Directed Evolution of APOX for Proximity Labeling Using Phenols with High Redox Potentials

Proximity labeling (PL) identifies endogenous proteins in specific subcellular regions. APEX2 is a peroxidase that enables PL with high temporal resolution by oxidizing biotin phenol (BP) into a radical that tags nearby proteins. However, the BP radical has a relatively large diffusion radius, limiting spatial resolution. Replacing the phenol in BP with nitrophenol (NP) could potentially increase spatial resolution by generating shorter-lived radicals, but APEX2 cannot efficiently oxidize phenols with high redox potentials. Here, we report the directed evolution of APOX, a quadruple mutant of APEX2 with a higher reduction potential that exhibits 6-fold and 2.5-fold faster oxidation of NPs and BP, respectively. Using APOX with a membrane-permeable alkyne-NP probe, we demonstrate PL in living mammalian cells, including proteomic mapping with excellent subcellular compartment specificity. APOX expands peroxidase PL by accessing high redox potential probes, opening opportunities to further tune the diffusion radius and enhance the tagging of biomolecules beyond proteins.