The objective of this renewal application is to continue the elucidation of the structure/function, and mechanisms of the ubiquinone (Q)- mediated electron transfer complexes of the mitochondrial respiratory chain. During the past support periods, the specific Q- binding proteins (domains) in succinate-Q-reductase (SQR) and ubiquinol- cytochrome c reductase (QCR) have been identified and characterized; several ubiquinone/ubiquinol binding proteins (subunits) in these complexes have been cloned and expressed; crystals of QCR suitable for structural analysis by X-ray diffraction have been obtained. In the next grant period the applicant will focus on the elucidation of the detailed structure of quinone/quinol binding sites in SQR and QCR complexes using multiple approaches, including protein/peptide chemistry, various spectroscopic measurements, organic synthesis of Q- derivatives, isolation and reconstitution, gene cloning, sequencing, expression and site directed mutagenesis, as well as three-dimensional analysis of the QCR complex by protein crystallography.
The specific aims are as follows: (a) to study Q-binding and succinate dehydrogenase- docking sites in QPs of SQR by chemical and molecular biological methods; (b) to elucidate Q-binding sites in QPc-9.5 kDa and in cytochrome b of QCR; (c) to determine the three-dimensional structure of QCR by X-ray diffraction analysis; (d) to elucidate protein:Q interactions in QCR an SCR using synthetic 19F and 13C labeled Q- derivatives. In addition, high field NMR studies of the three- dimensional structure of Q in protein-bound and free forms using 19F and 13C-Q derivatives, ENDOR investigation of QPs and QPC radicals, Resonance Raman spectroscopic characterization of QCR, and identification of heme ligands of native and mutated cytochrome b560 by IR-MCD will be carried out through collaboration with experts in these fields. The increasing acceptance of the chemiosmotic energy coupling hypothesis has given Q a central role in bioenergetics. Successful elucidation of the Q- binding site, Q:protein interactions in QCR and SQR, and three- dimensional structure of QCR will provide information crucial to understanding the electron transfer mechanisms and thus the energy conservation process. Also, since a quinone reactive sites are believed to be a significant source of superoxides and free Q can act as a scavenger for singlet oxygen, detailed knowledge of the structure/function relationships and reaction mechanisms of Q-mediated electron transfer should provide valuable information for understanding the mechanism of superoxide formation. This information will be useful in pharmaceutical investigations of cytotoxicity and the aging process.
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