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Journal of biological inorganic chemistry : JBIC : a publication of the Society of Biological Inorganic Chemistry

The binding of imidazole in an azurin-like blue-copper site.


PMID 10439070

Abstract

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.