David Hart Dec 12, 2006

1. Porphyrins David Hart Dec 12, 2006

2. Heme

3. Porphyrins • Cyclic compounds that bind metal ions • Chlorphyll (Mg2+) • Central to solar energy utilization • Heme (Fe2+) • Most prevalent metalloporphyrin in humans • Central to oxygen sensing and utilization • Cobalamin (Cobalt) Bacterial Coenzyme F430 (Nickel)

5. The Heme Pocket in Hemoglobin

6. Heme • One ferrous (Fe2+) atom in the center of the tetrapyrrole ring of ProtoporphyrinIX • Prosthetic group for • Hemoglobin and Myoglobin • The Cytochromes • Catalase and Tryptophan pyrrolase • Nitric Oxide Synthase • Turnover of Hemeproteins (Hemoglobin, etc) is coordinated with synthesis and degradation of porphyrins • Bound iron is recycled

7. Lecture Outline • Heme function • Heme synthesis and regulation • Iron metabolism • Porphyrias • Heme degradation

8. Heme Function • Oxygen sensing (heme and hemoproteins) • Oxygen transport (hemoglobin) • Oxygen storage (myoglobin) • Electron transport (cytochromes) • Oxidation (cyrochrome p450, tryptophan pyrrolase, guanylate cyclase …) • Decomposition and activation of H2O2 (catalase and peroxidase) • Nitric Oxide Synthesis • Regulation of cellular processes • Effector of apoptosis

9. Porphyrin: Cyclic molecule formed by linkage of four pyrrole rings through methenyl bridges B A N HN NH N C D

10. Porphyrin Side Chains • M = Methyl (-CH3) • V = Vinyl (-CH=CH2) • P = Propionyl (-CH2-CH2-COO-) • A = Acetyl (-CH2-COO-)

11. Biosynthesis of Heme • Synthesized in every human cell • Liver (15%): • 65% Cytochrome P450 • Synthesis fluctuates greatly • Alterations in cellular heme pool • Bone Marrow (80%) • Erythrocyte precursors: Hemoglobin • Synthesis relatively constant • Matched to rate of globin synthesis • Largely unaffected by other factors

12. All Carbon and Nitrogen atoms provided by 2 building blocks: COOH CH2 CH2 COSCoA SUCCINYL CoA CH2 NH2 COOH GLYCINE

13. COOH CH2 CH2 COSCoA SUCCINYL CoA CH2 NH2 COOH GLYCINE is Decarboxylated - CO2  AMINOLEVULINIC ACID SYNTHASE IN MITOCHONDRIA

14. COOH CH2 CH2 C=O CH2 NH2 Condense to form:  AMINOLEVULINIC ACID (ALA) MOVES OUT OF THE MITOCHONDRION

15. COOH CH2 CH2 C=O COOH CH2 CH2 C=O -2 H2O CH2 NH2 CH2 NH2 2 Molecules dehydrated by • ALA DEHYDRATASE

16. COOH CH2 CH2 C COOH CH2 C C C NH CH2 NH2 To form Porphobilinogen (PBG)

17. COOH CH2 CH2 Acetate CH2COO- Propionate CH2CH2COO- COOH CH2 N H CH2 NH2 Porphobilinogen (PBG)

18. A P N H CH2 NH2 Porphobilinogen (PBG)

19. A A A A P P P P N H N H N H N H CH2 NH2 CH2 NH2 CH2 NH2 CH2 NH2

20. Hydroxymethylbilane synthase& Uroporphyrinogen III synthase • Four PBG molecules condense • Ring closure • Isomerization

21. P A B A A P NH HN Uroporphyrinogen III NH HN A C D A P P

22. COOH CH2 CH2 COOH CH2 -CH2-CH2-COOH HOOC-H2C- NH HN Uroporphyrinogen III NH HN -CH2-COOH HOOC-H2C- CH2 CH2 COOH CH2 CH2 COOH

