Real-time monitoring of basal H2O2 levels with peroxiredoxin-based probes

Editorial Summary A fluorescent sensor combining a mutated form of the 2-Cys peroxiredoxin Tsa2 unable to undergo thioredoxin-mediated reduction with a redox-sensitive GFP protein allows real-time detection of baseline hydrogen peroxide levels in yeast cells. Genetically encoded probes based on the H 2 O 2 -sensing proteins OxyR and Orp1 have greatly increased the ability to detect elevated H 2 O 2 levels in stimulated or stressed cells. However, these proteins are not sensitive enough to monitor metabolic H 2 O 2 baseline levels. Using yeast as a platform for probe development, we developed two peroxiredoxin-based H 2 O 2 probes, roGFP2-Tsa2ΔC R and roGFP2-Tsa2ΔC P ΔC R , that afford such sensitivity. These probes are ∼50% oxidized under 'normal' unstressed conditions and are equally responsive to increases and decreases in H 2 O 2 . Hence, they permit fully dynamic, real-time measurement of basal H 2 O 2 levels, with subcellular resolution, in living cells. We demonstrate that expression of these probes does not alter endogenous H 2 O 2 homeostasis. The roGFP2-Tsa2ΔC R probe revealed real-time interplay between basal H 2 O 2 levels and partial oxygen pressure. Furthermore, it exposed asymmetry in H 2 O 2 trafficking between the cytosol and mitochondrial matrix and a strong correlation between matrix H 2 O 2 levels and cellular growth rate.