Using the heme peroxidase APEX2 to probe intracellular H₂O₂ flux and diffusion

Currently available genetically encoded H₂O₂ probes report on the thiol redox state of the probe, which means that they reflect the balance between probe thiol oxidation and reduction. Here we introduce the use of the engineered heme peroxidase APEX2 as a thiol-independent chemogenetic H₂O₂ probe that directly and irreversibly converts H₂O₂ molecules into either fluorescent or luminescent signals. We demonstrate sensitivity, specificity, and the ability to quantitate endogenous H₂O₂ turnover. We show how the probe can be used to detect changes in endogenous H₂O₂ generation and to assess the roles and relative contributions of endogenous H₂O₂ scavengers. Furthermore, APEX2 can be used to study H₂O₂ diffusion inside the cytosol. Finally, APEX2 reveals the impact of commonly used alkylating agents and cell lysis protocols on cellular H₂O₂ generation.