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

• Published in: Journal of biological inorganic chemistry 1999-06, Vol.4 (3), p.257-265
• Main Authors: van Gastel, M, Coremans, J W, Mol, J, Jeuken, L J, Canters, G W, Groenen, E J
• Format: Article
• Language: English
• Subjects: Azurin - chemistry; Azurin - genetics; Azurin - metabolism; Binding Sites; Computer Simulation; Copper - metabolism; Electron Spin Resonance Spectroscopy - methods; Histidine - genetics; Imidazoles - chemistry; Imidazoles - metabolism; Models, Molecular; Nitrogen; Protein Conformation
• ISSN: 0949-8257; 1432-1327
• DOI: 10.1007/s007750050311

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.