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However, the nonproductive oxidative damage caused by Cpd I compromises enzyme stability and limits catalytic efficiency. Here, we report a rational protein engineering strategy to address this bottleneck by constructing additional \u201chole traps\u201d on the enzyme surface. Guided by structural analysis, we introduced redox\u2010active tyrosine\/tryptophan residues at spatially defined positions on the surface of the P450BM3 F87A mutant. The optimal engineered variant M8 (F87A\/L215W\/H408W) significantly enhances turnover numbers (TONs) across a range of peroxide\u2010driven hydroxylation reactions of aliphatic and aromatic substrates. Steady\u2010state kinetics, reductant supplementation assays, and half\u2010life measurements consistently indicated that the introduced Trp residues play a crucial protective role, improving both catalytic output and enzyme robustness. The crystal structure of M8 revealed that the mutations facilitate an expanded local network of \u03c0\u2013\u03c0 interactions, forming efficient surface hole traps. This strategy was then demonstrated to efficiently improve the catalytic performance of two engineered CYP199A4 peroxygenase variants (F182A and D251Q\/T252E). These findings establish the rational construction of auxiliary hole traps as an effective strategy to enhance P450 peroxygenase performance, with potential applicability to other heme and even broad oxidizing enzymes.<\/jats:p>","DOI":"10.1002\/anie.3845288","type":"journal-article","created":{"date-parts":[[2026,10,24]],"date-time":"2026-10-24T07:27:27Z","timestamp":1792826847000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Engineering of Redox\u2010Active Residues Enhances P450 Peroxygenase Performance"],"prefix":"10.1002","author":[{"ORCID":"https:\/\/orcid.org\/0009-0002-3884-3508","authenticated-orcid":false,"given":"Zihan","family":"Wang","sequence":"first","affiliation":[{"name":"Key Laboratory of Photoelectric Conversion and Utilization of Solar Energy Qingdao New Energy Shandong Laboratory Qingdao Institute of Bioenergy and Bioprocess Technology Chinese Academy of Sciences  Qingdao 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Bioprocess Technology Chinese Academy of Sciences  Qingdao China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiangquan","family":"Qin","sequence":"additional","affiliation":[{"name":"Key Laboratory of Photoelectric Conversion and Utilization of Solar Energy Qingdao New Energy Shandong Laboratory Qingdao Institute of Bioenergy and Bioprocess Technology Chinese Academy of Sciences  Qingdao China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3353-9411","authenticated-orcid":false,"given":"Binju","family":"Wang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Physical Chemistry of Solid Surfaces and Fujian Provincial Key Laboratory of Theoretical and Computational Chemistry College of Chemistry and Chemical Engineering Xiamen University  Xiamen 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