| Abstract
| - The blue copper proteins (BCPs), pseudoazurin from Achromobacter cycloclastes and rusticyaninfrom Thiobacillus ferrooxidans, have been investigated by 1H NMR at a magnetic field of 18.8 T. Hyperfineshifts of the protons belonging to the coordinated ligands have been identified by exchange spectroscopy,including the indirect detection for those resonances that cannot be directly observed (the β-CH2 of theCys ligand, and the NH amide hydrogen bonded to the Sγ(Cys) atom). These data reveal that the Cu(II)−Cys interaction in pseudoazurin and rusticyanin is weakened compared to that in classic blue sites(plastocyanin and azurin). This weakening is not induced by a stronger interaction with the axial ligand, asfound in stellacyanin, but might be determined by the protein folding around the metal site. The averagechemical shift of the β-CH2 Cys ligand in all BCPs can be correlated to geometric factors of the metal site(the Cu−Sγ(Cys) distance and the angle between the CuNHisNHis plane and the Cu−Sγ(Cys) vector). It isconcluded that the degree of tetragonal distortion is not necessarily related to the strength of the Cu(II)−Sγ(Cys) bond. The copper−His interaction is similar in all BCPs, even for the solvent-exposed His ligand.It is proposed that the copper xy magnetic axes in blue sites are determined by subtle geometricaldifferences, particularly the orientation of the His ligands. Finally, the observed chemical shifts for β-CH2Cys and Ser NH protons in rusticyanin suggest that a less negative charge at the sulfur atom could contributeto the high redox potential (680 mV) of this protein.
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