How do substrates enter and products exit the buried active site of cytochrome P450cam? 2. Steered molecular dynamics and adiabatic mapping of substrate pathways.

Three possible channels by which substrates and products can exit from the buried active site of cytochrome P450cam have been identified by means of random expulsion molecular dynamics simulations. In the investigation described here, we computed estimates of the relative probabilities of ligand passage through the three channels using steered molecular dynamics and adiabatic mapping. For comparison, the same techniques are also applied to investigate substrate egress from cytochrome P450-BM3. The channel in cytochrome P450cam, for which there is the most supporting evidence from experiments (which we name pathway 2a), is computed to be the most probable ligand exit channel. It has the smallest computed unbinding work and force. For this channel, the ligand exits between the F/G loop and the B' helix. Two mechanistically distinct, but energetically similar routes through this channel were observed, showing that multiple pathways along one channel are possible. The probability of ligand exit via the next most probable channel (pathway 3), which is located between the I helix and the F and G helices, is estimated to be less than 1/10 of the probability of exit along pathway 2a. Low-frequency modes of the protein extracted from an essential dynamics analysis of a 1 ns duration molecular dynamics simulation of cytochrome P450cam with camphor bound, support the opening of pathway 2a on a longer timescale. On longer timescales, it is therefore expected that this pathway becomes more dominant than estimated from the present computations.