UPO‐Mimetic Engineering of CYP199A4 Enables Enhanced Peroxygenase Activity via the Hydrogen Peroxide Shunt Pathway

Engineering an acid–base dyad into the peroxygenase‐enabled mutant CYP199A4 T252E yielded four in silico‐designed double mutants, of which CYP199A4 F182R/T252E showed the best dyad‐like geometry and was characterized further. It delivered ∼10‐fold higher initial H 2 O 2 ‐driven O‐demethylation activity than wild type and CYP199A4 T252E , alongside reduced catalase activity and improved peroxide utilization. However, it was more prone to H 2 O 2 ‐induced heme bleaching and rapid inactivation under standard dosing; slow, controlled H 2 O 2 feeding sustained catalysis for hours. Overall, adding a second basic residue boosts per‐oxy‐gen‐ase‐like activity but reduces oxidative robustness, underscoring the trade‐off between efficiency and peroxide tolerance and guiding future engineering of robust P450 peroxygenases.