Rational Design of a Shortened Electron Transfer Pathway in P450BM3 for Enhanced Hydroxylation Catalysis

Steroid hormones, the second largest drug class after antibiotics, rely on cytochrome P450 enzymes for efficient and eco-friendly synthesis. However, its practical application is constrained by low electron transfer (ET) efficiency primarily due to an incomplete understanding of its intramolecular ET mechanism. Here, we utilized the newly resolved cryo-EM structures of two conformations (closed and open) of the P450BM3 catalytic dimer to propose a novel "interchain same-side" ET mechanism, where the NADPH-FAD binding domain of chain A (or chain B), the FMN domain of chain B (or chain A), and the heme domain of chain A (or chain B) are positioned on the same side. We also employed two strategies to enhance ET efficiency: (1) cofactor engineering and (2) shortened ET pathways. The mutant M5 (Q673A–A963M–N319A–A1047C–N489H) showed a 4.43-fold increase in enzyme activity, 3.94-fold increase in coupling efficiency (CE), 61.43-fold increase in ET rate (kET), and 11-fold increase in catalytic efficiency (kcat/Km) over the wild type. This study achieves the first elucidation of the authentic ET mechanism in P450BM3, and it demonstrates that the rational design of a shortened ET pathway can significantly enhance catalytic performance, thereby establishing a solid foundation for the efficient synthesis of hydroxylated steroid drugs.