Substrate Conformational Switch Enables the Stereoselective Dimerization in P450 NascB: Insights from Molecular Dynamics Simulations and Quantum Mechanical/Molecular Mechanical Calculations

P450 NascB catalyzes the coupling of cyclo-(l-tryptophan-l-proline) (1) to generate (−)-naseseazine C (2) through intramolecular C–N bond formation and intermolecular C–C coupling. A thorough understanding of its catalytic mechanism is crucial for the engineering or design of P450-catalyzed C–N dimerization reactions. By employing MD simulations, QM/MM calculations, and enhanced sampling, we assessed various mechanisms from recent works. Our study demonstrates that the most favorable pathway entails the transfer of a hydrogen atom from N7–H to Cpd I. Subsequently, there is a conformational change in the substrate radical, shifting it from the Re-face to the Si-face of N7 in Substrate 1. The Si-face conformation of Substrate 1 is stabilized by the protein environment and the π–π stacking interaction between the indole ring and heme porphyrin. The subsequent intermolecular C3–C6′ bond formation between Substrate 1 radical and Substrate 2 occurs via a radical attack mechanism. The conformational switch of the Substrate 1 radical not only lowers the barrier of the intermolecular C3–C6′ bond formation but also yields the correct stereoselectivity observed in experiments. In addition, we evaluated the reactivity of the ferric-superoxide species, showing it is not reactive enough to initiate the hydrogen atom abstraction from the indole NH group of the substrate. Our simulation provides a comprehensive mechanistic insight into how the P450 enzyme precisely controls both the intramolecular C–N cyclization and intermolecular C–C coupling. The current findings align with the available experimental data, emphasizing the pivotal role of substrate dynamics in governing P450 catalysis.