The goal of this project is to characterize the mechanisms by which cardiolipin (CL) binding affects the structure and function of two mitochondrial electron transport complexes: cytochrome C oxidase (COX) and cytochrome bc1 (Cyt. bc1). The specific goals during the project period are: Functional and Structural Association of Cardiolipin with Cytochrome C Oxidase 1. Identify amino acid sequences within COX subunits VIa, VIb, VIla and VIc that bind cardiolipin. 2. Determine the effect of CL removal upon COX conformational flexibility and/or binuclear center reactivity 3. Establish whether CL is Directly Involved in Subunit VIa & VIb Binding to COX 4. Determine the mechanism by which CL removal inhibits proton translocation by COX Functional and Structural Association of Cardiolipin with Cytochrome bci 1. Identify subunit(s) and amino acid sequence(s) of cyt. bc1 that participate in CL binding 2. Measure affinity and stoichiometry of cardiolipin binding to cytochrome bc1 These specific aims are each a logical extension of our previous studies. They also take advantage of new experimental approaches developed during the past funding period, i.e. 1) synthesis of CL derivatives that contain a variety of chemically reactive functional groups; 2) new sensitive HPLC and ESI/MS methods for analyzing subunit content and molecular masses of each subunit; 3) new methods for accurately measuring the self-association of each complex. With our unique CL analogues and approaches, we can map the CL binding sites within each complex and determine mechanisms by which tightly bound CL influences enzyme function and/or structure. Results obtained with these two complexes will allow us to determine whether CL has unique or universal roles in coupled electron transport.
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