PROTON & ELECTRON TRANSFER & ENERGY COUPLING IN SITE I

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DESCRIPTION: NADH-quinone oxidoreductase (Complex I) is the largest, most elaborate and least understood of the energy transducing enzymes. It has a uniquely high proton/electron stoichiometry (4-5 H+/2e-). Since Complex I encoding genes comprise more than half of the protein-encoding genes in mitochondrial DNA (7 out of 13), a majority of mitochondrial genetic diseases are associated with dysfunction of Complex I. Thus, the study of Complex I is of great medical importance. This research project places particular emphasis on the study of (I) the structure-function relationship in Complex I, combining state-of-the-art molecular genetic technology and cryogenic EPR spectroscopy, and (II) Complex I-related mitochondrial diseases. The P.I.'s group has shown that, contrary to a long-held view, Complex I consists of a large hydrophilic arm protruding out from the membrane (electron injector containing one flavin and several iron-sulfur clusters) and a hydrophobic part within the membrane (proton-pumping reactor containing three distinct quinone-binding sites). The P.I.'s group proposes a new quinone-gated proton-pump model for the energy coupling mechanism in Complex I. The following experimental strategies will be employed: (1) Determination of the spatial locations of cluster N2 and of quinone-binding sites; (2) further characterization of semiquinone-involving reactions using specific inhibitors, ionophores, and uncouplers; (3) identification of three different quinone-binding sites by isolation and characterization of inhibitor-resistant mutants: (4) targeting of the sequence motifs, which are in common with proton channel containing polypeptides by site-directed mutagenesis; (5) studies of Complex I-related diseases, such as Leber's Hereditary Optic Neuropathy and Parkinson's disease, using patient derived mitochondria propagated in cultured cells. The P.I. will collaborate on this project with a multi-disciplinary research team, consisting of renowned experts of molecular biology, mitochondrial disease, biochemistry and biophysics.