Protein Engineering for Enhancing Electron Transfer in P450‐Mediated Catalysis

Cytochrome P450 enzymes (P450s) are versatile biocatalysts with applications spanning pharmaceutical development and natural product biosynthesis. A critical bottleneck in P450-mediated reactions is the electron transfer process, which often limits catalytic efficiency and promotes uncoupling events leading to reactive oxygen species (ROS) formation. This review comprehensively examines recent protein engineering strategies aimed at enhancing electron transfer efficiency in P450 systems. We explore the design and application of different fusion constructs, which improve proximity between the P450 enzyme and its redox partners (RPs), as well as scaffold-mediated protein assembly, enabling precise spatial organization of P450s and RPs. Furthermore, we discuss targeted modifications at the P450-RP interaction interface and optimization of electron transfer pathways through site-directed mutagenesis and directed evolution. These strategies enhance catalytic activity, improve coupling efficiency, and reduce ROS formation. Finally, we address the remaining challenges in understanding and engineering P450 electron transfer, and discuss the future directions, emphasizing the need for advanced computational modeling, structural characterization, and integration of synthetic and systems biology approaches.