European Biophysics Journal

, Volume 23, Issue 3, pp 167–176 | Cite as

A spectroscopic characterization of a monomeric analog of copper, zinc superoxide dismutase

  • Ivano Bertini
  • Mario Piccioli
  • Maria Silvia Viezzoli
  • Choi Ying Chiu
  • Guy T. Mullenbach


A mutated protein of human Cu(II)2Zn(II)2 SOD in which residues Phe50 and Gly51 at the dimer interface were substituted by Glu's, thus producing a monomeric species, has been characterized by electronic absorption spectroscopy, EPR, relaxivity and1H NMR techniques. Such substitutions and/or accompanying remodeling and exposure of the dimer interface to solvent, alter the geometry of the active site: increases in the axiality of the copper chromophore and the Cu-OH2 distance have been observed. The affinity of both metal binding sites for Co(II) is also altered. The observed NMR parameters of the Co(II) substituted derivative have been interpreted as a function of the decrease of rotational correlation time as a consequence of the lower molecular weight of the mutated protein. Sharper NMR signals are also obtained for the reduced diamagnetic enzyme. Results are consistent with an active site structure similar to that observed for the dimeric analog Thr137Ile characterized elsewhere. An observed proportional decrease in enzymatic activity and affinity for the N3-anion suggests the importance of electrostatic forces during substrate docking and catalysis.

