Ligand binding and mobility in cytochrome P450cam: Crystallographic and computational studies

The binding and dynamics of nicotine and other non-native ligands in the active site of the heme-containing mono-oxygenase cytochrome P450cam have been characterized by X-ray crystallography and computer simulation. Cytochrome P450cam can bind many different compounds, making it a good candidate for structure-aided enzyme design.; Prior computational studies of nicotine binding to cytochrome P450cam had suggested an initial orientation that would lead directly to the observed product. Our crystallographic results, however, accounted for spectroscopic data that contradicted the computer model: the primary binding mode involves direct coordination of nicotine with the heme iron. To evaluate this non-productive orientation, crystals of the nicotine-P450cam complex were exposed to carbon monoxide to emulate the binding of oxygen, the next step in the reaction pathway. The crystal structure revealed that the nicotine, no longer coordinated with the heme iron, had shifted to a noticeably different orientation. In addition, a later data set collected on this crystal indicated further reorientation of nicotine. Cytochrome P450cam thus allows significant ligand mobility during its catalytic cycle.; This active site mobility is one of the properties that make cytochrome P450cam an attractive candidate for modification to carry out industrial syntheses. To this end, studies of the binding of the enantiomers of 2-ethylhexanoic acid were undertaken. Our crystallographic results suggest that (R)-2-ethylhexanoic acid binds in the active site in a more ordered fashion than (S)-2-ethylhexanoic acid, consistent with other researchers' binding assays and kinetics assays. However, the high apparent mobility of both ligands in the active site limited these crystallographic results.; To circumvent these difficulties, free energy perturbation calculations were performed on the ethylhexanoate-P450cam complexes. Perturbation of the (R) enantiomer in its crystallographically determined orientation into the (S) enantiomer enabled the difference in the binding free energies of the two compounds to be calculated. The results indicate that (R)-2-ethylhexanoic acid has a lower binding free energy than (S)-2-ethylhexanoic acid, consistent with our interpretation of the crystallographic data. These data could thus provide a starting point for the analysis of mutations useful for industrial synthesis.