MECHANISM OF RESPIRATION AND ENERGY TRANSDUCTION

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The cytochrome bc1 complex is an oligomeric membrane enzyme which participates in energy transduction linked to respiration in eukaryotic cells, and similarly participates in respiration, photosynthetic electron transfer, denitrification, nitrogen fixation, and sulfur metabolism in bacteria. Understanding the molecular basis of how the cytochrome bc1 complex performs this energy transduction is essential to understanding cellular energetics. The objectives of this research are to understand how the subunits of the cytochrome bc1 complex participate in the energy transduction activities of the complex, and to elucidate the pathway by which this oligomeric enzyme is assembled into the inner mitochondrial membrane. The experimentation is based on the rationale that the cytochrome bc1 complex operates by a protonmotive Q cycle mechanism, and that the mitochondrial forms of this enzyme contain multiple supernumerary subunits which have no obvious function in that mechanism. The experimentation is intended to answer three questions. 1. How does the iron-sulfur protein subunit of the bc1 complex catalyze the divergent oxidation of ubiquinol which is required in the Q cycle mechanism? 2. What are the functions of the supernumerary subunits in the mitochondrial bc1 complexes? 3. What is the pathway or sequence of events by which the 10 or 11 subunits of this enzyme complex are assembled into the inner mitochondrial membrane? We are using genetics and biochemical approaches to elucidate the answers to these questions in Saccharomyces cerevisiae. The approaches include isolation and manipulation of genes for individual subunits of the bc1 complex, characterization of electron transfer reactions within the complex following selected modifications to subunits, identification of intermediate steps in the assembly of the complex, and purification and characterization of partially assembled subcomplexes and possible factors involved in assembly of the complex.