Electrostatic control of the substrate access channel in cytochrome P-450cam.
Camphor binding to ferric cytochrome P-450cam 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-450cam 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, K1d, which decreases from 49.5 to 24 microM, 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 K1d is equal to 34 microM without sodium.(ABSTRACT TRUNCATED AT 250 WORDS)
Deprez
E
E
INSERM-INRA U310, Institut de Biologie Physico-Chimique, Paris, France.
Gerber
N C
NC
Di Primo
C
C
Douzou
P
P
Sligar
S G
SG
Hui Bon Hoa
G
G
D001665:Binding Sites; D002164:Camphor / Q000737:chemistry; D019475:Camphor 5-Monooxygenase; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000378:metabolism; D004058:Diffusion; D004563:Electrochemistry; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011994:Recombinant Proteins; D012964:Sodium / Q000737:chemistry; D013329:Structure-Activity Relationship; D013816:Thermodynamics
D011994:Recombinant Proteins; D002164:Camphor; D003577:Cytochrome P-450 Enzyme System; D012964:Sodium; D006899:Mixed Function Oxygenases; D019475:Camphor 5-Monooxygenase