Cytochrome oxidase, the terminal oxidase of the mitochondrial electron transport chain is a major site for the regulation of mitochondrial oxidative function. The enzyme complex from higher vertebrates contain 3 mitochondrial- and 10 nuclear-encoded subunits. With the long term objective of understanding the molecular basis of interaction between the nuclear and mitochondrial genes, we have characterized nuclear encoded murine COX IV and Vb genes with respect to basal transcription promoter elements. Preliminary results indicate that a novel negative enhancer region is involved in the vastly varying levels (over 10 fold) of expression of COX Vb in different tissue. The results also show a correlation between the subunit Vb content and catalytic activities of COX from liver, kidney and heart suggesting a possible role for this subunit in modulating enzyme activity or assembly. The objective of this grant is to determine the role of nuclear genes in cytochrome oxidase function; how the nuclear and mitochondrial COX genes respond to the oxidative demand of cells and how their regulation is altered during pathological conditions like hypoxia, ischemia, and cardiomyopathies. It is proposed to carry out the following specific experiments: 1) Further characterization of promoter/enhancer elements of COX IV, and Vb genes and the muscle specific COX VIII gene to determine the roles of various positive acting elements like the E-box, CArG and GTG motifs. The role of the negative regulatory region of COX Vb gene in determining its low expression in liver, and high expression in muscle and kidney will be investigated using in vivo and in vitro transcription and protein binding studies. Similarly, the mechanisms of induced expression of COX Vb and COX VIII genes during myogenesis will be investigated using skeletal and also cardiac muscle cells 2) The role of varying subunit Vb and VIII levels in the liver, heart and kidney enzymes will be studied using biochemical and immunochemical approaches. These studies will include different compartments of the heart tissue, and also an ischemic and a myopathic heart models. The role of nuclear encoded subunits in enzyme function will be investigated by negative dominant expression of different subunits in cultured hepatic and muscle cells also by a targeted expression in the transgenic mouse system. 3) The effects of physiological regulators such as inhibitors of heme biosynthesis, reversal by hemin, hypoxia, ischemia, etc. on COX Vb and VIII mRNAs will be studied. Finally, mechanisms of heme/O2 dependent regulation of the nuclear encoded Vb and also mitochondrial genes will be investigated to identify the putative cis DNA elements and trans acting factors involved in transcription regulation.
