Substrate mobility in thiocamphor-bound cytochrome P450cam: an explanation of the conflict between the observed product profile and the X-ray structure

Thiocamphor is an unusual substrate for P450_cam in that in theX-ray structure it binds in the active site pocket in two distinctorientations and neither of these orientations are consistentwith the 5-alcohol being the primary product. Other camphoranalogs such as norcamphor or camphane bind in a single orientationconsistent with the 5-alcohol being a major product. We presentan analysis of four 175 ps molecular dynamics trajectories ofthiocamphor-bound cytochrome P450_cam. The first two trajectorieswere calculated for cytochrome P450_cam with thiocamphor boundin both its major and minor crystallographic orientations. Inthe second set of simulations, a single oxygen atom was addedas a distal ligand to the heme group in order to model the putativeferryl oxygen reaction intermediate. Trajectories were againcalculated starting with thiocamphor in its major and minororientations. While the protein dynamics were quite similarin all four trajectories, the substrate showed distinctly differentmotions in each of the trajectories. In particular, the preferredsubstrate orientations were very different in the presence ofthe ferryl oxygen than in the absence of that oxygen. The preferredorientations in the absence of the distal oxygen were consistentwith the 3-akohol being the major product, while the preferredorientations in the presence of the distal oxygen were consistentwith the 5-alcohol being a major product. These simulationsoffer an explanation for the inconsistency between the X-raydata and the product profile.