Structure of metal site in Cd-substituted His117Gly mutant of azurin with and without addition of imidazole derivatives.
The present work uses 111mCd-perturbed angular correlations of gamma-rays (PAC) to investigate the structure of the metal site of the His117Gly mutant of Pseudomonas aeruginosa azurin in aqueous solution and the effect on the structure upon addition of the following exogenous ligands: imidazole, 4-methyl imidazole, 1-methyl imidazole, 2-methyl imidazole and histidine. The nuclear quadrupole interaction of cadmium bound to the mutant without addition of exogenous ligands shows a strong pH dependence with three different nuclear quadrupole interactions consistent with two pKa values at about 7.2 and 8.6 at 2 degrees C. Addition of the imidazole derivatives resulted in a significant change in the PAC spectrum showing that they coordinate. This is in accordance with observations by EPR for the same mutant with copper at the metal site [den Blaauwen, T. & Canters, G. W. (1993) J. Am. Chem. Soc. 115, 1121-1129]. However, whereas EPR and ultraviolet/visual absorption show that the characteristics of the wild-type copper protein are regained by addition of the imidazole derivatives with the exception of the possible bidentates (histidine and histamine), the comparison of the PAC results to model calculations shows that the cadmium ion must be fourfold coordinated in most cases, probably binding an additional water or hydroxide ligand. A fourfold coordination is in contrast to cadmium-substituted wild-type azurin where PAC data inferred a threefold coordination by a Cys and two His residues [Danielsen, E. Bauer, R., Hemmingsen, L., Andersen. M., Bjerrum, M. J., Butz, T., Tröger, W., Canters, G. W., Hoitink, C. W. G., Karlsson, G., Hansson, O. & Messerschmidt, A. (1995) J. Biol. Chem. 270, 573-580]
Danielsen
E
E
Physics laboratory, Royal Veterinary and Agricultural University, Frederiksberg, Denmark.
Bauer
R
R
Hemmingsen
L
L
Bjerrum
M J
MJ
Butz
T
T
Tröger
W
W
Canters
G W
GW
Den Blaauwen
T
T
Van Pouderoyen
G
G
D001400:Azurin / Q000737:chemistry*; D002104:Cadmium; D005998:Glycine; D006639:Histidine; D009154:Mutation
D002104:Cadmium; D001400:Azurin; D006639:Histidine; D005998:Glycine