Proteome-wide tagging with an H₂O₂ biosensor reveals highly localized and dynamic redox microenvironments

Hydrogen peroxide (H₂O₂) sensing and signaling involves the reversible oxidation of particular thiols on particular proteins to modulate protein function in a dynamic manner. H₂O₂ can be generated from various intracellular sources, but their identities and relative contributions are often unknown. To identify endogenous "hotspots" of H₂O₂ generation on the scale of individual proteins and protein complexes, we generated a yeast library in which the H₂O₂ sensor HyPer7 was fused to the C-terminus of all protein-coding open reading frames (ORFs). We also generated a control library in which a redox-insensitive mutant of HyPer7 (SypHer7) was fused to all ORFs. Both libraries were screened side-by-side to identify proteins located within H₂O₂-generating environments. Screening under a variety of different metabolic conditions revealed dynamic changes in H₂O₂ availability highly specific to individual proteins and protein complexes. These findings suggest that intracellular H₂O₂ generation is much more localized and functionally differentiated than previously recognized.