Origin of the photoacoustic signal in cytochrome P-450cam: role of the Arg186-Asp251-Lys178 bifurcated salt bridge.
The origin of the photoacoustic signal in ferrous CO-camphor-cytochrome P-450cam was investigated. Recently, the Arg186-Asp251-Lys178 bifurcated salt bridge, located above the heme pocket, has been shown to play a key role in the control of the diffusion step of camphor binding [Deprez, E., Gerber, N. C., Di Primo, C., Douzou, P., Sligar, S. G., & Hui Bon Hoa, G. (1994) Biochemistry 33, 14464-14468]. We considered the hypothesis that electrostriction resulting from the transient exposure of these charged residues to the solvent could be responsible for 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 decrease from -5 to -24 kcal/mol and from 11 to 5.4 mL/mol, respectively. The mutation has the same effect on the thermodynamic parameters as increasing the 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 strength whereas a small effect is observed on the volume change. The results indicate that electrostriction around the bifurcated salt bridge contributes to the photoacoustic signal and suggest a scheme in which, following photodissociation of CO and diffusion of the molecule through the protein matrix, the structure relaxes and the bifurcated salt bridge desolvates.
Di Primo
C
C
Institut de Biologie Physico-Chimique, INSERM-INRA U310, Paris, France.
Deprez
E
E
Sligar
S G
SG
Hui Bon Hoa
G
G
D002164:Camphor / Q000378:metabolism*; D002248:Carbon Monoxide / Q000378:metabolism; D055598:Chemical Phenomena; D002621:Chemistry; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry*; D007834:Lasers; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D009154:Mutation / Q000235:genetics; D009994:Osmolar Concentration; D016958:Pseudomonas putida / Q000737:chemistry*; D012492:Salts / Q000494:pharmacology; D013696:Temperature; D013816:Thermodynamics
D012492:Salts; D002164:Camphor; D002248:Carbon Monoxide; D003577:Cytochrome P-450 Enzyme System