The overall objective of this proposal is to determine the molecular basis of energyconserving electron and proton transfer in mitochondria. This complex, membraneassociated process presents unique problems for investigation because of the large, multisubunit proteins involved and their diverse array of peptides and prosthetic groups. Cytochrome c oxidase is by far the best characterized of the three major complexes, in terms of sequence and sturctural information, but the mechanism by which it transforms energy is still not understood, nor are many other aspects of its structure, function and control. Considerable controversy surrounds several fundamental issues, including: the number and role of required subunits; the involvement of lipid; the role of aggregation state; the number and nature of substrate interactions; and the mechanism of respiratory control. In this proposal we will address these issues using a variety of approaches.
The specific aims are: 1) to determine the role of aggregation state by separating monomer and dimer forms by FPLC, selecting for monoclonal antibodies to the monomer, and identifying the molecular form in reconstituted vesicles by electron microscopy; 2) to clarify the preparations, with and without subunit III, with respect to kinetic, spectral and physical properties and their proton pumping and respiratory control, capacities; 3) to examine several proposed mechanism of proton translocation, and the roles of nuclearcoded subunits, by utilizing the unusual spectral and structural features of plant cytochrome oxidase; 4) to investigate the role of cardiolipin using FPLC separation of lipidcontaining and depleted forms, and selection of cardiolipinspecific monoclonal antibodies; 5) to define the nature and functionality of cytochrome c binding sites by investigating the effects of high affinity monoclonal antibodies to subunit II on cytochrome c bining and kinetics; and 6) to analyze the properties of subunits I and II, by developing a system to permit expression of the cloned mitochondrial genes that have been converted to 'nuclear' forms. We expect that these studies will provide new insight into the mechanism of energy transduction in cytochrome oxidase, resulting in a more comprehensive understanding of the process and its regulation in the whole mitochondrial respiratory chain.
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