Using in vivo oxidation status of one- and two-component redox relays to determine H₂O₂ levels linked to signaling and toxicity.

BACKGROUND:Hydrogen peroxide (H₂O₂) is generated as a by-product of metabolic reactions during oxygen use by aerobic organisms, and can be toxic or participate in signaling processes. Cells, therefore, need to be able to sense and respond to H₂O₂ in an appropriate manner. This is often accomplished through thiol switches: Cysteine residues in proteins that can act as sensors, and which are both scarce and finely tuned. Bacteria and eukaryotes use different types of such sensors-either a one-component (OxyR) or two-component (Pap1-Tpx1) redox relay, respectively. However, the biological significance of these two different signaling modes is not fully understood, and the concentrations and peroxides driving those types of redox cascades have not been determined, nor the intracellular H₂O₂ levels linked to toxicity. Here we elucidate the characteristics, rates, and dynamic ranges of both systems. RESULTS:By comparing the activation of both systems in fission yeast, and applying mathematical equations to the experimental data, we estimate the toxic threshold of intracellular H₂O₂ able to halt aerobic growth, and the temporal gradients of extracellular to intracellular peroxides. By calculating both the oxidation rates of OxyR and Tpx1 by peroxides, and their reduction rates by the cellular redoxin systems, we propose that, while Tpx1 is a sensor and an efficient H₂O₂ scavenger because it displays fast oxidation and reduction rates, OxyR is strictly a H₂O₂ sensor, since its reduction kinetics are significantly slower than its oxidation by peroxides, and therefore, it remains oxidized long enough to execute its transcriptional role. We also show that these two paradigmatic H₂O₂-sensing models are biologically similar at pre-toxic peroxide levels, but display strikingly different activation behaviors at toxic doses. CONCLUSIONS:Both Tpx1 and OxyR contain thiol switches, with very high reactivity towards peroxides. Nevertheless, the fast reduction of Tpx1 defines it as a scavenger, and this efficient recycling dramatically changes the Tpx1-Pap1 response to H₂O₂ and connects H₂O₂ sensing to the redox state of the cell. In contrast, OxyR is a true H₂O₂ sensor but not a scavenger, being partially insulated from the cellular electron donor capacity.

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