Hypoxic adaptation mediated by HIF transcription factors has been shown to require mitochondria. Current models suggest that mitochondria regulate oxygen sensor (HIF prolyl hydroxylase) activity and HIF1a stability during hypoxia by either increasing mitochondrial peroxide as a second messenger or by serving as oxygen consumers that enhance the kinetics of cytoplasmic oxygen reduction. Here, we address the role of mitochondrial peroxide specifically in regulating HIF1a stability. We use state-of-the-art tools to evaluate the role of peroxide and other reactive oxygen species (ROS) in regulating HIF1a stability. We show that antioxidant enzymes are not homeostatically induced nor are peroxide levels increased in hypoxia. Forced expression of diverse antioxidant enzymes, all of which diminish peroxide, had disparate effects on HIF1a protein stability. Reduction of lipid peroxides by glutathione peroxidase-4 or superoxide by mitochondrial SOD failed to influence HIF1a protein stability. These data showed that mitochondrial, cytosolic and lipid ROS are dispensable for HIF1a stability and should affirm therapeutic efforts to activate the HIF pathway in disease states by HIF prolyl hydroxylase inhibition.

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