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Nickel superoxide dismutase: structural and functional roles of Cys2 and Cys6

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Abstract

Nickel superoxide dismutase (NiSOD) is unique among the family of superoxide dismutase enzymes in that it coordinates Cys residues (Cys2 and Cys6) to the redox-active metal center and exhibits a hexameric quaternary structure. To assess the role of the Cys residues with respect to the activity of NiSOD, mutations of Cys2 and Cys6 to Ser (C2S-NiSOD, C6S-NiSOD, and C2S/C6S-NiSOD) were carried out. The resulting mutants do not catalyze the disproportionation of superoxide, but retain the hexameric structure found for wild-type NiSOD and bind Ni(II) ions in a 1:1 stoichiometry. X-ray absorption spectroscopic studies of the Cys mutants revealed that the nickel active-site structure for each mutant resembles that of C2S/C6S-NiSOD and demonstrate that mutation of either Cys2 or Cys6 inhibits coordination of the remaining Cys residue. Mutation of one or both Cys residue(s) in NiSOD induces the conversion of the low-spin Ni(II) site in the native enzyme to a high-spin Ni(II) center in the mutants. This result indicates that coordination of both Cys residues is required to generate the native low-spin configurations and maintain catalytic activity. Analysis of the quaternary structure of the Cys mutants by differential scanning calorimetry, mass spectrometry, and size-exclusion chromatography revealed that the Cys ligands, particularly Cys2, are also important for stabilizing the hexameric quaternary structure of the native enzyme.

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Abbreviations

CuZnSOD:

Copper- and zinc-containing superoxide dismutase

DFT:

Density functional theory

DSC:

Differential scanning calorimetry

EPR:

Electron paramagnetic resonance

ESI-MS:

Electrospray ionization mass spectrometry

EXAFS:

Extended X-ray absorption fine structure

FeSOD:

Iron-containing superoxide dismutase

LIC:

Ligation-independent cloning

MnSOD:

Manganese-containing superoxide dismutase

NHE:

Normal hydrogen electrode

Ni–NTA:

Nickel nitrilotriacetic acid

NiSOD:

Nickel-containing superoxide dismutase

PCR:

Polymerase chain reaction

SOD:

Superoxide dismutase

Tris:

Tris(hydroxymethyl)aminomethane

XANES:

X-ray absorption near-edge spectroscopy

XAS:

X-ray absorption spectroscopy

References

  1. Cabelli DE, Riley D, Rodriguez JA, Valentine JS, Zhu H (1998) In: Meunier B (eds) Biomimetic oxidations catalyzed by transition metal complexes. Imperial College Press, London, pp 461–508

  2. Fridovich I (1995) Ann Rev Biochem 64:97–112

    Article  PubMed  CAS  Google Scholar 

  3. Miller AF, Sorkin DL (1997) Comments Mol Cell Biophys 9(1):1–48

    CAS  Google Scholar 

  4. Touati D (1997) In: Scandalios JG (eds) Oxidative stress and the molecular biology of antioxidant defenses. Cold Spring Harbor Laboratory Press, Plainview, pp 447–493

  5. Bannister JV, Bannister WH, Rotilio G (1987) CRC Crit Rev Biochem 22(2):111–180

    Article  PubMed  CAS  Google Scholar 

  6. Fridovich I (1975) Ann Rev Biochem 44:147–159

    Article  PubMed  CAS  Google Scholar 

  7. Kim EJ, Chung HJ, Suh B, Hah YC, Roe JH (1998) Mol Microbiol 27(1):187–195

    Article  PubMed  CAS  Google Scholar 

  8. Lee J-W, Roe J-H, Kang SO (2002) Methods Enzymol 349:90–101

    Google Scholar 

  9. Uudsemaa M, Tamm T (2003) J Phys Chem A 107(46):9997–10003

    Article  CAS  Google Scholar 

  10. Fee JA, Valentine JS (1977) In: Michelson AM, McCord JM, Fridovich I (eds) Superoxide and superoxide dismutases. Academic Press, London, pp 25–28

