Year: 2026
Type: article
Abstract: Cytochrome P450 peroxygenases depend on the high-valent iron-oxo intermediate Compound I (Cpd I) to activate C-H bonds. 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 "hole traps" on the enzyme surface. Guided by structural analysis, we introduced redox-active 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-driven hydroxylation reactions of aliphatic and aromatic substrates. Steady-state kinetics, reductant supplementation assays, and half-life 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 π-π 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.
DOI: 10.1002/anie.3845288
Language: English
Subfield: Inorganic Chemistry
Field: Chemistry
Domain: Physical Sciences
Open Access status: bronze
OpenAlex ID: W7223471404