Dose-dependent mitochondrial H2O2 signaling drives toxicity or stress adaptation and longevity in fission yeast

The spatiotemporal dynamics of hydrogen peroxide (H 2 O 2 ) signaling and its effects on gene expression and cell fitness remain unclear. Using fission yeast, we applied genetic tools to control and monitor intracellular H 2 O 2 levels. We expressed the H 2 O 2 biosensor HyPer7 in four subcellular compartments, overexpressed D-amino acid oxidase (Dao1) in specific locations to induce localized H 2 O 2 production, and modulated H 2 O 2 detoxification or sensing. H 2 O 2 concentrations showed 2- to 5-fold reductions across membranes, and D-amino acid treatments generated nanomolar H 2 O 2 levels in Dao1-expressing cells. Mitochondrial-targeted Dao1 produced H 2 O 2 fluxes capable of reaching the nucleus. While high mitochondrial H 2 O 2 disrupted mitochondrial morphology and respiration, lower levels triggered antioxidant defenses, enhancing stress resistance and longevity. These findings establish a quantitative framework for mitochondrial H 2 O 2 signaling and demonstrate that localized redox signals from mitochondria can either promote or impair cellular fitness depending on their intensity.