23. Series of decarboxylations & oxidations • Porphyrinogens: • Chemically reduced • Colorless intermediates • Porphyrins: • Intensely colored • Fluorescent • Uroporphyrinogen III • Coproporphyrinogen III Moves back into Mitochondrion • Protoporphyrinogen IX • Protoporphyrin IX

24. CH=CH2 CH3 -CH=CH2 H3C- NH N Protoporphyrin IX N HN -CH3 H3C- CH2 CH2 COOH CH2 CH2 COOH

25. HEME Fe2+ chelated by Protoporphyrin IX Assisted by Ferrochelatase CH3-

26. Regulation of Heme Synthesis

27. AMINOLEVULINIC ACID SYNTHASE • Two tissue-specific isozymes • Coded on separate genes • InLiver, heme represses synthesis and activity of ALAS • Heme can be used for treatment of acute porphyric attack • In RBC heme synthesis regulation is more complex • Coordinated with globin synthesis

28. IN MITOCHONDRIA COOH CH2 CH2 COSCoA COOH CH2 CH2 C=O SUCCINYL CoA ALA CH2 NH2 COOH CH2 NH2 GLYCINE • AMINOLEVULINIC ACID SYNTHASE RATE-CONTROLLING STEP IN HEPATIC HEME SYNTHESIS

29. Bonkovsky ASH Education Book December 2005

30. Disorders of Heme Synthesis • X-linked Sideroblastic Anemia • Lead Poisoning • Iron Deficiency Anemia • The Porphyrias

31. X-linked Sideroblastic Anemia ALAS Requires Pyridoxal Phosphate as Coenzyme Some Sideroblastic Anemias improve with Pyridoxine (B6)

32. ALA moves out of the mitochondrion COOH CH2 CH2 C=O COOH CH2 CH2 C=O A P -2 H2O N H CH2 NH2 CH2 NH2 PBG CH2 NH2 • ALA DEHYDRATASE Inhibited by Heavy Metal: LEAD POISONING

33. Lead Poisoning

34. Lead Poisoning Lead Poisoning ALAD and Ferrochelatase Are particularly sensitive to Lead inhibition Ferrochelatase Heme Fe + PPIX

35. Iron Metabolism • Reactive Transition Metal (Fe2+ Fe3+) • Normally present complexed with proteins that limit its reactivity • Both iron deficiency and iron overload cause cellular defects and disease • Most available iron generated by macrophages that recycle red cell iron • Dietary Fe3+ in duodenum converted to Fe2+ and absorbed by duodenal enterocyte

36. Iron 35% of Earth’s mass nasa

37. Hepatocyte Macrophage Erythroid Cell Blood Apical Duodenal Enterocyte GUT Contents Fe3+ Heme diFe3+ Transferrin Fe2+ Fe2+ Mitochondrial Heme Synthesis

38. NEJM June 2004

39. Blood Macrophage RBC Hemoglobin Haptoglobin Heme Hemopexin Fe2+ Fe2+

40. ? Syed, Hemoglobin 2006

41. http://walz.med.harvard.edu

42. Hentze, Muckenthaler & Andrews Cell, Vol 117, 285-297, April 30, 2004 Hepcidin

43. Hepcidin: 25 Amino Acids J Med Genet 2004

44. Nemeth et al, Science, Dec 2004

45. Beutler, Science Dec 2004

46. Hentze, Muckenthaler & Andrews Cell, Vol 117, 285-297, April 30, 2004 Ferroportin

47. Genetic HemochromatosisDisruption of Hepcidin / Ferroportin • Autosomal Recessive • HFE C282Y/C282Y • TfR2 • Hemojuvelin • Hepcidin • Autosomal Dominant • Ferroportin

48. Normal Liver medlib.med.utah.edu

49. Granular, Dark Reddish Brown Surface of Liver in Hemochromatosis www.med.niigata-u.ac.j

50. Iron Accumulation in Chronic Disease http://eduserv.hscer.washington.edu