ORIGIN OF THE PHOTOACOUSTIC SIGNAL IN CYTOCHROME P-450(CAM) - ROLE OFTHE ARG186-ASP251-LYS178 BIFURCATED SALT BRIDGE

The origin of the photoacoustic signal in ferrous CO-camphor-cytochrom e P-450(cam) was investigated. Recently, the Arg186-Asp251-Lys178 bifu rcated salt bridge. located above the heme pocket, has been shown to p lay a key role in the control of the diffusion step of camphor binding [Deprez, E., Gerber. N. C., Di Prime, C., Douzou, P., Sligar, S, G,, & Kui Bon Hoa, G. (1994) Biochemistry 33, 14464-14468]. We considered the hypothesis that electrostriction resulting from the transient expo sure of these charged residues to the solvent could bit responsible fo r part of the photoacoustic signal, We thus examined the effects of a site-directed mutation of these linkages and ionic strength increases. Upon replacement of the Asp251 residue by an asparagine residue, the overall enthalpy and volume change of the CO dissociation reaction dec rease from -5 to -24 kcal/mol and from 11 to 5.4 mL/mol, respectively, The mutation has the same effect oil the thermodynamic parameters as increasing tile ionic strength of the medium over a range of potassium or sodium concentrations from 0 to 500 mM. For the D251N mutant, the overall enthalpy of the reaction does not change with the ionic streng th whereas a small effect is observed on the volume change. The result s indicate that electrostriction around the bifurcated salt bridge con tributes to the photoacoustic signal and suggest a scheme in which, fo llowing photodissociation of CO and diffusion of tile molecule through the protein matrix, the structure relaxes and the bifurcated salt bri dge desolvates.