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

Frozen solutions of the azurin mutant His117Gly in the presence of excess o f methyl-substituted imidazoles have been investigated by electron spin-ech o envelope modulation (ESEEM) spectroscopy at 9 GHz. The addition of imidaz ole is known to reconstitute a blue-copper site and variation of the non-pr otein 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 t he methyl-substituted imidazoles in the mutant adopt the same orientation r elative to copper as the histidine-117 in the wild-type protein. Analysis o f the hyperfine tensors in terms of spin densities reveals that the spin de nsity on the remote nitrogen of the substituted imidazole has sigma and a v ariable pi character, depending on the position of the methyl group. For az urin the corresponding spin density is of virtually pure a character. In co nclusion, blue copper sites show subtle variations as regards the histidine /imidazole centred part of the wavefunction of the unpaired electron.