Unlocking Aromatic Amine Oxidation in APEX3 by Engineering the Heme Axial Ligand with Genetically Encoded Nδ Substituted Histidine

The axial ligand of heme is a critical determinant of reactivity in heme-dependent enzymes, yet its systematic engineering remains challenging due to the limited chemical diversity of natural amino acids. Here, we demonstrate that replacing the native histidine axial ligand with a nonnatural analog provides an effective strategy to regulate heme enzyme activity. By site-specifically incorporating δVin-H into APEX2, we reprogrammed the electronic structure of the heme center and unlocked the catalytic activity toward aromatic amine substrates. Directed evolution of the APEX2-VinH enzyme yielded APEX3-VinH, which exhibits a pronounced enhancement in catalytic efficiency and robustness for aromatic amine oxidation. Subsequently, systematic screening of an axial ligand library identified an optimized variant, APEX3-MeH, which maximizes catalytic efficiency and robustness. Furthermore, APEX3-MeH enables highly efficient proximity labeling on tissue sections at low hydrogen peroxide concentrations, significantly enhancing biocompatibility for complex biological environments. Notably, the APEX3 scaffold exhibited enhanced catalytic efficiency toward aromatic amine substrates relative to wild-type APEX2 even without unnatural amino acid incorporation, providing a new variant for chemical biology applications. This work establishes axial ligand engineering via genetic code expansion as a general and powerful strategy to expand the catalytic scope of heme enzymes, with potential implications for biocatalysis and the development of proximity labeling tools.