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. Available evidence suggests the existence of specific ubiquinone/ubiquinol binding proteins (sites) in succinate-Q reductase (SQR) and ubiquinol-cytochrome c reductase (QCR). The specific Q-binding proteins (domains) have been identified and characterized during past support periods. In the next grant period we will focus on the elucidation of detailed structure of quinone/quinol binding sites in SQR and QCR 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 in addition to crystallization and three dimensional analysis of QCR and SQR complexes.
The specific aims are as follows: (1) to continue studying the Q-binding proteins (QPs) in SQR with emphasis on cloning, sequencing and expression of two QPs subunits (cytochrome b560 and 13 kDa protein); (2) to continue elucidating the Q-binding sites in QPc-9.5 kDa and QPc-cytochrome b in QCR by site-directed mutagenesis, mass spectrometry, and immunoinhibition using antibodies against synthetic Q-peptides; (3) to determine the role of subunit VI (13.4 kDa) in QCR; (4) to determine the three dimensional structures of QCR and SQR by crystallization; (5) to synthesize ubiquinone derivatives labeled with fluorine or substituted with bulky substitutents in order to elucidate the protein: Q interaction; and (6) to establish the singlet oxygen scavenger role of Q. In addition, single crystal EPR studies of QCR, ENDOR investigation of QPs radicals, Resonance Raman spectroscopic characterization of QCR and identification of heme ligands of 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, the Q:protein interaction and the three dimensional structure of electron transfer complexes will provide information crucial to understanding the electron transfer mechanism and thus the energy conservation process.
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