Spatial and temporal control of mitochondrial H₂ O₂ release in intact human cells

Hydrogen peroxide (H₂ O₂ ) has key signaling roles at physiological levels, while causing molecular damage at elevated concentrations. H₂ O₂ production by mitochondria is implicated in regulating processes inside and outside these organelles. However, it remains unclear whether and how mitochondria in intact cells release H₂ O₂ . Here, we employed a genetically encoded high-affinity H₂ O₂ sensor, HyPer7, in mammalian tissue culture cells to investigate different modes of mitochondrial H₂ O₂ release. We found substantial heterogeneity of HyPer7 dynamics between individual cells. We further observed mitochondria-released H₂ O₂ directly at the surface of the organelle and in the bulk cytosol, but not in the nucleus or at the plasma membrane, pointing to steep gradients emanating from mitochondria. Gradient formation is controlled by cytosolic peroxiredoxins, which act redundantly and with a substantial reserve capacity. Dynamic adaptation of cytosolic thioredoxin reductase levels during metabolic changes results in improved H₂ O₂ handling and explains previously observed differences between cell types. Our data suggest that H₂ O₂ -mediated signaling is initiated only in close proximity to mitochondria and under specific metabolic conditions.