramagnetic resonance quartz tube. Anal Biochem 406:89-90
Ohnishi, S Tsuyoshi; Salerno, John C; Ohnishi, Tomoko (2010) Possible roles of two quinone molecules in direct and indirect proton pumps of bovine heart NADH-quinone oxidoreductase (complex I). Biochim Biophys Acta 1797:1891-3
Nakamaru-Ogiso, Eiko; Han, Hongna; Matsuno-Yagi, Akemi et al. (2010) The ND2 subunit is labeled by a photoaffinity analogue of asimicin, a potent complex I inhibitor. FEBS Lett 584:883-8
Ohnishi, S Tsuyoshi; Shinzawa-Itoh, Kyoko; Ohta, Kazuhiro et al. (2010) New insights into the superoxide generation sites in bovine heart NADH-ubiquinone oxidoreductase (Complex I): the significance of protein-associated ubiquinone and the dynamic shifting of generation sites between semiflavin and semiquinone radicals. Biochim Biophys Acta 1797:1901-9
Fato, Romana; Bergamini, Christian; Bortolus, Marco et al. (2009) Differential effects of mitochondrial Complex I inhibitors on production of reactive oxygen species. Biochim Biophys Acta 1787:384-92
Ohnishi, Tomoko; Nakamaru-Ogiso, Eiko (2008) Were there any ""misassignments"" among iron-sulfur clusters N4, N5 and N6b in NADH-quinone oxidoreductase (complex I)? Biochim Biophys Acta 1777:703-10
Nakamaru-Ogiso, Eiko; Matsuno-Yagi, Akemi; Yoshikawa, Shinya et al. (2008) Iron-sulfur cluster N5 is coordinated by an HXXXCXXCXXXXXC motif in the NuoG subunit of Escherichia coli NADH:quinone oxidoreductase (complex I). J Biol Chem 283:25979-87

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