Imidazole binding to horse metmyoglobin: dependence upon pH and ionic strength.
The reaction between metmyoglobin and imidazole has been studied as a function of pH between pH 4.2 and 11.5 and as a function of ionic strength at integral pH values (5 to 10) between 0.001 and 1.0 M ionic strength. The reaction between metmyoglobin and 1-methylimidazole has also been investigated as a function of pH. Comparison of the pH dependence of the association rate constants for the two ligands indicates that the negatively charged imidazolate ion does not contribute to the observed rate of imidazole binding at pH < or = 11.5. At all pH values between pH 4.2 and pH 11.5 the initial complex formed involves the neutral form of bound imidazole. At pH 11.5, the neutral imidazole complex is converted slowly (t1/2 approximately 10 s) into an imidazolate complex. The kinetic data were analyzed according to two mechanisms, one involving the binding of neutral imidazole only and one involving the direct binding of both imidazole and the imidazolium ion to metmyoglobin. Although secondary kinetic salt effects account for the ionic strength dependence of the association rate constant, evidence which indicates that metmyoglobin reacts with imidazole and with the imidazolium ion with similar rates is provided. A self-consistent analysis indicates that the rate constants for imidazole and imidazolium ion binding to metmyoglobin are 350 and 230 M-1 s-1, respectively, at neutral pH and 0.1 M ionic strength. Imidazole can react directly with hydroxymetmyoglobin with a rate of 56 M-1 s-1 at 0.1 M ionic strength, about sixfold slower than binding to aquometmyoglobin. Protonation of a second heme-linked group, thought to be His-97, has little influence on the binding of imidazole but does decrease the rate of imidazolium binding by about eightfold to 29 M-1 s-1 at 0.1 M ionic strength.
Lin
J
J
Department of Chemistry and Biochemistry, Northern Illinois University, DeKalb 60115, USA.
Vitello
L B
LB
Erman
J E
JE
D000818:Animals; D006736:Horses; D006863:Hydrogen-Ion Concentration; D007093:Imidazoles / Q000378:metabolism*; D007700:Kinetics; D008786:Metmyoglobin / Q000378:metabolism*; D009994:Osmolar Concentration; D011485:Protein Binding / Q000502:physiology; D013053:Spectrophotometry
D007093:Imidazoles; D008786:Metmyoglobin; C029899:imidazole; C018100:1-methylimidazole