Physiological Modulators of Cytochrome Oxidase Function

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DESCRIPTION (provided by applicant): Cytochrome c oxidase (CytOX) is the terminal oxidase of the mitochondrial respiratory chain. Altered CytOX structure and activity are associated with a wide spectrum of degenerative diseases, and myocardial injury. Preliminary results show that CytOX activity in hypoxia and ischemia/reperfusion is markedly inhibited through PKA mediated phosphorylation of subunits IV, and Vb and to a lesser extent subunit I. An exciting observation is that hypoxia mediated changes in cells and ischemia mediated myocardial injury can be substantially reversed by pre-treating cells, and or, heart with PKA specific inhibitor, H89. Based on this, the renewal application is focused to test the hypothesis that cAMP mediated hyperphosphorylation of CytOX alters the enzyme activity and exerts deleterious effects on cells/tissues due to overproduction of ROS. It is proposed to use a combination of biochemical, cell biological, and transgenic approaches to test the hypothesis as follows: 1). CytOX from murine macrophage cell line subjected to hypoxia, and rabbit heart subjected to experimental ischemia/reperfusion, will be characterized with respect to kinetic parameters (Km and TN), proton pumping, and ROS production in a proteoliposome system, and the effects of PKA inhibitors in reversing these changes will be studied. The sites of phosphorylation of subunits I, IV and Vb will be mapped by a combination of fingerprint analysis and tandem MS-MS analysis of peptides. 2). The effects of conditional depletion of CytOX IV and Vb subunits and replacement with phosphorylation site mutated subunits in macrophage cells on hypoxia induced injury will be studied. 3). Finally, the role of nuclear subunits in CytOX assembly and function will be further studied by generating mouse lines with phosphorylation site mutated CytOX IV and Vb genes. The overall goal is to determine the molecular basis of H89 mediated protection against ischemic injury to the myocardium.