Key words

Cu Zn Superoxide Dismutase 1H NMR Monomeric analog 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Banci L, Bertini I, Luchinat C, Monnanni R, Scozzafava A, Salvato B (1986) A spectroscopic investigation of Co2Zn2- and Co2Co2-superoxide dismutase. Gazz Chim Ital 116:51–54Google Scholar
  2. Banci L, Bertini I, Luchinat C, Scozzafava A (1987) Nuclear relaxation in the magnetic coupled system Cu2Co2SOD. J Am Chem Soc 109:2328–2334Google Scholar
  3. Banci L, Bertini I, Luchinat C, Hallewell RA (1988) An investigation of superoxide dismutase Lys-143, Ile-143, and Glu-143 mutants: Cu2Co2SOD derivatives. J Am Chem Soc 110:3629–3633Google Scholar
  4. Banci L, Bertini I, Luchinat C, Piccioli M, Scozzafava A, Turano P (1989 a)1H NOE studies on dicopper(II) dicobalt(II) superoxide dismutase. Inorg Chem 28:4650–4656Google Scholar
  5. Banci L, Bertini I, Luchinat C, Piccioli M, Scozzafava A (1989 b) Cyanide and azide behave in a similar fashion versus cupro-zinc superoxide dismutase. J Biol Chem 264:9742–9744Google Scholar
  6. Banci L, Bertini I, Luchinat C, Piccioli M (1990a) Spectroscopic studies on Cu2Zn2SOD: a continous advancement of investigation tools. Coord Chem Rev 100:67–103Google Scholar
  7. Banci L, Bertini I, Cabelli DE, Hallewell RA, Luchinat C, Viezzoli MS (1990b) Investigation of copper-zinc superoxide dismutase Ser-137 and Ala-137 mutants. Inorg Chem 29:2398–2403Google Scholar
  8. Banci L, Bencini A, Bertini I, Luchinat C, Viezzoli MS (1990 c) The angular overlap analysis of the spectroscopic parameters of copper-zinc SOD and its mutants. Gazz Chim Ital 120:179–185Google Scholar
  9. Banci L, Bencini A, Bertini I, Luchinat C, Piccioli M (1990d)1H NOE and ligand field studies of superoxide dismutase with anions. Inorg Chem 29:4867–4873Google Scholar
  10. Banci L, Bertini I, Cabelli DE, Hallewell RA, Tung JW, Viezzoli MS (1991 a) A characterization of copper/zinc superoxide dismutase mutants at position 124-Zinc deficient proteins. Eur J Biochem 196:123–128Google Scholar
  11. Banci L, Bertini I, Cabelli DE, Hallewel RA, Luchinat C, Viezzoli MS (1991 b) Advances in understanding of the structure-function relationship in Cu, Zn superoxide dismutase. Free Rad Res Commun 12–13:239–251Google Scholar
  12. Banci L, Bertini I, Luchinat C, Piccioli M (1991 c) Frontiers in NMR of paramagnetic molecules:1H NOE and related experiments. In: NMR and Macromolecular Structure (Bertini I, Niccolai N and Molinari H, eds.) Ch. 2, VCH, WeinheimGoogle Scholar
  13. Banci L, Carloni P, La Penna G, Orioli PL (1992) Molecular dynamics studies on superoxide dismutase and its mutants: the structural and functional role of Arg143. J Am Chem Soc 114: 6994–7001Google Scholar
  14. Banci L, Bertini I, Luchinat C, Piccioli M, Scozzafava A (1993) 1D versus 2D1H NMR experiments in dicopper, dicobalt superoxide dismutase: a further mapping of the active site. Gazz Chim Ital 123:95–100Google Scholar
  15. Bertini I, Luchinat C (1986) NMR of paramagnetic molecules in biological systems. Benjamin/Cummings, Menlo Park, CAGoogle Scholar
  16. Bertini I, Scozzafava A (1981) Copper(II) as probe in substituted metalloproteins. In: Metal ions in biological systems, Vol 12. Siegel H (ed) Marcel Dekker, New York, pp 31–74Google Scholar
  17. Bertini I, Lanini G, Luchinat C, Messori L, Monnanni R, Scozzafava A (1985 a) An investigation of Cu2Co2SOD and its anion derivatives. J Am Chem Soc 107:4391–4396Google Scholar
  18. Bertini I, Luchinat C, Mancini M, Spina G (1985b) The dipolar coupling between unpaired electrons and resonanting nuclei. In: Magneto-structural correlations in exchanged coupled systems. Gatteschi D, Kahn O, Willett RD (eds) Reidel, Dordrecht, pp 421–461Google Scholar
  19. Bertini I, Luchinat C, Viezzoli MS (1986) Metal substitution as a tool for the investigation of zinc proteins. In: Zinc enzymes. Bertini I, Luchinat C, Maret W, Zeppezauer M (eds) Birkhauser, Cambridge MA, pp 27–47Google Scholar
  20. Bertini I, Banci L, Luchinat C, Bielski BHJ, Cabelli DE, Mullenbach GT, Hallewell RA (1989) An investigation of a human erythrocyte SOD modified at the position 137. Inorg Chem 111:714–719Google Scholar
  21. Bertini I, Capozzi F, Luchinat C, Piccioli M, Viezzoli MS (1991 a) Assignment of active site protons in the1H NMR spectrum of reduced human Cu, ZN superoxide dismutase. Eur J Biochem 197:691–697Google Scholar
  22. Bertini I, Lepori A, Luchinat C, Turano P (1991 b) Role of Arg-143 in human Cu2Zn2SOD studied through anion binding. Inorg Chem 30:3363–3364Google Scholar
  23. Bertini I, Luchinat C, Piccioli M (1994) Copper zinc superoxide dismutase: a paramagnetic protein that provides a unique frame for the NMR investigations. Progr Nucl Magn Reson Spectrosc 26:91–140Google Scholar
  24. Beyer WF, Fridovich I, Mullenbach GT, Hallewell RA (1987) Examination of the role of arginine-143 in the human copper and zinc superoxide dismutase by site-specific mutagenesis. J Biol Chem 262:11182–11187Google Scholar
  25. Blackburn NJ, Hasnain SS, Binsted N, Diakun GP, Garner CD, Knowles PF (1984) An extended X-ray absorption fine-structure study of bovine superoxide dismutase in aqueous solution. Biochem J 219:985–990Google Scholar
  26. Cass AEG, Hill HAO, Smith BE, Bannister JV, Bannister WH (1977a) Carbon-2 proton exchange at histidine-41 in bovine erythrocyte superoxide dismutase. Biochem J 165:587–589Google Scholar
