JBIC Journal of Biological Inorganic Chemistry

, Volume 13, Issue 6, pp 981–991 | Cite as

“Iron priming” guides folding of denatured aporubredoxins

  • Francesco Bonomi
  • Stefania Iametti
  • Pasquale Ferranti
  • Donald M. KurtzJr
  • Anna Morleo
  • Enzio Maria Ragg
Original Paper


The relationship between iron uptake by aporubredoxins (apoRds) and formation of native holorubredoxins (holoRd), including their Fe(SCys)4 sites, was studied. In the absence of denaturants, apoRds exhibited spectroscopic features consistent with structures very similar to those of the folded holoRds. However, additions of either ferric or ferrous salts to the apoRds in the absence of denaturants gave less than 40% recovery of the native holoRd circular dichroism and UV–vis spectroscopic features. In the presence of either 6 M urea or 6 M guanidine hydrochloride, the nativelike structural features of the apoRds were absent. Nevertheless, nearly quantitative recoveries of the native holoRd spectroscopic features were achieved by addition of either ferric or ferrous salts to the denatured apoRds without diluting the denaturant. Consistent with this observation, the native spectroscopic features were unaffected by addition of the same denaturant concentrations to the as-isolated holoRds. Denaturing concentrations of urea or guanidine hydrochloride also increased the rates of holoRd recoveries from apoRds and ferrous salts. Mass spectrometry confirmed that ferric iron binding to the denatured apoRds precedes the recoveries of protein secondary structures and Fe(SCys)4 sites. Thus, iron binding to the apoRds guides, both kinetically and thermodynamically, refolding to the native holoRd structures. Our results imply that the ferrous oxidation state would more efficiently drive formation of the native holoRd structure from the nascent apoprotein in vivo, but that the Fe(SCys)4 site must attain the ferric state in order to achieve its native structure.


Rubredoxin Metalloproteins folding Iron uptake Chaotropes Hyperthermophilic 



Circular dichroism


Clostridium pasteurianum


Electrospray ionization


Guanidine hydrochoride


Mass spectrometry


Pyrococcus furiosus





This work was supported in part by funds from the University of Milan (FIRST, to F.B.) and US National Institutes of Health grant GM040388 (to D.M.K.). A.M. is the grateful recipient of a postdoctoral fellowship from the University of Milan.

Supplementary material

775_2008_385_MOESM1_ESM.pdf (236 kb)
Supplementary materials (PDF 236 kb)


