MECHANISM OF RESPIRATION AND ENERGY TRANSDUCTION

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The objective of this project is to elucidate the mechanism of electron transfer and proton translocation in the cytochrome bc1 complex of the mitochondrial respiratory chain. Available evidence indicates that electron transfer through the cytochrome bc1 complex is by a protonmotive Q cycle mechanism. We are particularily interested in defining the two ubiquinol redox sites ("center o and center i" in Q cycle terminology), and in establishing the functions of non-redox proteins of the bc1 complex. We are using molecular genetics and membrane biochemistry to address these questions in saccharomyces cerevisiae. During the current project period our specific aims include locating a putative ubiquinol oxidation site on the Rieske iron-sulfur cluster. In addition, we plan to identify the regions of the Rieske iron-sulfur protein which, together with protein domains from cytochrome b, form the ubiquinol oxidizing site at center o. To identify polypeptides possibly contributing to the ubiquinone reducing site at center i, we plan to examine the roles of the 14 kDa subunit 7 and the 11 kDa subunit 8 in stabilizing ubisemiquinone. We also plan to identify the interfaces of the 14 kDa and 11 kDa subunits which interact with cytochrome b We will test whether the 7.3 kDa subunit 9 and the acidic 17 kDa subunit 6 may modulate the activity or assembly of the complex in some currently unrecognized manner. These two "supernumerary" polypeptides, which appear to be unique to eukaryotic cytochrome bc1 complexes, have no obvious obligatory function in the electron transfer or proton translocation activities of the mitochondrial bc1 complex. The possible functions of these proteins will be tested in yeast strains from which the genes for these proteins have been deleted, or in which the wild type genes have been replaced by mutants generated in vitro.