Structural evidence for a functionally relevant second camphor binding site in P450cam: model for substrate entry into a P450 active site.

P450cam has long served as a prototype for the cytochrome P450 (CYP) gene family. But, little is known about how substrate enters its active site pocket, and how access is achieved in a way that minimizes exposure of the reactive heme. We hypothesize that P450cam may first bind substrate transiently near the mobile F-G helix that covers the active site pocket. Such a two-step binding process is kinetically required if P450cam rarely populates an open conformation-as suggested by previous literature and the inability to obtain a crystal structure of P450cam in an open conformation. Such a mechanism would minimize exposure of the heme by allowing P450cam to stay in a closed conformation as long as possible, since only brief flexing into an open conformation would be required to allow substrate entry. To test this model, we have attempted to dock a second camphor molecule into the crystal structure of camphor-bound P450cam. The docking identified only one potential entry site pocket, a well-defined cavity on the F-helix side of the F-G flap, 16 A from the heme iron. Location of this entry site pocket is consistent with our NMR T1 relaxation-based measurements of distances for a camphor that binds in fast exchange (active site camphor is known to bind in slow exchange). Presence of a second camphor binding site is also confirmed with [(1)H-(13)C] HSQC titrations of (13)CH3-threonine labeled P450cam. To confirm that camphor can bind outside of the active site pocket, (13)CH3-S-pyridine was bound to the heme iron to physically block the active site, and to serve as an NMR chemical shift probe. Titration of this P450cam-pyridine complex confirms that camphor can bind to a site outside the active site pocket, with an estimated Kd of 43 microM. The two-site binding model that is proposed based on these data is analogous to that recently proposed for CYP3A4, and is consistent with recent crystal structures of P450cam bound to tethered-substrates, which force a partially opened conformation.