Mapping the Substrate Recognition Pathway in Cytochrome P450

Abstract 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, despite the breadth of cytochrome P450 mediated reactions, a long-standing question is: How does the substrate, in the first place, access the catalytic center of cytochrome P450? The prevalence of conflicting crystallographic evidences of both closed and open catalytic center in the substrate-free and substrate-bound cytochrome P450 has given rise to a notion of conformational heterogeneity, which makes the plausible mechanism of substrate recognition by cytochrome P450 puzzling from structural point of view. Here we report multi-microsecond-long unbiased molecular dynamics simulations, which are able to capture the spontaneous process of binding of substrate from bulk solvent to the occluded catalytic center of an archetypal system cytochrome P450cam, at an atomistic precision. In all binding trajectories, the substrate enters through a single channel, where it makes its first contact with the protein-surface and subsequently dwells in a highly long-lived intermediate state, before sliding into the catalytic center of P450cam. The simulated substrate-bound pose and crystallographic pose are in excellent agreement. Contrary to the prevalent hypotheses, our results indicate that a large-scale opening of F/G loop of P450cam is not required for passage of substrate to the catalytic center. Rather, we find that a substrate-induced side-chain displacement of Phe87 residue, coupled with a complex array of dynamical interconversions of multiple metastable substrate conformations along the entry channel, drives the substrate recognition in P450cam. By reconciling multiple precedent investigations, this work put forward an unambiguous view of the substrate recognition mechanism in deep buried cavity of cytochrome P450.