Mapping the Substrate Recognition Pathway in Cytochrome P450

Cytochrome P450s are ubiquitous metalloenzymes involved in the metabolism and detoxification of foreign components via catalysis of the hydroxylation reactions of a vast array of organic substrates. However, the mechanism underlying the pharmaceutically critical process of substrate access to the catalytic center of cytochrome P450 is a long-standing puzzle, further complicated by the crystallographic evidence of a closed catalytic center in both substrate-free and substrate-bound cytochrome P450. Here, we address a crucial question whether the conformational heterogeneity prevalent in cytochrome P450 translates to heterogeneous pathways for substrate access to the catalytic center of these metalloenzymes. By atomistically capturing the full process of spontaneous substrate association from bulk solvent to the occluded catalytic center of an archetypal system P450cam in multi-microsecond-long continuous unbiased molecular dynamics simulations, we here demonstrate that the substrate recognition in P450cam always occurs through a single well-defined dominant pathway. The simulated final bound pose resulting from these unguided simulations is in striking resemblance with the crystallographic bound pose. Each individual binding trajectory reveals that the substrate, initially placed at random locations in bulk solvent, spontaneously lands on a single key channel on the protein-surface of P450cam and resides there for an uncharacteristically long period, before correctly identifying the occluded target-binding cavity. Surprisingly, the passage of substrate to the closed catalytic center is not accompanied by any large-scale opening in protein. Rather, the unbiased simulated trajectories (∼57 μs) and underlying Markov state model, in combination with free-energy analysis, unequivocally show that the substrate recognition process in P450cam needs a substrate-induced side-chain displacement coupled with a complex array of dynamical interconversions of multiple metastable substrate conformations. Further, the work reconciles multiple precedent experimental and theoretical observations on P450cam and establishes a comprehensive view of substrate-recognition in cytochrome P450 that only occurs via substrate-induced structural rearrangements.