  11. Bordo D, Matak D, Djinovic-Carugo K, Rosano C, Pesce A, Bolognesi M, Stroppolo ME, Falconi M, Battistoni A, Desideri A (1999) J Mol Biol 285(1):283–296

    Article  PubMed  CAS  Google Scholar 

  12. Borgstahl GE, Parge HE, Hickey MJ, Beyer WF Jr, Hallewell RA, Tainer JA (1992) Cell 71(1):107–118

    Article  PubMed  CAS  Google Scholar 

  13. Lah MS, Dixon MM, Pattridge KA, Stallings WC, Fee JA, Ludwig ML (1995) Biochemistry 34(5):1646–1660

    Article  PubMed  CAS  Google Scholar 

  14. Tierney DL, Fee JA, Ludwig ML, Pennerhahn JE (1995) Biochemistry 34(5):1661–1668

    Article  PubMed  CAS  Google Scholar 

  15. Barondeau DP, Kassmann CJ, Bruns CK, Tainer JA, Getzoff ED (2004) Biochemistry 43(25):8038–8047

    Article  PubMed  CAS  Google Scholar 

  16. Wuerges J, Lee JW, Yim YI, Yim HS, Kang SO, Carugo KD (2004) Proc Natl Acad Sci USA 101(23):8569–8574

    Article  PubMed  CAS  Google Scholar 

  17. Darnault C, Volbeda A, Kim EJ, Legrand P, Vernede X, Lindahl PA, Fontecilla-Camps JC (2003) Nat Struct Biol 10(4):271–279

    Article  PubMed  CAS  Google Scholar 

  18. Doukov TI, Iverson TM, Seravalli J, Ragsdale SW, Drennan CL (2002) Science 298(5593):567–572

    Article  PubMed  CAS  Google Scholar 

  19. Dobbek H, Svetlitchnyi V, Gremer L, Huber R, Meyer O (2001) Science 293(5533):1281–1285

    Article  PubMed  CAS  Google Scholar 

  20. Volbeda A, Charon MH, Piras C, Hatchikian EC, Frey M, Fontecilla-Camps JC (1995) Nature 373(6515):580–587

    Article  PubMed  CAS  Google Scholar 

  21. Ermler U, Grabarse W, Shima S, Goubeaud M, Thauer RK (1997) Science 278(5342):1457–1462

    Article  PubMed  CAS  Google Scholar 

  22. Jabri E, Carr MB, Hausinger RP, Karplus PA (1995) Science 268(5213):998–1004

    Article  PubMed  CAS  Google Scholar 

  23. He MM, Clugston SL, Honek JF, Matthews BW (2000) Biochemistry 39(30):8719–8727

    Article  PubMed  CAS  Google Scholar 

  24. Al-Mjeni F, Ju T, Pochapsky TC, Maroney MJ (2002) Biochemistry 41(21):6761–6769

    Article  PubMed  CAS  Google Scholar 

  25. Pochapsky TC, Pochapsky SS, Ju T, Mo H, Al-Mjeni F, Maroney MJ (2002) Nat Struct Biol 9(12):966–972

    Article  PubMed  CAS  Google Scholar 

  26. Fiedler AT, Bryngelson PA, Maroney MJ, Brunold TC (2005) J Am Chem Soc 127(15):5449–5462

    Article  PubMed  CAS  Google Scholar 

  27. Pelmenschikov V, Siegbahn PEM (2006) J Am Chem Soc 128(23):7466–7475

    Article  PubMed  CAS  Google Scholar 

  28. Prabhakar R, Morokuma K, Musaev DG (2006) J Comput Chem 27(12):1438–1445

    Article  PubMed  CAS  Google Scholar 

  29. Jackson TA, Brunold TC (2004) Acc Chem Res 37(7):461–470

    Article  PubMed  CAS  Google Scholar 

  30. Miller AF (2008) Acc Chem Res 41(4):501–510

    Article  PubMed  CAS  Google Scholar 

  31. Rulisek L, Jensen KP, Lundgren K, Ryde U (2006) J Comput Chem 27(12):1398–1414

    Article  PubMed  CAS  Google Scholar 

  32. Vance CK, Miller AF (1998) J Am Chem Soc 120(3):461–467

    Article  CAS  Google Scholar 

  33. Carrasco R, Morgenstern-Badarau I, Cano J (2007) Inorg Chim Acta 360(1):91–101

    Article  CAS  Google Scholar 

  34. Miller AF, Padmakumar K, Sorkin DL, Karapetian A, Vance CK (2003) J Inorg Biochem 93(1–2):71–83

    Article  PubMed  CAS  Google Scholar 

  35. Herbst RW, Guce A, Bryngelson PA, Higgins KA, Ryan KC, Cabelli DE, Garman SC, Maroney MJ (2009) Biochemistry 48(15):3354–3369

