Phenoxazinone synthase-like activity of rationally designed heme enzymes based on cytochrome P450BM3

The development of functional metalloenzymes provides a promising strategy for the sustainable synthesis of heterocyclic compounds such as phenazines and phenoxazinones. Here, we establish an H 2 O 2 -driven P450BM3 peroxygenase system enabled by dual-functional small molecules (DFSMs) to efficiently oxidize aromatic amines. Mutational engineering at positions 78, 87, and 264 within the heme active site significantly modulated catalytic performance. Among these mutants, F87G/V78A exhibited a high k cat /K m of 175.69 mM −1 s −1 toward 2, 5-diaminobenzenesulfonic acid (2,5-DABSA), while F87G displayed exceptional activities in the oxidation of o -aminophenol (OAP) and o -phenylenediamine (OPD), with k cat /K m values of 276.92 and 79.13 mM −1 s −1 respectively. These catalytic efficiencies surpass those of commonly used enzymes, including laccase, dye-decolorizing peroxidase, o -aminophenol oxidase, and myoglobin. Overall, this study highlights engineered P450BM3 as a versatile and environmentally friendly biocatalyst, offering an attractive alternative to conventional chemical methods and enabling the rapid, selective synthesis of valuable heterocyclic scaffolds relevant to pharmaceuticals and materials science. The DFSM-P450BM3 system has successfully achieved green bioconversion of phenazine and phenoxazinone compounds with high reaction rates and yields.