Unlocking Aromatic Amine Oxidation in APEX2 by Engineering the Heme Axial Ligand with Genetically Encoded Nδ-Vinyl Histidine

The axial ligand of heme is a key 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 non-natural 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 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. Notably, optimization of the δVin-H synthetase enabled highly efficient incorporation. This work establishes axial ligand engineering via genetic code expansion as a general and powerful strategy to reprogram the catalytic repertoire of heme enzymes, with broad implications for biocatalysis and the development of next-generation proximality labeling tools.