Increasing extracellular H₂O₂ produces a bi-phasic response in intracellular H₂O₂, with peroxiredoxin hyperoxidation only triggered once the cellular H₂O₂-buffering capacity is overwhelmed

Reactive oxygen species, such as H₂O₂, can damage cells but also promote fundamental processes, including growth, differentiation and migration. The mechanisms allowing cells to differentially respond to toxic or signaling H₂O₂ levels are poorly defined. Here we reveal that increasing external H₂O₂ produces a bi-phasic response in intracellular H₂O₂. Peroxiredoxins (Prx) are abundant peroxidases which protect against genome instability, ageing and cancer. We have developed a dynamic model simulating in vivo changes in Prx oxidation. Remarkably, we show that the thioredoxin peroxidase activity of Prx does not provide any significant protection against external rises in H₂O₂. Instead, our model and experimental data are consistent with low levels of extracellular H₂O₂ being efficiently buffered by other thioredoxin-dependent activities, including H₂O₂-reactive cysteines in the thiol-proteome. We show that when extracellular H₂O₂ levels overwhelm this buffering capacity, the consequent rise in intracellular H₂O₂ triggers hyperoxidation of Prx to thioredoxin-resistant, peroxidase-inactive form/s. Accordingly, Prx hyperoxidation signals that H₂O₂ defenses are breached, diverting thioredoxin to repair damage.