Cytochrome P450, the ubiquitous metalloenzyme involved in detoxification of foreign components, has remained one of the most popular systems for substrate-recognition process. However, despite being known for its high substrate specificity, the mechanistic basis of substrate-binding by archetypal system cytochrome P450cam has remained at odds with the contrasting reports of multiple diverse crystallographic structures of its substrate-free form. Here we address this issue by elucidating the probability of mutual dynamical transition to the other crystallographic pose of cytochrome P450cam and vice versa via unbiased all-atom computer simulation. A robust Markov state model (MSM), constructed using adaptively sampled multi microsecond-long Molecular dynamics simulation trajectories, maps the broad and heterogenous P450cam conformational landscape into five key sub-states. In particular, the MSM identifies an intermediate-assisted dynamic equilibrium between a pair of conformations of P450cam, in which the substrate-recognition sites remain `closed' and `open' respectively. However, the estimate of a significantly high stationary population of closed conformation, coupled with faster rate of open → closed transition than its reverse process, dictates that the net conformational equilibrium would be swayed in favour of `closed' conformation. Together, the investigation quantitatively infers that while a potential substrate of cytochrome P450cam would in principle explore a diverse array of conformations of substrate-free protein, it would mostly encounter a `closed' or solvent-occluded conformation and hence would follow an induced-fit based recognition process. Overall, the work reconciles multiple precedent crystallographic, spectroscopic investigations and establishes how a statistical elucidation of conformational heterogeneity in protein would provide crucial insights in the mechanism of potential substrate-recognition process.

Reconciling conformational heterogeneity and substrate recognition in cytochrome P450

ACS Spring Meeting 2021

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