  1. 1.
    Johnson DC, Dean DR, Smith AD, Johnson MK (2005) Ann Rev Biochem 74:247–281PubMedCrossRefGoogle Scholar
  2. 2.
    Lill R, Muhlenhoff U (2006) Ann Rev Cell Dev Biol 22:457–486CrossRefGoogle Scholar
  3. 3.
    O’Halloran TV, Culotta VC (2000) J Biol Chem 275:25057–25060PubMedCrossRefGoogle Scholar
  4. 4.
    Kuchar J, Hausinger RP (2004) Chem Rev 104:509–525PubMedCrossRefGoogle Scholar
  5. 5.
    Lakowski TM, Lee GM, Okon M, Reid RE, McIntosh LP (2007) Protein Sci 16:1119–1132PubMedCrossRefGoogle Scholar
  6. 6.
    Tanaka S, Saito K, Chon H, Matsumura H, Koga Y, Takano K, Kanaya S (2007) J Biol Chem 282:8246–8255PubMedCrossRefGoogle Scholar
  7. 7.
    Leckner J, Bonander N, Wittung-Stafshede P, Malmstrom BG, Karlsson BG (1997) Biochim Biophys Acta 1342:19–27PubMedGoogle Scholar
  8. 8.
    Pozdnyakova I, Wittung-Stafshede P (2001) Biochemistry 40:13728–13733PubMedCrossRefGoogle Scholar
  9. 9.
    Wilson CJ, Apiyo D, Wittung-Stafshede P (2004) Q Rev Biophys 37:285–314PubMedCrossRefGoogle Scholar
  10. 10.
    Blindauer CA, Razi MT, Campopiano DJ, Sadler PJ (2007) J Biol Inorg Chem 12:393–405PubMedCrossRefGoogle Scholar
  11. 11.
    Blindauer CA, Sadler PJ (2005) Acc Chem Res 38:62–69PubMedCrossRefGoogle Scholar
  12. 12.
    Reddi AR, Gibney BR (2007) Biochemistry 46:3745–3758PubMedCrossRefGoogle Scholar
  13. 13.
    Duncan KE, Stillman MJ (2006) J Inorg Biochem 100:2101–2107CrossRefGoogle Scholar
  14. 14.
    Duncan KE, Ngu TT, Chan J, Salgado MT, Merrifield ME, Stillman MJ (2006) Exp Biol Med 231:1488–1499Google Scholar
  15. 15.
    Chen C-J, Lin Y-H, Huang Y-C, Liu M-Y (2006) Biochem Biophys Res Commun 349:79–90PubMedCrossRefGoogle Scholar
  16. 16.
    Bertini I, Kurtz DM Jr, Eidsness MK, Liu G, Luchinat C, Rosato A, Scott RA (1998) J Biol Inorg Chem 3:401–410CrossRefGoogle Scholar
  17. 17.
    Richie KA, Teng Q, Elkin CJ, Kurtz DM Jr 1996 Protein Sci 5:883–894PubMedCrossRefGoogle Scholar
  18. 18.
    Taylor PK, Parks BA, Kurtz DM. Jr, Amster IJ (2001) J Biol Inorg Chem 6:201–206PubMedCrossRefGoogle Scholar
  19. 19.
    Bonomi F, Fessas D, Iametti S, Kurtz DM Jr, Mazzini S (2000) Protein Sci 9:2413–2426PubMedCrossRefGoogle Scholar
  20. 20.
    Bonomi F, Burden AE, Eidsness MK, Fessas D, Iametti S, Kurtz DM Jr, Mazzini S, Scott RA, Zeng Q (2002) J Biol Inorg Chem 7:427–436PubMedCrossRefGoogle Scholar
  21. 21.
    Lin IJ, Gebel EB, Machonkin TE, Westler WM, Markley JL (2003) J Am Chem Soc 125:1464–1465PubMedCrossRefGoogle Scholar
  22. 22.
    Park IY, Eidsness MK, Lin IJ, Gebel EB, Youn B, Harley JL, Machonkin TE, Frederick RO, Markley JL, Smith ET, Ichiye T, Kang C (2004) Proteins 57:618–625PubMedCrossRefGoogle Scholar
  23. 23.
    Bonomi F, Eidsness MK, Iametti S, Kurtz DM Jr, Mazzini S, Morleo A (2004) J Biol Inorg Chem 9:297–306PubMedCrossRefGoogle Scholar
  24. 24.
    Tan ML, Kang C, Ichiye T (2006) Proteins 62:708–714PubMedCrossRefGoogle Scholar
  25. 25.
    Bonomi F, Iametti S, Kurtz DM Jr, Ragg E, Richie KA (1998) J Biol Inorg Chem 3:595–606CrossRefGoogle Scholar
  26. 26.
    Zartler ER, Jenney FE Jr, Terrell M, Eidsness MK, Adams MW, Prestegard JH (2001) Biochemistry 40:7279–7290PubMedCrossRefGoogle Scholar
  27. 27.
    Strop P, Mayo SL (2000) Biochemistry 39:1251–1255PubMedCrossRefGoogle Scholar
  28. 28.
    Strop P, Mayo SL (1999) J Am Chem Soc 121:2341–2345CrossRefGoogle Scholar
  29. 29.
    Eidsness MK, Burden AE, Richie KA, Kurtz DM Jr, Scott RA, Smith ET, Ichiye T, Beard B, Min T, Kang C (1999) Biochemistry 38:14803–14809PubMedCrossRefGoogle Scholar
  30. 30.
    Eidsness MK, Richie KA, Burden AE, Kurtz DM Jr, Scott RA (1997) Biochemistry 36:10406–10413PubMedCrossRefGoogle Scholar
  31. 31.
    Lowery MD, Guckert JA, Gebhard MS, Solomon EI (1993) J Am Chem Soc 115:3012–3013CrossRefGoogle Scholar
  32. 32.
    Christensen HEM, Hammerstad-Pedersen JM, Holm A, Iversen G, Tensen MH, Ulstrup J (1994) Eur J Biochem 224:97–101PubMedCrossRefGoogle Scholar
  33. 33.
    Coulter ED, Kurtz DM Jr (2001) Arch Biochem Biophys 394:76–86PubMedCrossRefGoogle Scholar
  34. 34.
    Dauter Z, Wilson KS, Sieker LC, Moulis JM, Meyer J (1996) Proc Natl Acad Sci USA 93:8836–8840PubMedCrossRefGoogle Scholar

Copyright information

© SBIC 2008

Authors and Affiliations

  • Francesco Bonomi
    • 1
  • Stefania Iametti
    • 1
  • Pasquale Ferranti
    • 2
  • Donald M. KurtzJr
    • 3
  • Anna Morleo
    • 1
  • Enzio Maria Ragg
    • 1
  1. 1.DISMAUniversity of MilanMilanItaly
  2. 2.DSAUniversity of NaplesPorticiItaly
  3. 3.Department of ChemistryUniversity of Texas at San AntonioSan AntonioUSA

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