Functions of Iron- Overview e - donor and acceptor Enzyme-Fe 2+ + substrate Enz-Fe 3+ + product ( plus 1 e - )

1. Functions of Iron- Overview e- donor and acceptor Enzyme-Fe2+ + substrate Enz-Fe3+ + product ( plus 1 e-) Enzyme- Fe3+ + substrate Enz-Fe2+ + product (minus 1 e-) Three Fe enzyme flavors- heme, Fe-S, non-heme

2. Uses of Iron I - Heme Proteins • Oxygen Carriers Hemoglobin, myoglobin • Electron Transfer (ETC/ox phos) Cytochromes a, b, c (convert NADH, FADH2 into ATP energy) • Metabolism of O2 & Peroxides (R-OOH) Cytochrome P450 (drug metabolism) Catalase (degrades H2O2) NADPH oxidase (phagocytes)

3. Uses of Iron II – Iron-Sulfur Cluster Proteins • Redox enzymes Succinate dehydrogenase (TCA) Xanthine oxidase (purine degradation) • Non-redox Enzymes Aconitase (TCA)

4. Uses of Iron III – Single Fe-Containing Enzymes • Monooxygenases • Add –OH to aromatic amino acids (neurotransmitter synth) • tyrosine dopamine • tryptophan serotonin • Dioxygenases • Synthesis of OH-lysine, OH-proline in collagen • Used to crosslink collagen fibrils in connective tissue • Enzymes containing Fe-O-Fe • Ribonucleotide Reductase (makes deoxyribose)

5. Iron toxicity • Insoluble in body Fe+3 (ferric) 10-17 M; Fe+2 (ferrous) 10-9 M • Strong oxidizing agent (Fenton reaction) Fe+2 + H2O2 Fe+3 + • OH + OH- Fe+3 + O2 • - Fe+2 + O2 (& repeat) • Must be kept protein bound to keep it soluble & reduce its toxicity • Toxicity risk- hemochromatosis

6. Fe in serum • Regulated tightly by Fe status • Transferrin is major carrier in the blood • Cell must have Transferrin Receptor to take up & use Fe • Normal status: Tf is 30% saturated with Fe • Iron also found as serum ferritin

7. Transferrin

8. The transferrin cycle Dmt1

9. The transferrin cycle Dmt1

10. Iron Stores • Major tissue sites are liver & bone marrow/spleen • Storage varies w/gender, age, diet, disease • Storage form: ferritin Cytosolic multimeric protein • Reversible storage form

11. Ferritin x 24

12. Regulation of cellular iron metabolism • Post-transcriptional control • mRNA stability • translation • Regulated by the Iron Regulatory Proteins (IRPs)

14. Iron metabolism • Functions and toxicity of Fe • Fe in serum- transferrin • Cellular Fe metabolism • Regulation of cellular iron • Intestinal iron absorption • Macrophage iron metabolism • The Fe cycle • Systemic control of iron metabolism- hepcidin • Hemochromatosis

15. Iron in the diet • Heme and nonheme iron • meats- primarily heme iron • vegetables- primarily non-heme iron • Bound to proteins • Released by proteases (e.g. pepsin) and HCl • Heme is taken up intact • Non-heme iron is transported as Fe(II)

16. Intestinal iron uptake Ferroportin Dmt1 Fe2+ Fe3+ Fe2+ Fe2+ e- Hcp1 DcytB Transferrin Fe3+ Heme Heme Hephaestin Ferritin apical basolateral

17. Zn, Cu, and Fe Papers 1. Components of zinc transport. Liuzzi et al. 2005, Eide 2006 2. Levels and trafficking of intracellular metal ions. Outten and O’Halloran, 2001 3. Copper transporters and their function. Puig and Thiele 2002, Nose et al. 2006 • 4. Regulation of mineral homeostasis-regulated protein trafficking. • Petris et al. 2002 5. Use of model systems (e.g. yeast, nematodes, zebrafish) to identify mammalian iron transporters. Hentze et al. 2004, Donovan et al. 2000 6. Hormonal regulation of iron status- Hepcidin. Nemeth et al. 2004

18. Fig. 1. The phenotype of weissherbst (weh) Reduced hemoglobin Hypochromic anemia Not due to globin protein defects

19. Fig. 2. The protein product of weissherbst (weh)- ferroportin1

20. Fig.3. Ferroportin1 is an iron exporter protein

21. Fig. 4. Expression of the mammalianferroportin protein

22. Intestinal iron uptake Ferroportin Dmt1 Fe2+ Fe3+ Fe2+ Fe2+ e- Hcp1 DcytB Transferrin Fe3+ Heme Heme Hephaestin Ferritin apical basolateral

23. Macrophage iron metabolism Cerulo- plasmin Ferroportin Fe Fe2+ Heme Tf Fe3+ Fe Ferritin

24. Iron metabolism • Functions and toxicity of Fe • Fe in serum- transferrin • Cellular Fe metabolism • Regulation of cellular iron • Intestinal iron absorption • Macrophage iron metabolism • The Fe cycle • Systemic control of iron metabolism- hepcidin • Hemochromatosis

25. Where’s the Iron in Body?(~3 gm total) • Hemoglobin 2000 mg • Storage Fe 1000 mg • Tissue Fe • Myoglobin Fe 130 mg • Enzyme Fe 8 mg • Transport Fe (blood) 3 mg Values are for a 70 kg adult male; female values lower

27. Zn, Cu, and Fe Papers 1. Components of zinc transport. Liuzzi et al. 2005, Eide 2006 2. Levels and trafficking of intracellular metal ions. Outten and O’Halloran, 2001 3. Copper transporters and their function. Puig and Thiele 2002, Nose et al. 2006 • 4. Regulation of mineral homeostasis-regulated protein trafficking. • Petris et al. 2002 5. Use of model systems (e.g. yeast, nematodes, zebrafish) to identify mammalian iron transporters. Hentze et al. 2004, Donovan et al. 2000 6. Hormonal regulation of iron status- Hepcidin. Nemeth et al. 2004

28. Systemic control of iron status • Systemic regulators: • Stores • Erythroid • Hypoxia • Inflammatory • Sites of action • control intestinal uptake • control release of iron from macrophages Hepicidin

30. Previous hepcidin studies • Hepcidin is a 25 amino acid serum peptide • Hep KO have increased Fe accumulation • Hep overexpressors have decreased Fe • Hepcidin is expressed in liver • Hepcidin expression is induced by: iron infection & inflammation (IL-6) hypoxia erythropoiesis

31. Peptide sequencing EST C B A Park et al. JBC 276: 7806

37. Hereditary hemochromatosis • Disease of Fe overload Majority caused by mutation in HFE gene Most common genetic defect of Caucasians (~1/400) Carrier frequency of 1/10 • People absorb & store Fe too efficiently Dysregulation of intestinal Fe absorption • Symptoms are of Fe toxicity Oxidative damage to tissues (esp heart and liver failure) Incr risk of atherosclerosis & CVD • Treat w/phlebotomy, Fe-chelators • Why is this mutation so common?

38. Liver iron deposition Normal Hemochromatosis

39. Genes causing hemochromatosis or other iron overload conditions • HFE • Surface protein, function unknown • Interacts with TfR • Ferroportin • Alleles with decreased hepcidin response? • Tfr2 • Liver specific TfR • Signaling to hepcidin expression? • Hemojuvelin • GPI-linked cell surface protein • Activity affects hepcidin expression • Hepcidin • Ferritin H (IRE mutant)- hyperferritinemia • Ceruloplasmin

40. JBC 281: 28494 (2006)