EXCEED THE SPACE PROVIDED. Cytochrome c oxidase is the key enzyme in energy transduction in aerobic organisms. As the terminal electron sink in the respiratory chain of microbes, plants and animals, it is one of the more complex of the energy conversion enzymes in terms of subunit structure and metal content. A number of crystal structures have been obtained, but the mechanism of energy conversion remains unclear. The research proposed extends a long term effort to dissect the structural and kinetic properties of cytochrome oxidase using the enzyme from Rhodobacter sphaeroides as a model system that is highly homologous to the mammalian enzyme and accessible by mutagenic, spectral, kinetic, computational and crystallographic techniques. In the past several years we have made progress on refining methods of purification and crystallization, emphasizing the importance of lipid in maintaining the native structure. To this end we have developed sensitive mass spectrometry techniques for assessing molecular homogeneity and lipid content. These methods will be applied, along with spectral and kinetic measurements of proton and electron transfer, to study native and mutant forms of cytochrome c oxidase. Our major emphasis is on: 1) Producing high resolution crystal structures of different forms of the enzyme in intermediate catalytic states; 2) Establishing sensitive rapid kinetic techniques for proton and membrane potential measurements inside and outside reconstituted oxidase vesicles, to determine the structural basis of proton pumping and back leak in energy generation and regulation; 3) Exploring how protein structure affects electron transfer into and within the oxidase, by kinetic, computational and crystallographic approaches. We expect that understanding the mechanism and coupling of electron and proton translocation will lead to new insight into how rate and efficiency is controlled and how the process may lose (or gain) efficiency in disease, obesity and aging. PERFORMANCE SITE ========================================Section End===========================================
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