Mechanism of Respiration and Energy Transduction

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The cytochrome bc1 complex is an energy-transducing enzyme that participates in cell respiration in oxygen utilizing eukaryotic cells and is located in the inner mitochondrial membrane. A similar bc1 complex is located in the plasma membrane of many bacteria, where it takes part in respiration, denitrification, nitrogen fixation, and cyclic photosynthetic electron transfer, depending on the species. In all of these organisms the bc1 complex oxidizes a membrane-localized quinol and reduces a water-soluble, c-type cytochrome and links this electron transfer reaction to translocation of protons across the membrane in which the bc1 complex resides. The bc1 complexes from mitochondria of several species have been crystallized and the mechanism of the enzyme, the protonmotive Q cycle, is partly understood. The long term objectives of this research are to understand the mechanism of the cytochrome bc1 complex and to elucidate the pathway by which the subunits of this oligomeric enzyme complex are assembled into the inner mitochondrial membrane. The specific aims of the current research project are to answer the following questions regarding the mechanism. - Is ubiquinol oxidation a concerted or sequential reaction? - What is the structural basis of negative cooperativity in the binding of ubiquinol? - How are protons conducted to and from the ubiquinone and ubiquinol reaction sites? - How does ubisemiquinone stability affect the rate of ubiquinone reduction? We plan to investigate these questions in the cytochrome bc1 complex of the yeast Saccharomyces cerevisiae, since this enzyme can be modified by molecular genetics methods and isolated in quantities sufficient for biochemical and biophysical characterization.