Using the heme peroxidase APEX2 to probe intracellular H 2 O 2 flux and diffusion.

Currently available genetically encoded H 2 O 2 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 H 2 O 2 probe that directly and irreversibly converts H 2 O 2 molecules into either fluorescent or luminescent signals. We demonstrate sensitivity, specificity, and the ability to quantitate endogenous H 2 O 2 turnover. We show how the probe can be used to detect changes in endogenous H 2 O 2 generation and to assess the roles and relative contributions of endogenous H 2 O 2 scavengers. Furthermore, APEX2 can be used to study H 2 O 2 diffusion inside the cytosol. Finally, APEX2 reveals the impact of commonly used alkylating agents and cell lysis protocols on cellular H 2 O 2 generation.