JBIC Journal of Biological Inorganic Chemistry

, Volume 20, Issue 6, pp 957–969 | Cite as

Fe2+ substrate transport through ferritin protein cage ion channels influences enzyme activity and biomineralization

  • Rabindra K. Behera
  • Rodrigo Torres
  • Takehiko Tosha
  • Justin M. Bradley
  • Celia W. Goulding
  • Elizabeth C. Theil
Original Paper


Ferritins, complex protein nanocages, form internal iron-oxy minerals (Fe2O3·H2O), by moving cytoplasmic Fe2+ through intracage ion channels to cage-embedded enzyme (2Fe2+/O2 oxidoreductase) sites where ferritin biomineralization is initiated. The products of ferritin enzyme activity are diferric oxy complexes that are mineral precursors. Conserved, carboxylate amino acid side chains of D127 from each of three cage subunits project into ferritin ion channels near the interior ion channel exits and, thus, could direct Fe2+ movement to the internal enzyme sites. Ferritin D127E was designed and analyzed to probe properties of ion channel size and carboxylate crowding near the internal ion channel opening. Glu side chains are chemically equivalent to, but longer by one –CH2 than Asp, side chains. Ferritin D127E assembled into normal protein cages, but diferric peroxo formation (enzyme activity) was not observed, when measured at 650 nm (DFP λ max). The caged biomineral formation, measured at 350 nm in the middle of the broad, nonspecific Fe3+–O absorption band, was slower. Structural differences (protein X-ray crystallography), between ion channels in wild type and ferritin D127E, which correlate with the inhibition of ferritin D127E enzyme activity include: (1) narrower interior ion channel openings/pores; (2) increased numbers of ion channel protein–metal binding sites, and (3) a change in ion channel electrostatics due to carboxylate crowding. The contributions of ion channel size and structure to ferritin activity reflect metal ion transport in ion channels are precisely regulated both in ferritin protein nanocages and membranes of living cells.


Ferritin Iron oxidation Ion channels Crystal structure Electrostatics Di-iron center Diferric peroxo Caged iron-oxy biomineral Oxidoreductase enzyme reactivity 



Diferric peroxo


Wild type

di-Fe2+/O2 center

Oxidoreductase or ferroxidase center (Fox Center)



We thank Dr. Francesco Oteri, Dr. Ho Leung Ng and Dr. Anshuman Dixit for helpful discussions. We also thank the Advanced Light Source (ALS) at Berkeley National Laboratories and Stanford Synchrotron Radiation Light source (SSRL) for their invaluable help in data collection. This work was supported by the CHORI Partners and National Institutes of Health Grants DK20251 (to E. C. T), AI081161 (to C. W. G) and DST-INSPIRE Faculty Award (to R. K. B).

Supplementary material

775_2015_1279_MOESM1_ESM.pdf (463 kb)
Supplementary material 1 (PDF 463 kb)


