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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

Abstract

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.

Keywords

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

Abbreviations

DFP

Diferric peroxo

WT

Wild type

di-Fe2+/O2 center

Oxidoreductase or ferroxidase center (Fox Center)

Notes

Acknowledgments

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)

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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

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