Aim: Mitochondria are major sites of reactive oxygen species (ROS) generation, and adaptive mitochondrial ROS signaling extends longevity. We aim at linking the genetic manipulation of mitochondrial H2O2 sensing in live cells to mechanisms driving aging in the model organism, Saccharomyces cerevisiae. To this end, we compare in vivo ROS (O2•−, H2O2 and •OH) accumulation, antioxidant enzyme activities, labile iron levels, GSH depletion, and protein oxidative damage during the chronological aging of three yeast strains: ccp1Δ that does not produce the mitochondrial H2O2 sensor protein, cytochrome c peroxidase (Ccp1); ccp1W191F that produces a hyperactive variant of this sensor protein (Ccp1W191F); and the isogenic wild-type strain. Results: Since they possess elevated manganese superoxide dismutase (Sod2) activity, young ccp1Δ cells accumulate low mitochondrial superoxide (O2•−) levels but high H2O2 levels. These cells exhibit stable aconitase activity and contain low amounts of labile iron and hydroxyl radicals (•OH). Furthermore, they undergo late glutathione (GSH) depletion, less mitochondrial protein oxidative damage and live longer than wild-type cells. In contrast, young ccp1W191F cells accumulate little H2O2, possess depressed Sod2 activity, enabling their O2•− level to spike and deactivate aconitase, which, ultimately, leads to greater mitochondrial oxidative damage, early GSH depletion, and a shorter lifespan than wild-type cells. Innovation: Modulation of mitochondrial H2O2 sensing offers a novel interventional approach to alter mitochondrial H2O2 levels in live cells and probe the pro- versus anti-aging effects of ROS. Conclusion: The strength of mitochondrial H2O2 sensing modulates adaptive mitochondrial ROS signaling and, hence, lifespan. Antioxid. Redox Signal. 21, 1490–1503.
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