Two-dimensional NMR assignment of hyperfine-shifted resonances of very fast relaxing metal binding sites of proteins by NOE spectroscopy. The case of Cu, Co superoxide dismutase.

The isotropically shifted NMR resonances of bovine Cu, Co superoxide dismutase, arising from the imidazole protons of the metal binding site of the native Cu, Zn enzyme when Zn(II) is replaced by Co(II), were investigated by two-dimensional NMR. NOE spectra have been measured at different mixing times in order to obtain a good set of interproton dipolar connectivities. A number of NOEs were obtained for very fast relaxing resonances close to the paramagnetic copper ion and new isotropically shifted resonances were detected in the proximity of the diamagnetic region. The recently obtained original X-ray coordinates of the Cu, Co enzyme were used in order to give the appropriate values to the interproton distances of the imidazole rings of the copper-coordinated histidines. The data allowed an unequivocal and definitive assignment of the isotropically shifted resonances to be done, with respect to previous results obtained using the coordinates of the Cu, Zn enzyme and monodimensional NMR techniques, which contain a potential source of artifacts in the selective excitation required prior to acquisition of spectra.