<p>Since their discovery in the late 1980s as peroxidases that eliminate hydrogen peroxide (H₂O₂), peroxiredoxins (Prxs) have been recognized not only for detoxification but also for roles in redox signaling. In fission yeast, the main Prx, Tpx1, maintains steady-state H₂O₂ below toxic levels through its scavenging activity. Under excess peroxides, Tpx1 switches to a signaling role, transmitting oxidative signals to the transcription factor Pap1. Dissecting the relative contributions of Tpx1’s peroxidase and sensor functions is challenging. To address this, we used a synthetic biology strategy to separate these dual roles. Tpx1 or catalase (Ctt1) was expressed in distinct subcellular compartments, and their capacity to remove H₂O₂ and activate Pap1 was assessed. Both scavengers can complement the growth defects of cells lacking Tpx1 in high oxygen. Combined with H₂O₂ biosensors, our results show that Tpx1 efficiently controls H₂O₂ levels from any compartment, whereas Ctt1 does not. However, cells expressing mitochondrial- or nuclear-targeted Tpx1 display reduced oxidative stress tolerance because Pap1 activation requires cytosolic Tpx1. Overall, Tpx1 is a highly effective peroxidase throughout the cell, but its subcellular localization is essential for proper redox signaling regulation.</p>