  27. Cass AEG, Hill HAO, Smith BE, Bannister JV, Bannister WH (1977b) Investigation of the structure of bovine erythrocyte superoxide dismutase by1H nuclear magnetic resonance spectroscopy. Biochemistry 16:3061–3066Google Scholar
  28. Dugad LB, La Mar GN, Unger SW (1990) The influence of molecular correlation time on the homonuclear Overhauser effect in paramagnetic proteins. J Am Chem Soc 112:1386–1392Google Scholar
  29. Ernst RR, Bodenhausen G, Wokaun A (1987) Principles of NMR in one and two dimensions. Clarendon Press, OxfordGoogle Scholar
  30. Fee JA (1973) Studies on the reconstitution of bovine erythrocyte superoxide dismutase. IV: Preparation and some properties of the enzyme in which Co(II) is substituted by Zn(II). J Biol Chem 248:4229–4234Google Scholar
  31. Forman JH, Evans HJ, Hill RL, Fridovich I (1973) Histidine at the active site of superoxide dismutase. Biochemistry 12:823–827Google Scholar
  32. Fridovich I (1974) Superoxide dismutase. Adv Enzymol 41:35–97Google Scholar
  33. Fridovich I (1987) Superoxide dismutase. Adv Enzymol Relat Areal Mol Biol 58:61–97Google Scholar
  34. Getzoff ED, Cabelli DE, Fisher CL, Parge HE, Viezzoli MS, Banci L, Hallewell RA (1992) Faster superoxide dismutase mutants designed by enhancing electrostatic guidance. Nature 358:347–351Google Scholar
  35. Inubushi T, Becker ED (1983) Efficient detection of paramagnetically shifted NMR resonances by optimizing the WEFT pulse sequence. J Magn Reson 512:128–133Google Scholar
  36. Koenig SH, Brown RD III (1987) In: NMR spectroscopy of cells and organisms. Gupta RK (ed) CRC Press, Boca Raton, pp 75–85Google Scholar
  37. Koenig SH, Brown RD III (1990) Field-cycling relaxometry of protein solutions and tissue. Implication for MRI. Progn Nucl Magn Reson Spectrosc 22:487–567Google Scholar
  38. Lecomte JTJ, Unger SW, La Mar GN (1991) Practical considerations for the measurements of the mononuclear Overhauser effect on strongly relaxed protons in paramagnetic proteins. J Magn Reson 94:112–122Google Scholar
  39. Lever ABP (1984) Inorganic electronic spectroscopy, 2nd edn. Elsevier, AmsterdamGoogle Scholar
  40. Lippard SJ, Burger AR, Ugurbil K, Pantoliano MW, Valentine JS (1987) Nuclear magnetic resonance and chemical modification studies of bovine erythrocyte superoxide dismutase: evidence for zinc-promoted organization of the active site structure. Biochemistry 16:1136–1141Google Scholar
  41. Marion D, Wutrich K (1983) Application of phase sensitive 2-dimensional correlated spectroscopy (COSY) for measurements of1H-1H spin-spin constants in proteins. Biochem Biophys Res Commun 113:967–974Google Scholar
  42. McCord JM, Fridovich I (1969) Superoxide dismutase. An enzymatic function for erythrocuprein (hemocuprein) J Biol Chem 244: 6049–6055Google Scholar
  43. Mota de Freitas D, Ming L-J, Ramasamy R, Valentine JS (1990)13Cl and1H NMR study of anion binding to reduced bovine copper-zinc superoxide dismutase. Inorg Chem 29:3512–3518Google Scholar
  44. Paci M, Desideri A, Sette M, Ciriolo MR, Rotilio G (1990) Assignment of imidazole resonances from two-dimensional proton NMR spectra of bovine Cu, Zn superoxide dismutase. Evidence for similar active site conformation in the oxidized and reduced enzyme. FEBS Lett 263:127–130Google Scholar
  45. Pantoliano MW, Valentine JS, Mammone RJ, Scholler DM (1979) Reversible loss of metal ions from the zinc binding site of copperzinc superoxide dismutase. The low pH transition. J Am Chem Soc 101:6454–6456Google Scholar
  46. Pantoliano MW, Valentine JS, Nafie LA (1982) Spectroscopic studies of copper(II) bound at the native copper site or substituted at the native zinc site of bovine erythrocuprein (superoxide dismutase). J Am Chem Soc 104:6310–6317Google Scholar
  47. Rotilio G, Morpurgo L, Giovagnoli C, Calabrese L, Mondovï B (1972a) Metal sites of copper proteins. III. Simmetry of copper in bovine superoxide dismutase and its function significance. Biochemistry 11:2187–2192Google Scholar
  48. Rotilio G, Calabrese L, Bossa F, Barra D, Finazzi Agró A, Mondovï B (1972b) Properties of the apoprotein and role of copper and zinc in protein conformation and enzyme activity of bovine superoxide dismutase. Biochemistry 11:2182–2187Google Scholar
  49. Sklenar V, Bax A (1987) Spin-echo water suppression for the generation of pure-phase two dimensional NMR spectra. J Magn Reson 74:469–479Google Scholar
  50. Stoesz JD, Malinowski DP, Redfield AG (1979) Nuclear magnetic resonance study of solvent exchange and nuclear Overhauser effect of the histidine protons of bovine superoxide dismutase. Biochemistry 18:4669–4675Google Scholar
  51. Tainer JA, Getzoff ED, Beem KM, Richardson JS, Richardson DC (1982) Determination and analysis of the 2-Å structure of copper, zinc superoxide dismutase. J Mol Biol 160:181–217Google Scholar
  52. Valentine JS, Pantoliano MW (1982) Protein-metal ion interaction in cuprozinc protein (superoxide dismutase). In: Copper proteins, Vol 3. Spiro G (ed). Wiley, New York, pp 291–358Google Scholar

Copyright information

© Springer-Verlag 1994

Authors and Affiliations

  • Ivano Bertini
    • 1
  • Mario Piccioli
    • 1
  • Maria Silvia Viezzoli
    • 1
  • Choi Ying Chiu
    • 2
  • Guy T. Mullenbach
    • 2
  1. 1.Department of ChemistryUniversity of FlorenceFlorenceItaly
  2. 2.Chiron CorporationEmeryvilleUSA

Personalised recommendations