    Article  PubMed  CAS  Google Scholar 

  36. Fisher CL, Cabelli DE, Hallewell RA, Beroza P, Lo TP, Getzoff ED, Tainer JA (1997) Proteins Struct Funct Bioinf 29(1):103–112

    Article  CAS  Google Scholar 

  37. Miller AF, Sorkin DL, Padmakumar K (2005) Biochemistry 44(16):5969–5981

    Article  PubMed  CAS  Google Scholar 

  38. Tabares LC, Cortez N, Un S (2007) Biochemistry 46(32):9320–9327

    Article  PubMed  CAS  Google Scholar 

  39. Allan CB, Davidson G, Choudhury SB, Gu ZJ, Bose K, Day RO, Maroney MJ (1998) Inorg Chem 37(17):4166–4167

    Article  PubMed  CAS  Google Scholar 

  40. Chohan BS, Shoner SC, Kovacs JA, Maroney MJ (2004) Inorg Chem 43(24):7726–7734

    Article  PubMed  CAS  Google Scholar 

  41. Grapperhaus CA, Darensbourg MY (1998) Acc Chem Res 31(8):451–459

    Article  CAS  Google Scholar 

  42. Shoner SC, Olmstead MM, Kovacs JA (1994) Inorg Chem 33(1):7–8

    Article  CAS  Google Scholar 

  43. Bryngelson PA, Arobo SE, Pinkham JL, Cabelli DE, Maroney MJ (2004) J Am Chem Soc 126(2):460–461

    Article  PubMed  CAS  Google Scholar 

  44. Leitch S, Bradley MJ, Rowe JL, Chivers PT, Maroney MJ (2007) J Am Chem Soc 129(16):5085–5095

    Article  PubMed  CAS  Google Scholar 

  45. Padden KM, Krebs JF, MacBeth CE, Scarrow RC, Borovik AS (2001) J Am Chem Soc 123(6):1072–1079

    Article  PubMed  CAS  Google Scholar 

  46. Webb SM (2005) Phys Scr T 115:1011–1014

    Article  Google Scholar 

  47. Ankudinov AL, Ravel B, Rehr JJ, Conradson SD (1998) Phys Rev B 58(12):7565–7576

    Article  CAS  Google Scholar 

  48. Johnson OE, Ryan KC, Maroney MJ, Brunold TC (2009) J Biol Inorg Chem

  49. Colpas GJ, Maroney MJ, Bagyinka C, Kumar M, Willis WS, Suib SL, Baidya N, Mascharak PK (1991) Inorg Chem 30(5):920–928

    Article  CAS  Google Scholar 

  50. Bielski BH, Cabelli DE (1991) Int J Radiat Biol 59(2):291–319

    Article  PubMed  CAS  Google Scholar 

  51. Fridovich I (1998) J Exp Biol 201(8):1203–1209

    PubMed  CAS  Google Scholar 

  52. Choudhury SB, Lee JW, Davidson G, Yim YI, Bose K, Sharma ML, Kang SO, Cabelli DE, Maroney MJ (1999) Biochemistry 38(12):3744–3752

