The binding of imidazole in an azurin-like blue-copper site.
Frozen solutions of the azurin mutant His117Gly in the presence of excess of methyl-substituted imidazoles have been investigated by electron spin-echo envelope modulation (ESEEM) spectroscopy at 9 GHz. The addition of imidazole is known to reconstitute a blue-copper site and variation of the non-protein bound ligand [N-methyl-, 2-methyl-, 4(5)-methylimidazole] has allowed the study of the copper-imidazole binding as a model for histidine binding in such sites. Quadrupole and hyperfine tensors of the remote nitrogen of the imidazoles have been determined. The quadrupole tensors indicate that the methyl-substituted imidazoles in the mutant adopt the same orientation relative to copper as the histidine-117 in the wild-type protein. Analysis of the hyperfine tensors in terms of spin densities reveals that the spin density on the remote nitrogen of the substituted imidazole has sigma and a variable pi character, depending on the position of the methyl group. For azurin the corresponding spin density is of virtually pure sigma character. In conclusion, blue-copper sites show subtle variations as regards the histidine/imidazole centred part of the wavefunction of the unpaired electron.
van Gastel
M
M
Centre for the Study of Excited States of Molecules, University of Leiden, Huygens Laboratory, The Netherlands.
Coremans
J W
JW
Mol
J
J
Jeuken
L J
LJ
Canters
G W
GW
Groenen
E J
EJ
D001400:Azurin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D003198:Computer Simulation; D003300:Copper / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D006639:Histidine / Q000235:genetics; D007093:Imidazoles / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D009584:Nitrogen; D011487:Protein Conformation
D007093:Imidazoles; D001400:Azurin; D006639:Histidine; D003300:Copper; C029899:imidazole; D009584:Nitrogen; C023439:4-methylimidazole; C032655:2-methylimidazole