Complex I (NADH-ubiquinone oxidoreductase) serves as the main entry point for reoxidation NADH produced by the anaerobic phase of metabolism; it is the most complicated and least studied energy transducing device of the mitochondrial respiratory chain. The P.I.s propose to continue studying the molecular mechanism of electron/vectorial proton transfer in mammalian Complex I. The structure/function rearrangements involved in the """"""""active"""""""" (pulsed) - """"""""inactive"""""""" (resting) state transition of Complex I will also be studied. This work constitutes one of the physiologically most important research areas, since many diseases in highly energy-demanding tissues are associated with inherited or induced defects in Complex I. Specific new directions of the proposed work include: (1) studies on the substrate (pyridine nucleotide binding site(s), which will be conducted by measuring kinetic parameters of NADH dehydrogenation utilizing competitive NAD+ and NADH analogues. The dependence of the binding site affinities for catalytically inert nucleotides on the enzyme redox state(s) will also be investigated; (2) determination of the spatial organization of EPR-detectable redox components relative to each other and to the surfaces of the energy transducing membrane, with a special emphasis on the two distinct species of delta-mu(H+)-dependent, complex I-associated ubisemiquinones, which have recently been discovered in the P.I. and Co-P.I.'s collaborative endeavor; (3) identification of the polypeptide(s) involved in the slow inactive/active transition of Complex I utilizing specific labelling with fluorescent and spectrally- detectable SH-reagents; (4) development of a procedure for solubilization and purification of the enzyme stabilized in the active conformation by using specific inhibitors which bind exclusively or preferentially to active Complex I. The U.S. P.I. has extensive experience in cryogenic EPR (the only technique which is presently available for studies of Complex I redox components in situ) and in thermodynamic analysis of the respiratory chain components. The P.I. has made significant contributions in the analysis of iron-sulfur proteins, flavin, and semiquinone free radicals. The Co-P.I. has extensive experience in preparative biochemistry of redox enzymes and in enzyme kinetic analysis. The collaborative project between these investigators will greatly enhance the progress in understanding the molecular mechanisms of energy-transduction in Complex I and its physiologically relevant regulation.
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