  1. 1.
    Treffry A, Zhao Z, Quail MA, Guest JR, Harrison PM (1995) Biochemistry 34:15204–15213CrossRefPubMedGoogle Scholar
  2. 2.
    Zhao Z, Treffry A, Quail MA, Guest JR, Harrison PM (1997) J Chem Soc Dalton Trans 3977–3978Google Scholar
  3. 3.
    Chasteen ND, Harrison PM (1999) J Struct Biol 126:182–194CrossRefPubMedGoogle Scholar
  4. 4.
    de Val N, Declercq JP, Lim CK, Crichton RR (2012) J Inorg Biochem 112:77–84CrossRefPubMedGoogle Scholar
  5. 5.
    Granier T, d’Estaintot BL, Gallois B, Chevalier JM, Precigoux G, Santambrogio P, Arosio P (2003) J Biol Inorg Chem 8:105–111CrossRefPubMedGoogle Scholar
  6. 6.
    Theil EC, Behera RK, Tosha T (2013) Coord Chem Rev 257:579–586PubMedCentralCrossRefPubMedGoogle Scholar
  7. 7.
    Haldar S, Bevers LE, Tosha T, Theil EC (2011) J Biol Chem 286:25620–25627PubMedCentralCrossRefPubMedGoogle Scholar
  8. 8.
    Jutz G, van Rijn P (2015) Santos Miranda B, Boker A. Chem Rev 115:1653–1701CrossRefPubMedGoogle Scholar
  9. 9.
    Pozzi C, Di Pisa F, Lalli D, Rosa C, Theil E, Turano P, Mangani S (2015) Acta Crystallogr A 71:941–953Google Scholar
  10. 10.
    Ebrahimi KH, Hagedoorn PL, Hagen WR (2015) Chem Rev 115:295–326CrossRefGoogle Scholar
  11. 11.
    Fetter J, Cohen J, Danger D, Sanders-Loehr J, Theil EC (1997) J Biol Inorg Chem 2:652–661CrossRefGoogle Scholar
  12. 12.
    Hwang J, Krebs C, Huynh BH, Edmondson DE, Theil EC, Penner-Hahn JE (2000) Science (N Y) 287:122–125CrossRefGoogle Scholar
  13. 13.
    Pereira AS, Small W, Krebs C, Tavares P, Edmondson DE, Theil EC, Huynh BH (1998) Biochemistry 37:9871–9876CrossRefPubMedGoogle Scholar
  14. 14.
    Watt RK (2013) ChemBioChem 14:415–419CrossRefPubMedGoogle Scholar
  15. 15.
    Wong SG, Grigg JC, Le Brun NE, Moore GR, Murphy ME, Mauk AG (2015) J Biol Chem 290:3732–3739PubMedCentralCrossRefPubMedGoogle Scholar
  16. 16.
    Moenne-Loccoz P, Krebs C, Herlihy K, Edmondson DE, Theil EC, Huynh BH, Loehr TM (1999) Biochemistry 38:5290–5295Google Scholar
  17. 17.
    Chiancone E, Ceci P (2010) Biochim Biophys Acta 1800:798–805CrossRefPubMedGoogle Scholar
  18. 18.
    Lalli D, Turano P (2013) Acc Chem Res 46:2676–2685CrossRefPubMedGoogle Scholar
  19. 19.
    Ferreira C, Bucchini D, Martin ME, Levi S, Arosio P, Grandchamp B, Beaumont C (2000) J Biol Chem 275:3021–3024CrossRefPubMedGoogle Scholar
  20. 20.
    Ceci P, Di Cecca G, Falconi M, Oteri F, Zamparelli C, Chiancone E (2011) J Biol Inorg Chem 16:869–880CrossRefPubMedGoogle Scholar
  21. 21.
    Khare G, Gupta V, Nangpal P, Gupta RK, Sauter NK, Tyagi AK (2011) PLoS One 6:e18570PubMedCentralCrossRefPubMedGoogle Scholar
  22. 22.
    Tosha T, Ng HL, Bhattasali O, Alber T, Theil EC (2010) J Am Chem Soc 132:14562–14569PubMedCentralCrossRefPubMedGoogle Scholar
  23. 23.
    Behera RK, Theil EC (2014) Proc Natl Acad Sci USA 111:7925–7930PubMedCentralCrossRefPubMedGoogle Scholar
  24. 24.
    Barnes CM, Theil EC, Raymond KN (2002) Proc Natl Acad Sci USA 99:5195–5200PubMedCentralCrossRefPubMedGoogle Scholar
  25. 25.
    Bertini I, Lalli D, Mangani S, Pozzi C, Rosa C, Theil EC, Turano P (2012) J Am Chem Soc 134:6169–6176PubMedCentralCrossRefPubMedGoogle Scholar
