How do substrates enter and products exit the buried active site of cytochrome P450cam? 1. Random expulsion molecular dynamics investigation of ligand access channels and mechanisms

Cytochrome P450s form a ubiquitous protein family with functions including the synthesis and degradation of many physiologically important compounds a nd the degradation of xenobiotics. Cytochrome P450cam from Pseudomonas puti da has provided a paradigm for the structural understanding of cytochrome P 450s. However, the mechanism by which camphor, the natural substrate of cyt ochrome P450cam, accesses the buried active site is a long-standing puzzle. While there is recent crystallographic and simulation evidence for opening of a substrate-access channel in cytochrome P450BM-3, for cytochrome P450c am, no such conformational changes have been observed either in different c rystal structures or by standard molecular dynamics simulations. Here, a no vel simulation method, random expulsion molecular dynamics, is presented, i n which substrate-exit channels from the buried active site are found by im posing an artificial randomly oriented force on the substrate, in addition to the standard molecular dynamics force field. The random expulsion molecu lar dynamics method was tested in simulations of the substrate-bound struct ure of cytochrome P450BM-3, and then applied to complexes of cytochrome P45 0cam with different substrates and with product. Three pathways were identi fied, one of which corresponds to a channel proposed earlier on the basis o f crystallographic and site-directed mutagenesis data. Exit via the water-f illed channel, which was previously suggested to be a product exit channel, was not observed. The pathways obtained by the random expulsion molecular dynamics method match well with thermal motion pathways obtained by an anal ysis of crystallographic B-factors. in contrast to large backbone motions ( up to 4 Angstrom) observed in cytochrome P450BM-3 for the exit of palmitole ic acid, passage of camphor through cytochrome P450cam only requires small backbone motions (less than 2.4 Angstrom) in conjunction with side-chain ro tations. Concomitantly, in almost all the exit trajectories, salt-links tha t have been proposed to act as ionic tethers between secondary structure el ements of the protein, are perturbed. (C) 2000 Academic Press.