2-DIMENSIONAL H-1-NMR STUDIES OF THE PARAMAGNETIC METALLOENZYME COPPER-NICKEL SUPEROXIDE-DISMUTASE

Normally, copper(II)containing metalloproteins are unsuitable for IH NMR spectral studies because the relatively long electronic relaxation times result in broad signals. However, for the copper(I1)-containing protein copper-zinc superoxide dismutase (Cu2Zn2SOD), substitution of Zn2+ by either Co2+ or Ni2+ provides derivatives in which the electronic relaxation times are dramatically shorter, due to interaction between the Cu2+ and Co2+ or Ni2+ mediated by the imidazolate bridge that links them (see Figure 1). We have found the Cu2Ni2SOD derivative to be particularly suitable for 2D IH NMR studies, and we report here such studies (NOESY") of this derivative which have allowed us to assign almost all ofthe isotropically shifted resonances attributable to the metal-coordinated histidine residues. This report presents the first example of the application of 2D NMR techniques to the assignment of isotropically shifted signals due to metal-coordinated histidine residues.