ELECTROSTATIC CONTROL OF THE SUBSTRATE ACCESS CHANNEL IN CYTOCHROME P-450(CAM)

Camphor binding to ferric cytochrome P-450(cam) is a two-step process. The first step corresponds to the diffusion of camphor into the heme pocket, and the second one corresponds to an observable spin transition of the heme iron. In this paper, electrostatic interactions that may control the opening of the structure to allow substrate access to the buried and not solvent-exposed active site were examined. The electrostatic interactions occurring at the protein surface were weakened by increasing the ionic strength of the medium with sodium salts and strengthened by decreasing the dielectric constant of the medium with ethylene glycol as a cosolvent. The results obtained with the wild-type protein were compared to those obtained with the site-directed mutant of cytochrome P-450(cam) in which the Arg 186-Asp 251 and Lys 178-Asp 251 salt bridges, located at the entrance of the proposed access channel, were suppressed by replacing Asp 251 with an asparagine residue. Over a range of sodium chloride concentrations from 0 to 400 mM, camphor binding is favored, as seen in the variation in the first step dissociation equilibrium constant, K-ld, which decreases from 49.5 to 24 mu M, respectively. Addition of ethylene glycol favors the dissociation of the substrate-bound complex. The addition of sodium to the ethylene glycol-containing samples reverses the effect of the cosolvent. Removal of the Arg 186-Asp 251 and Lys 178-Asp 251 salt bridges results in an alteration in camphor binding in which K-ld is equal to 34 mu M Without sodium. The thermodynamic analysis of the overall binding step shows that the thermodynamic parameters for the diffusion step are equal when salt bridges are disrupted by the addition of sodium to the wild-type protein sample or by complete removal as in the D251N mutant. These results suggest a model in which the Arg 186-Asp 251-Lys 178 bifurcated salt bridge plays a key role in the control of camphor access to the buried active site of cytochrome P-450(cam).