    Article  PubMed  CAS  Google Scholar 

  53. Cowan JA (1997) Inorganic biochemistry: an introduction, 2nd edn. Wiley-VCH, New York

    Google Scholar 

  54. Szilagyi RK, Bryngelson PA, Maroney MJ, Hedman B, Hodgson KO, Solomon EI (2004) J Am Chem Soc 126(10):3018–3019

    Article  PubMed  CAS  Google Scholar 

  55. Ray M, Hammes BS, Yap GPA, Rheingold AL, Borovik AS (1998) Inorg Chem 37(7):1527–1532

    Article  CAS  Google Scholar 

  56. Melnik M, Sramko T, Dunajjurco M, Sirota A, Jona E, Holloway CE (1994) Rev Inorg Chem 14(1–4):1–300

    CAS  Google Scholar 

  57. Rosenfield SG, Armstrong WH, Mascharak PK (1986) Inorg Chem 25(17):3014–3018

    Article  CAS  Google Scholar 

  58. Baidya N, Olmstead M, Mascharak PK (1991) Inorg Chem 30(5):929–937

    Article  CAS  Google Scholar 

  59. Marganian CA, Vazir H, Baidya N, Olmstead MM, Mascharak PK (1995) J Am Chem Soc 117(5):1584–1594

    Article  CAS  Google Scholar 

  60. Rosenfield SG, Berends HP, Gelmini L, Stephan DW, Mascharak PK (1987) Inorg Chem 26(17):2792–2797

    Article  CAS  Google Scholar 

  61. Iwig JS, Leitch S, Herbst RW, Maroney MJ, Chivers PT (2008) J Am Chem Soc 130(24):7592–7606

    Article  PubMed  Google Scholar 

  62. Calatayud ML, Castro I, Sletten J, Cano J, Lloret F, Faus J, Julve M, Seitz G, Mann K (1996) Inorg Chem 35(10):2858–2865

    Article  Google Scholar 

  63. Halonen P, Tammenkoski M, Niiranen L, Huopalahti S, Parfenyev AN, Goldman A, Baykov A, Lahti R (2005) Biochemistry 44(10):4004–4010

    Article  PubMed  CAS  Google Scholar 

  64. Niemoth-Anderson JD, Rodriguez JA, Lee J-W, Roe J, Yim YI, Cabelli DE, Valentine JS, Kang S-O, Maroney MJ (2000) Book of abstracts, 219th ACS national meeting, San Francisco, CA, INOR-515

  65. Youn HD, Kim EJ, Roe JH, Hah YC, Kang SO (1996) Biochem J 318:889–896

    PubMed  CAS  Google Scholar 

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Acknowledgments

This work was supported by grants from the National Science Foundation (CHE-0809188 to M.J.M) and the National Institutes of Health (GM 64631 to T.C.B.) and by a National Institutes of Health Chemistry–Biology Interface Training Grant (T32 GM008505 to O.E.J.). The US Department of Energy, Division of Materials Sciences and Division of Chemical Sciences, supported XAS data collection at the National Synchrotron Light Source (NSLS) at Brookhaven National Laboratory. The National Institutes of Health supports beamline X3B (formerly X9B) at NSLS. Pulse radiolysis studies were carried out at the Center for Radiation Chemical Research, which is funded under contract DE-AC02-98CH10886 with the US Department of Energy. The authors also acknowledge Peter A. Bryngelson for contributing information regarding wild-type NiSOD and for help in mutagenesis, and Robert W. Herbst for assistance in EPR and ESI-MS data collection.

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Authors and Affiliations

  1. Department of Chemistry, University of Massachusetts at Amherst, 104 Lederle Graduate Research Tower A, 710 North Pleasant Street, Amherst, MA, 01003, USA

    Kelly C. Ryan & Michael J. Maroney

  2. Department of Chemistry, University of Wisconsin-Madison, 1101 University Avenue, Madison, WI, 53706, USA

    Olivia E. Johnson & Thomas C. Brunold

  3. Department of Chemistry, Building 555A Brookhaven National Laboratory, P.O. Box 5000, Upton, NY, 11973, USA

    Diane E. Cabelli

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  1. Kelly C. Ryan
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  2. Olivia E. Johnson
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  3. Diane E. Cabelli
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Correspondence to Michael J. Maroney.

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Ryan, K.C., Johnson, O.E., Cabelli, D.E. et al. Nickel superoxide dismutase: structural and functional roles of Cys2 and Cys6. J Biol Inorg Chem 15, 795–807 (2010). https://doi.org/10.1007/s00775-010-0645-y

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