Structural evidence for a second camphor binding site in P450cam from NMR and docking studies

Abstract

P450cam was the first cytochrome P450 (CYP) enzyme to be structurally characterized, and has since served as a prototype for the CYP gene family. But, little is known about how substrate gains access to the active site, and how access is accomplished in a way that minimizes exposure of the reactive heme. We present here our initial NMR T1 relaxation measurements and the computational docking studies that suggest a second camphor binding site, which is 15~16 Å from the heme-iron for both the P450cam (Fe3+)/camphor and the P450cam (Fe3+)/cyanide/camphor complexes. Furthermore, titration of 13C-threonine labeled P450cam (Fe3+) indicates that one threonine residue experiences chemical shift changes when a second equivalent of camphor is added. These NMR measured distances and chemical shift changes suggested that P450cam undergoes certain conformational change upon binding the second camphor. Located above helix I and in the helix F region, Thr151 and Asp251 may play an important role in this structural rearrangement - since Thr151 may hydrogen bond to the carbonyl of the second camphor and Asp251 is located between the second binding site and the active site. Our data suggest that if there is already a camphor or product in the active site, the second camphor may bind at the remote binding site first, until the camphor in the active site is released, at which point Asp251 may move to open the access to the active site. Such a two-step binding mechanism would minimize exposure of the heme to solvent by allowing P450cam to stay in a closed conformation as long as possible.