Synergistic Tuning of Conformational Dynamics, Electron Tunneling, and Substrate Positioning Enhances Electron Transfer in a P450 Chimera for Calcifediol Biosynthesis

ABSTRACT Electron transfer (ET) efficiency dictates catalytic performance in multi‐domain self‐sufficient cytochrome P450s. Conventional engineering, however, predominantly focuses on localized optimization of either the active‐site pocket or linker regions, overlooking inter‐domain conformational transitions and ET chain integrity. Herein, we report a holistic ET‐optimization strategy integrating conformational dynamics modulation, ET pathway engineering, and substrate positioning tuning, which was applied to enhance ET in a chimeric P450 VK1‐CYP116B46‐L21 (L21) for calcifediol biosynthesis. [2Fe‐2S]→Heme ET pathway engineering yielded variant L21‐M2 (F346K/R354M), which decreased the conformational transition barrier by 4.5 kcal/mol and shortened the ET pathway by 4.05 Å, leading to a 72‐fold enhancement in ET rate. Heme domain engineering generated variant L21‐M3 (P83A/A177M/K180F), which shortened the near‐attack conformations distance to 4.19 Å (from 4.62 Å) and increased reactive conformation to 38 % (from 25 %). The pentuple variant L21‐M5 combined both improvements, which demonstrated exceptional catalytic performance: an 8.2‐ fold higher catalytic efficiency, a coupling efficiency of 56.78 %, and a total turnover number (TTN) of 3222. In a semi‐preparative‐scale biotransformation, L21‐M5 achieved 3.26 g/L production of calcifediol with 82 % conversion, underscoring its strong industrial potential. These results highlight the efficacy of the proposed ET‐optimization strategy and provide a transferable workflow for engineering multi‐domain redox biocatalysts.