  26. 26.
    Ruvinsky AM, Vakser IA, Rivera M (2014) J Chem Phys 140:115104PubMedCentralCrossRefPubMedGoogle Scholar
  27. 27.
    Laghaei R, Kowallis W, Evans DG, Coalson RD (2014) J Phys Chem 118:7442–7453CrossRefGoogle Scholar
  28. 28.
    Tosha T, Behera RK, Ng HL, Bhattasali O, Alber T, Theil EC (2012) J Biol Chem 287:13016–13025PubMedCentralCrossRefPubMedGoogle Scholar
  29. 29.
    Schwartz JK, Liu XS, Tosha T, Theil EC, Solomon EI (2008) J Am Chem Soc 130:9441–9450PubMedCentralCrossRefPubMedGoogle Scholar
  30. 30.
    Liu X, Theil EC (2004) Proc Natl Acad Sci USA 101:8557–8562PubMedCentralCrossRefPubMedGoogle Scholar
  31. 31.
    Otwinowski Z, Minor W (1997) Method Enzymol 276:307–326CrossRefGoogle Scholar
  32. 32.
    Adams PD, Afonine PV, Bunkoczi G, Chen VB, Davis IW, Echols N, Headd JJ, Hung LW, Kapral GJ, Grosse-Kunstleve RW, McCoy AJ, Moriarty NW, Oeffner R, Read RJ, Richardson DC, Richardson JS, Terwilliger TC, Zwart PH (2010) Acta Crystallogr A 66:213–221CrossRefGoogle Scholar
  33. 33.
    Emsley P, Lohkamp B, Scott WG, Cowtan K (2010) Acta Crystallogr A 66:486–501Google Scholar
  34. 34.
    DeLano WL (2010) version 1.3r1. Schrödinger, LLC, New YorkGoogle Scholar
  35. 35.
    Jones T, Spencer R, Walsh C (1978) Biochemistry 17:4011–4017CrossRefPubMedGoogle Scholar
  36. 36.
    Takahashi T, Kuyucak S (2003) Biophys J 84:2256–2263PubMedCentralCrossRefPubMedGoogle Scholar
  37. 37.
    Bellapadrona G, Stefanini S, Zamparelli C, Theil EC, Chiancone E (2009) J Biol Chem 284:19101–19109PubMedCentralCrossRefPubMedGoogle Scholar
  38. 38.
    Liu X, Theil EC (2005) Acc Chem Res 38:167–175CrossRefPubMedGoogle Scholar
  39. 39.
    Bou-Abdallah F, Zhao G, Biasiotto G, Poli M, Arosio P, Chasteen ND (2008) J Am Chem Soc 130:17801–17811PubMedCentralCrossRefPubMedGoogle Scholar
  40. 40.
    Allen TW, Andersen OS, Roux B (2004) J Gen Physiol 124:679–690PubMedCentralCrossRefPubMedGoogle Scholar
  41. 41.
    Liu XS, Patterson LD, Miller MJ, Theil EC (2007) J Biol Chem 282:31821–31825CrossRefPubMedGoogle Scholar
  42. 42.
    Ha Y, Shi D, Small GW, Theil EC, Allewell NM (1999) J Biol Inorg Chem 4:243–256CrossRefPubMedGoogle Scholar

Copyright information

© SBIC 2015

Authors and Affiliations

  • Rabindra K. Behera
    • 1
    • 5
  • Rodrigo Torres
    • 2
  • Takehiko Tosha
    • 1
    • 6
  • Justin M. Bradley
    • 1
    • 7
  • Celia W. Goulding
    • 2
    • 3
  • Elizabeth C. Theil
    • 1
    • 4
  1. 1.Children’s Hospital Oakland Research Institute (CHORI)OaklandUSA
  2. 2.Department of Molecular Biology and BiochemistryUniversity of California-IrvineIrvineUSA
  3. 3.Department of Pharmaceutical SciencesUniversity of California-IrvineIrvineUSA
  4. 4.Department of Molecular and Structural BiochemistryNorth Carolina State UniversityRaleighUSA
  5. 5.Department of ChemistryNational Institute of TechnologyRourkelaIndia
  6. 6.Biometal Science LaboratoryRIKEN SPring-8 CenterSayoJapan
  7. 7.School of ChemistryUniversity of East AngliaNorwichUK

Personalised recommendations