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The effects of physiologically relevant hypoxia on the catalytic activity of cytochrome c oxidase (CytOX), mitochondrial gene expression, and both nuclear and mitochondrial encoded CytOX mRNA levels were investigated in murine monocyte macrophages, mouse C2C12 skeletal myocytes and rat adrenal pheochromocytoma PC12 cells. Our results suggest a coordinated down regulation of mitochondrial genome-coded CytOX I and II and nuclear genome-coded CytOX IV and Vb mRNAs during hypoxia. Hypoxia also caused a severe decrease in mitochondrial transcription rates, and associated decrease in mitochondrial transcription factor A. The enzyme from hypoxia exposed cells exhibited altered subunit content as revealed by blue native gel electrophoresis. There was a generalized decline in mitochondrial function that led to a decrease in total cellular heme and ATP pools. We also observed a decrease in mitochondrial heme aa3 content and decreased levels of CytOX subunit I, IV and Vb, though the catalytic efficiency of the enzyme (TN for cytochrome c oxidase) remained nearly the same. Increased glycolytic flux and alterations in the kinetic characteristics of the CytOX might be the two mechanisms by which hypoxic cells maintain adequate ATP levels to sustain life processes. Reoxygenation nearly completely reversed hypoxia-mediated changes in CytOX mRNA contents, rate of mitochondrial transcription, and the catalytic activity of CytOX enzyme. Our results show adaptive changes in CytOX structure and activity during physiological hypoxia.

The in vivo effects of heme biosynthesis inhibitors, succinylacetone and CoCl2 on the cytochrome c oxidase (COX) gene expression and enzyme activity in different mouse tissues were investigated. Succinylacetone and CoCl2 showed tissue-specific differences in their ability to modulate heme aa3 content. A single dose of succinylacetone treatment for 8 h reduced the heme aa3 content of kidney mitochondria with no effect on the liver. CoCl2 treatment for 8 h, however, selectively affected the heme aa3 level in the liver. Reduced mitochondrial heme aa3 with both treatments was accompanied by approximately 50% reduced, mitochondrial genome-encoded COX I and II mRNAs and nuclear genome-encoded COX Vb mRNAs, but no change in COX IV mRNA level. Use of isolated mouse liver and brain mitochondrial systems showed a 50-80% reduction in mitochondrial transcription and translation rates in heme-depleted tissues. Blue native gel electrophoresis followed by immunoblot analysis showed that the complex from heme-depleted tissues contained a 30-50% reduction in levels of subunits I, IV, Vb and near normal levels of subunit VIc, indicating altered subunit content. Treatment of submitochondrial particles with protein kinase A and ATP resulted in partial dissociation of COX, suggesting a mechanistic basis for the reduced subunit content of the complex from heme-depleted tissues. Surprisingly, the enzyme from heme-depleted tissues showed twofold to fourfold higher turnover rates for cytochrome c oxidation, suggesting alterations in the kinetic characteristics of the enzyme following heme reduction. This is probably the first evidence that the tissue heme level regulates not only the mammalian COX gene expression, but also the catalytic activity of the enzyme, probably by affecting its stability.