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.
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