Rational Engineeringof Self-Sufficient P450s to BoostCatalytic Efficiency of Carbene-Mediated C–S Bond Formation

Intermolecular C–S bond formation is a key step in the construction of sulfur-containing compounds in organic chemistry. As versatile biocatalysts, P450-catalyzed radical reactions are compatible with a diverse range of functional groups. Here, to boost the catalytic efficiency of carbene-mediated C–S bond formation, self-sufficient P450_TT was rationally engineered using multiple AI models (SaProt, ProSST, EVmutation). Employing purified enzymes, a triple variant P450_TT-M3 (V118A/C385H/F424P) demonstrated a significantly higher TOF of 6.1 min^–1 than that of P450_TT-M1­(C385H) (1.5) in 1 h. Furthermore, with remarkable adaptability to a diverse range of aryl mercaptans, it exhibits high versatility in catalyzing the formation of intermolecular C–S bonds. Computational studies have shown that C–S bond formation involved H atom transfer from the thiol group of thiophenol to Fe-carbene, which differs from P450-catalyzed N–H insertion reactions. In addition, QM/MM simulations suggested that the variant enables a further approach of ethyl diazoacetate to the iron center, thereby enhancing the catalytic efficiency.