The goal is to understand the essential hydrophobic and subunit interactions with the mitochondrial electron transport system that enable it to couple electron transport to ATP synthesis. Four specific projects will probe functionally important interactions within cytochrome c oxidase, which is the terminal electron transport complexes in the inner mitochondiral membrane. The first objective will be to determine the phospholipid specificity and the functionally important high affinity lipid binding sites on cytochrome c oxidase. The approaches to be used include measurement of functional binding of synthetic derivatives of cardiolipin that contain defined abnormal fatty acid tails, and photoaffinity labeling the cardiolipin binding sites with cardiolipin analogs. The second objective will be to determine the involvement of the tightly bound cardiolipin in dimerization of cytochrome c oxidase. The techniques to be used will utilize high speed sedimentation velocity to measure the aggregation state of the protein as a function of bound cardiolipin. The third goal is to structurally locate the redox centers within the protein relative to the membrane interior and one of the protein subunits, subunit III. The approach will be measurement of intrinsic protein fluorescence. The last goal will be a direct investigation of the structure of one of the subunits of cytochrome c oxidase, subunit III. The approaches in this project utilize detergent binding methods to measure surface hydrophobicity and hydrodynamic measurements to determine overall size and shape of the purified subunit. Together, these four approaches will probe the importance of specific lipids and subunits in the structure and function of this inner mitochondiral membrane protein.
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