Engineering of Redox-Active Residues Enhances P450 Peroxygenase Performance.

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.