caa a22 4500 60550752X CHVBK 20210128100632.0 cr unu---uuuuu 210128e20151201xx s 000 0 eng 10.1007/s00775-015-1304-0 doi (NATIONALLICENCE)springer-10.1007/s00775-015-1304-0 Histidine residues are important for preserving the structure and heme binding to the C. elegans HRG-3 heme-trafficking protein [Elektronische Daten] [Ortal Marciano, Yoni Moskovitz, Iqbal Hamza, Sharon Ruthstein] 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. SBIC, 2015 HRG-3 nationallicence Heme transfer nationallicence EPR nationallicence Site-directed spin labeling nationallicence Absorption spectroscopy nationallicence CD : Circular dichroism nationallicence CW : Continuous wave nationallicence EPR : Electron paramagnetic resonance nationallicence MTSSL : 1-Oxyl-2,2,5,5-tetramethyl-2,5-pyrroline-3-methyl) methanesulfonothioate nationallicence SDSL : Site-directed spin labeling nationallicence Marciano Ortal Department of Chemistry, Faculty of Exact Sciences, Bar Ilan University, 5290002, Ramat-Gan, Israel aut Moskovitz Yoni Department of Chemistry, Faculty of Exact Sciences, Bar Ilan University, 5290002, Ramat-Gan, Israel aut Hamza Iqbal Department of Animal and Avian Sciences, University of Maryland, 20742, College Park, MD, USA aut Ruthstein Sharon Department of Chemistry, Faculty of Exact Sciences, Bar Ilan University, 5290002, Ramat-Gan, Israel aut JBIC Journal of Biological Inorganic Chemistry Springer Berlin Heidelberg 20/8(2015-12-01), 1253-1261 0949-8257 20:8<1253 2015 20 775 https://doi.org/10.1007/s00775-015-1304-0 text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00775-015-1304-0 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Marciano Ortal Department of Chemistry, Faculty of Exact Sciences, Bar Ilan University, 5290002, Ramat-Gan, Israel aut NATIONALLICENCE 700 1- Moskovitz Yoni Department of Chemistry, Faculty of Exact Sciences, Bar Ilan University, 5290002, Ramat-Gan, Israel aut NATIONALLICENCE 700 1- Hamza Iqbal Department of Animal and Avian Sciences, University of Maryland, 20742, College Park, MD, USA aut NATIONALLICENCE 700 1- Ruthstein Sharon Department of Chemistry, Faculty of Exact Sciences, Bar Ilan University, 5290002, Ramat-Gan, Israel aut NATIONALLICENCE 773 0- JBIC Journal of Biological Inorganic Chemistry Springer Berlin Heidelberg 20/8(2015-12-01), 1253-1261 0949-8257 20:8<1253 2015 20 775