The NADH-quinone oxidoreductase (complex I) is the largest (M.W. = approximately 1 mega-daltons) and most complicated (43 subunits) energy-transducing system in mitochondria. Many mitochondria-linked genetic diseases have been discovered, and the majority of them originate from a complex I defect(s). The elucidation of the structure-function relationship of complex I is vital not only for the study of bioenergetics, but also for the understanding of the nature of these diseases, in order to develop therapies. Based upon our previous findings, we will extend our studies in the following directions: (1) The NADH-binding site, one FMN molecule, and a majority of iron-sulfur clusters with low midpoint potential are localized in the hydrophilic promontory domain of complex I. In contrast, the iron-sulfur cluster N2 (which has the highest midpoint redox potential) and three distinct ubisemiquinone species are located within the membrane domain. We hypothesized that cluster N2 and these semiquinones play key roles in the proton and electron transfer in complex I. We found that cluster N2 resides in either of TYKY or PSST subunits. Both subunits are at least partially buried within the membrane. Determining the subunit location and ligand structure of cluster N2 has been one of the most important yet difficult tasks in complex I study. Recently, we have developed systems with much simpler bacterial complex I counterparts in which these two candidate subunits can be separated. Using these systems, we will identify which subunit harbors cluster N2. Furthermore, we will study the unique functions of cluster N2 employing site-directed mutagenesis techniques. (2) The subunit PSST (not TYKY) contains a specific and tight binding site for various complex I inhibitors. We have discovered that the distinct ubisemiquinone species respond differently to these inhibitors. Using vari9us inhibitors with different specificity, we will study the functional roles of both cluster N2 and the three quinone species in the energy-coupling mechanism in complex I. (3) We have found that the complex I counterpart in Thermus thermophilus has extreme thermo-stability and that its purified subunits are very stable. We will use this bacterium for crystallization and X-ray crystallographic studies. (4) We will determine physicochemical properties and spatial organization of all important redox components by combining state-of-the-art molecular genetic technology with sophisticated physical techniques such as EPR, ENDOR, ESEEM, and cyclic voltammetry as collaborative efforts. (5) We have developed an exciting bacterial model system, which allows us to study mechanisms of mitochondria-linked diseases by making clinically significant point mutations.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM030736-21
Application #
6628796
Study Section
Special Emphasis Panel (ZRG1-SSS-B (01))
Program Officer
Preusch, Peter C
Project Start
1983-02-01
Project End
2005-01-31
Budget Start
2003-02-01
Budget End
2004-01-31
Support Year
21
Fiscal Year
2003
Total Cost
$352,976
Indirect Cost
Name
University of Pennsylvania
Department
Biochemistry
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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