Cytosolic PRDX1 acts as an extramitochondrial sink to set mitochondrial H2O2 levels and enable resilience to chronic mitochondrial oxidative stress

Hydrogen peroxide (H 2 O 2 ) plays a dual role as both a signalling molecule and a mediator of oxidative stress. Although mitochondria are major producers of H 2 O 2 , the relative contributions of mitochondrial versus cytosolic antioxidant systems to mitochondrial H 2 O 2 homeostasis in intact cells remain poorly defined. Here, we combined compartment-resolved live-cell imaging using HyPer7, inducible mitochondrial H 2 O 2 generation (matrix-targeted d -amino acid oxidase), kinetic modelling, and a targeted CRISPR/Cas9 screen to dissect determinants of mitochondrial H 2 O 2 dynamics in HEK293 cells. Unexpectedly, we found that the cytosolic peroxiredoxin PRDX1 is a dominant regulator of mitochondrial matrix H 2 O 2 levels. Loss of cytosolic PRDXs markedly enhanced matrix Hyper7 signals under both exogenous and mitochondria-intrinsic H 2 O 2 production, exceeding the effects of deleting mitochondrial peroxiredoxins. Modelling and transport experiments indicated a very high permeability of the mitochondrial inner membrane to H 2 O 2 enabling rapid efflux and the establishment of steep concentration gradients. This permits the cytosol to function as a major sink to limit matrix H 2 O 2 accumulation. PRDX1 deficiency sensitized cells to chronic mitochondrial oxidative stress. A targeted CRISPR screen identified the Rab7 GAP TBC1D5, linking mitophagy to cellular survival under these conditions. Consistently, PRDX1/2-deficient cells exhibited elevated mitophagic flux, indicating mitochondrial quality control as a compensatory response. Our study reveals that cytosolic PRDXs critically impact mitochondrial redox homeostasis and provides a systems-level framework for understanding compartmental redox control and stress adaptation.