The endosymbiotic theory posits that ancient eukaryotic cells engulfed O₂-consuming prokaryotes, which protected them against O₂ toxicity. Previous studies have shown that cells lacking cytochrome c oxidase (COX), required for respiration, have increased DNA damage and reduced proliferation, which could be improved by reducing O₂ exposure. With recently developed fluorescence lifetime microscopy-based probes demonstrating that the mitochondrion has lower [O₂] than the cytosol, we hypothesized that the perinuclear distribution of mitochondria in cells may create a barrier for O₂ to access the nuclear core, potentially affecting cellular physiology and maintaining genomic integrity. To test this hypothesis, we utilized myoglobin-mCherry fluorescence lifetime microscopy O₂ sensors without subcellular targeting ("cytosol") or with targeting to the mitochondrion or nucleus for measuring their localized O₂ homeostasis. Our results showed that, similar to the mitochondria, the nuclear [O₂] was reduced by ∼20 to 40% compared with the cytosol under imposed O₂ levels of ∼0.5 to 18.6%. Pharmacologically inhibiting respiration increased nuclear O₂ levels, and reconstituting O₂ consumption by COX reversed this increase. Similarly, genetic disruption of respiration by deleting SCO2, a gene essential for COX assembly, or restoring COX activity in SCO2^-/- cells by transducing with SCO2 cDNA replicated these changes in nuclear O₂ levels. The results were further supported by the expression of genes known to be affected by cellular O₂ availability. Our study reveals the potential for dynamic regulation of nuclear O₂ levels by mitochondrial respiratory activity, which in turn could affect oxidative stress and cellular processes such as neurodegeneration and aging.

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