JBIC Journal of Biological Inorganic Chemistry

, Volume 20, Issue 8, pp 1253–1261 | Cite as

Histidine residues are important for preserving the structure and heme binding to the C. elegans HRG-3 heme-trafficking protein

  • Ortal Marciano
  • Yoni Moskovitz
  • Iqbal Hamza
  • Sharon RuthsteinEmail author
Original Paper


C. elegans is a heme auxotroph that requires environmental heme for sustenance. As such, worms utilize HRG-3, a small heme-trafficking protein, to traffic heme from the intestine to extra-intestinal tissues and embryos. However, how HRG-3 binds and delivers heme remains unknown. In this study, we utilized electron paramagnetic resonance spectroscopy together with site-directed spin labeling, absorption spectroscopy, circular dichroism, and mutagenesis to gain structural and molecular insights into HRG-3. We showed that HRG-3 is a dimer, whereas H9 and H10 are significant residues that preserve a specific conformational state in the HRG-3 dimer. In the absence of H9 and H10, HRG-3 can still bind heme, although with a different affinity. Furthermore, the heme-binding site is closer to the N-termini than to the C-termini. Taken together, our results lay the groundwork for future mechanistic and structural studies of HRG-3 and inter-tissue heme trafficking in metazoans.


HRG-3 Heme transfer EPR Site-directed spin labeling Absorption spectroscopy 



Circular dichroism


Continuous wave


Electron paramagnetic resonance


1-Oxyl-2,2,5,5-tetramethyl-2,5-pyrroline-3-methyl) methanesulfonothioate


Site-directed spin labeling



This work was supported by funds received from Bar Ilan University.


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Copyright information

© SBIC 2015

Authors and Affiliations

  • Ortal Marciano
    • 1
  • Yoni Moskovitz
    • 1
  • Iqbal Hamza
    • 2
    • 3
  • Sharon Ruthstein
    • 1
    Email author
  1. 1.Department of Chemistry, Faculty of Exact SciencesBar Ilan UniversityRamat-GanIsrael
  2. 2.Department of Animal and Avian SciencesUniversity of MarylandCollege ParkUSA
  3. 3.Department of Cell Biology and Molecular GeneticsUniversity of MarylandCollege ParkUSA

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