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Abstract

Multiple ligand binding modes are possible in many enzyme active sites; their presence in cytochrome P450cam (P450cam) is evident from crystallographic studies of the binding of thiocamphor and phenylimidazoles. Here, we use multicopy molecular dynamics simulations to compare the binding modes of (1R)- and (1S)-camphor in the active site of P450cam. Simulations with (1R)-camphor, the natural substrate, serve to calibrate our protocol: 19 out of 20 copies of (1R)-camphor converged to coordinates very close to those observed for (1R)-camphor in its crystallographic complex with P450cam during the simulations. Simulations with the (1S)-camphor enantiomer showed greater mobility of the substrate, consistent with spectroscopic data, and resulted in 3 major binding modes. One of these is similar to the major conformation (of the two conformations assigned) in a recently determined crystal structure, but this conformation is not correctly oriented for regiospecific hydroxylation at C-5. The simulations, however, provide evidence for reorientation of (1S)-camphor upon formation of the reactive Fe-O intermediate to an orientation suitable for hydroxylation. The simulations thus permit rationalisation of the apparent inconsistency between the crystal structure and the reaction products.