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

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.