Increasing extracellular H(2)O(2) produces a bi-phasic response in intracellular H(2)O(2), with peroxiredoxin hyperoxidation only triggered once the cellular H(2)O(2)-buffering capacity is overwhelmed

Reactive oxygen species, such as H(2)O(2), can damage cells but also promote fundamental processes, including growth, differentiation and migration. The mechanisms allowing cells to differentially respond to toxic or signaling H(2)O(2) levels are poorly defined. Here we reveal that increasing external H(2)O(2) produces a bi-phasic response in intracellular H(2)O(2). 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 H(2)O(2). Instead, our model and experimental data are consistent with low levels of extracellular H(2)O(2) being efficiently buffered by other thioredoxin-dependent activities, including H(2)O(2)-reactive cysteines in the thiol-proteome. We show that when extracellular H(2)O(2) levels overwhelm this buffering capacity, the consequent rise in intracellular H(2)O(2) triggers hyperoxidation of Prx to thioredoxin-resistant, peroxidase-inactive form/s. Accordingly, Prx hyperoxidation signals that H(2)O(2) defenses are breached, diverting thioredoxin to repair damage.