Reconciling Conformational Heterogeneity and Substrate Recognition in Cytochrome P450

Abstract

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 unbiasedall-atom computer simulation. A robust Markov state model (MSM), constructed using adaptively sampled 84 microsecond-longMolecular dynamics simulation trajectories, maps the broad and heterogenous P450cam conformational landscape into five keysub-states. In particular, the MSM identifies an intermediate-assisted dynamic equilibrium between a pair ofconformations ofP450cam, 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 investigationquantitatively infers that while a potential substrate of cytochrome P450cam would in principle explore a diverse array ofconformations of substrate-free protein, it would mostly encounter a ‘closed’ or solvent-occluded conformation and hence wouldfollow an induced-fit based recognition process. Overall, the work reconciles multiple precedent crystallographic, spectroscopicinvestigations and establishes how a statistical elucidation of conformational heterogeneity in protein would provide crucialinsights in the mechanism of potential substrate-recognition process