Superoxide dismutases (SOD) are a group of metalloenzymes (containing Fe, Mn, or Cu and Zn) that catalyze the disproportionation of superoxide free radical (O2) to form hydrogen peroxide and dioxygen as shown below (Eq. 1) [1].

 2 O2 + 2 H+   <==>  H2O2 + O2             (1)

These enzymes have been considered the defense system against the cytotoxic superoxide free radical.  Moreover, mutants of Cu,Zn-SOD have recently been found to associate with the Gehrig's disease (familial amytrophic lateral sclerosis) [2].   Cu,Zn-SOD is a prototypical dinuclear metalloprotein that has been subjected to extensive mechanistic and spectroscopic studies.  The structure of the enzyme from bovine erythrocytes (dimer with MW = 32,000 Da) has been determined at 2.0 Å resolution by the use of X-ray crystallography which revealed a distorted square pyramidal Cu2+ and a distorted tetrahedral Zn2+ in the active site [3].   The Cu2+ site is responsible of the catalysis and is coordinated by four His residues (His-44, His-46, His-61, and His-118) and a water molecule, and the Zn2+ site is bound to the protein through His-61, His-69, His-78, and Asp-81 (Figure 1).  The two metal ions are 6.3 Å away from each other and bridged by the imidazolate of the His-61 residue.  The positively charged Arg-141 located in the active site channel about 5 Å away from the Cu has been proposed to play a key role in the catalysis by providing an electric gradient to attract the negatively charged O2 substrate.  Studies focusing on this residue by means of chemical modification [4]  and inhibitor (phosphate) binding [5]  also indicate the significance of this residue in Cu,Zn-SOD catalysis.

 Figure 1.  Active site configuration of Cu,Zn-SOD based on crystal structure.  The sequence follows the bovine enzyme.
    Nearly two dozens of different metal-substituted derivatives of this enzyme have been prepared, including Cu2+/+, Ag+, Zn2+, Co2+, Cd2+, and Hg2+ derivatives [1] and several recently prepared Ni2+ derivatives [6,7]   All the derivatives with Cu2+ in the copper site, such as Cu,Cu-SOD, Cu,Co-SOD, and Cu,Ni-SOD, show high activities thus can serve as good models for the study of the molecular and mechanistic properties of the native enzyme by the use of different physical methods [1].  When both metal sites in the protein are occupied by paramagnetic metal ions (e.g., Co2+, Ni2+, or Cu2+), magnetic interaction occurs between the two sites mediated by the bridging imidazolate.  As a consequence, the slowly relaxing unpaired electron on the Cu2+ is significantly enhanced which enables the detection of sharp isotropically shifted proton NMR signals due to protons near the Cu2+ (Figure 2).  The signals A and A' are from the bridging His-61 ring protons.  The signals B-L in these two derivatives have similar chemical shifts and are attributable to His ring protons in the Cu2+ site.  The signals a-e are due to His ring protons in the Ni2+ site.  The isotropically shifted signals in the derivatives Cu,Ni-SOD have been assigned by means of  the NOESY technique ([8] see abstract and Figure).
    Figure 2.  The proton NMR spectra of magnetically coupled dinuclear metalloproteins Cu,Ni-SOD (Top) and Cu,Co-SOD (Bottom [9]).  The asterisked signals are solvent exchangeable His ring NH protons.   Assignments of the isotropically shifted signals can be achieved by means of anion titrations and NOESY.

    [1] Valentine, J. S., and Pantoliano, M. W. 1981, in Copper Proteins, Spiro, T. G., (Ed.), Wiley: NY, Vol. 3, Chapter 8.
    [2] (a) Radunovic, A., Shaw, C. E., Akman-Demir, G., Idrisoglu, H., and Leigh, P. N. 1997, "CuZnSOD-associated amyotrophic lateral sclerosis" Ann. Neurol., 42, 273-274.  (b) Hart, P. J., Liu, H., Pellegrini, M., Nersissian, A. M., Gralla, E. B., Valentine, J. S., and Eisenberg, D. 1998, "Subunit asymmetry in the three-dimensional structure of a human CuZnSOD mutant found in familial amyotrophic lateral sclerosis" Protein Sci., 7, 545-555
    [3] Tainer, J. A., Getzoff, E. D., Beem, K. M., Richardson, J. S., and Richardson, D. C. 1982, "Determination and analysis of the 2 Å-structure of copper, zinc superoxide dismutase" J. Mol. Biol., 160, 181-217
    [4] Mota de Freitas, D., Ming, L.-J., Ramasamy, R., and Valentine, J. S. 1990, "35Cl and 1H NMR Study of Anion Binding to Reduced Bovine Copper-Zinc Superoxide Dismutase" Inorg. Chem., 29, 3512-3518.
    [5] (a) Mota de Freitas, D., and Valentine, J. S. 1984, "Phosphate is an inhibitor of copper-zinc superoxide dismutase" Biochemistry, 23, 2079-2082.  (b) Mota de Freitas, D., Luchinat, C., Banci, L., Bertini, I., and Valentine, J. S. 1987, "31P NMR study of the interaction of inorganic phosphate with bovine copper-zinc superoxide dismutase" Inorg. Chem., 26, 2788-2791.
    [6] (a) Ming, L.-J., and Valentine, J. S. 1987, "Preparation and characterization of Cu2Ni2 and Ag2Ni2 superoxide dismutase, two new metal-substituted derivatives" J. Am. Chem. Soc., 109, 4426-4428.  (b) Ming, L.-J., Banci, L., Luchinat, C., Bertini, I., and Valentine, J. S. 1988, "Characterization of copper-nickel and silver-nickel bovine superoxide dismutase by 1H NMR spectroscopy" Inorg Chem., 27, 4458-4463.
    [7] Ming, L.-J., and Valentine, J. S. 1990, "NMR studies of nickel(II)-substituted derivatives of bovine copper-zinc superoxide dismutase with nickel(II) bound in the copper site"  J. Am. Chem. Soc., 112, 6374-6383.
    [8] Bertini, I., Luchinat, C., Ming, L.-J., Piccioli, M., Sola, M., and Valentine, J. S. 1992, "Two-dimensional 1H NMR studies of the paramagnetic metalloenzyme copper-nickel superoxide dismutase" Inorg. Chem., 31, 4433-4435.
    [9] Bertini, I., Lanini, G., Luchinat, C., Messori, L., Monnanni, R., Scozzafava, A. 1985, "Investigation of Cu2Co2SOD and its anion derivatives. 1H NMR and electronic spectra" J. Am. Chem. Soc., 107, 4391-4396.