David Hart Dec 12, 2006

Heme

Porphyrins Cyclic compounds that bind metal ions Chlorphyll (Mg 2+ ) –Central to solar energy utilization Heme (Fe 2+ ) –Most prevalent metalloporphyrin in humans –Central to oxygen sensing and utilization Cobalamin (Cobalt)

The Heme Pocket in Hemoglobin

Heme One ferrous (Fe 2+ ) atom in the center of the tetrapyrrole ring of Protoporphyrin IX 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

Lecture Outline Heme function Heme synthesis and regulation Iron metabolism Porphyrias Heme degradation

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 H 2 O 2 (catalase and peroxidase) Nitric Oxide Synthesis Regulation of cellular processes Effector of apoptosis

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

Porphyrin Side Chains M = Methyl (-CH 3 ) V = Vinyl (-CH=CH 2 ) P = Propionyl (-CH 2 -CH 2 -COO - ) A = Acetyl (-CH 2 -COO - )

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

COOH CH 2 COSCoA CH 2 NH 2 COOH SUCCINYL CoA GLYCINE All Carbon and Nitrogen atoms provided by 2 building blocks:

COOH CH 2 COSCoA CH 2 NH 2 COOH SUCCINYL CoA GLYCINE is Decarboxylated IN MITOCHONDRIA  AMINOLEVULINIC ACID SYNTHASE - CO 2

COOH CH 2 C=O CH 2 NH 2 Condense to form:  AMINOLEVULINIC ACID (  ALA) MOVES OUT OF THE MITOCHONDRION

COOH CH 2 C=O CH 2 NH 2 COOH CH 2 C=O CH 2 NH 2 2 Molecules dehydrated by  ALA DEHYDRATASE -2 H 2 O

COOH CH 2 C NH COOH CH 2 C CH 2 NH 2 To form Porphobilinogen (PBG)

COOH CH 2 NHNH COOH CH 2 NH 2 Porphobilinogen (PBG) Acetate CH 2 COO - Propionate CH 2 CH 2 COO -

Porphobilinogen (PBG) NHNH CH 2 NH 2 A P

NHNH CH 2 NH 2 A P NHNH CH 2 NH 2 A P NHNH CH 2 NH 2 A P NHNH CH 2 NH 2 A P

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

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

NH HN HOOC-H 2 C- -CH 2 -CH 2 -COOH -CH 2 -COOH CH 2 COOH CH 2 COOH CH 2 COOH CH 2 Uroporphyrinogen III

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

NH NHN N H 3 C- -CH=CH 2 -CH 3 CH 2 COOH CH 2 COOH CH 3 Protoporphyrin IX CH=CH 2

HEME Fe 2+ chelated by Protoporphyrin IX Assisted by Ferrochelatase CH 3 -

Regulation of Heme Synthesis

 AMINOLEVULINIC ACID SYNTHASE Two tissue-specific isozymes Coded on separate genes In Liver, 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

COOH CH 2 COSCoA CH 2 NH 2 COOH SUCCINYL CoA GLYCINE IN MITOCHONDRIA  AMINOLEVULINIC ACID SYNTHASE RATE-CONTROLLING STEP IN HEPATIC HEME SYNTHESIS COOH CH 2 C=O CH 2 NH 2  ALA

Bonkovsky ASH Education Book December 2005

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

X-linked Sideroblastic Anemia X-linked Sideroblastic Anemia  ALAS Requires Pyridoxal Phosphate as Coenzyme Some Sideroblastic Anemias improve with Pyridoxine (B 6 )

COOH CH 2 C=O CH 2 NH 2 COOH CH 2 C=O CH 2 NH 2 -2 H 2 O  ALA moves out of the mitochondrion  ALA DEHYDRATASE Inhibited by Heavy Metal: LEAD POISONING PBG NHNH CH 2 NH 2 A P

Lead Poisoning

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

Iron Metabolism Reactive Transition Metal (Fe 2+  Fe 3+ ) 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 Fe 3+ in duodenum converted to Fe 2+ and absorbed by duodenal enterocyte

Iron 35% of Earth’s mass nasa

Fe 3+ Heme Fe 2+ Blood Apical Duodenal Enterocyte Mitochondrial Heme Synthesis Hepatocyte Macrophage Erythroid Cell diFe 3+ Transferrin GUT Contents

NEJM June 2004

Fe 2+ Blood Macrophage RBC Hemoglobin Haptoglobin Heme Hemopexin

Syed, Hemoglobin 2006 ?

Hentze, Muckenthaler & Andrews Cell, Vol 117, , April 30, 2004 Hepcidin

Hepcidin: 25 Amino Acids J Med Genet 2004

Nemeth et al, Science, Dec 2004

Beutler, Science Dec 2004

Hentze, Muckenthaler & Andrews Cell, Vol 117, , April 30, 2004 Ferroportin

Genetic Hemochromatosis Disruption of Hepcidin / Ferroportin Autosomal Recessive –HFE C282Y/C282Y –TfR2 –Hemojuvelin –Hepcidin Autosomal Dominant –Ferroportin

medlib.med.utah.edu Normal Liver

Granular, Dark Reddish Brown Surface of Liver in Hemochromatosis

Iron Accumulation in Chronic Disease

Ring Sideroblast Prussian Blue stains Iron In Mitochondria

Iron Deficiency Anemia Hypochromic, Microcytic

Normal Red Blood Cells

Spinach: Non-Heme Iron Less Readily Absorbed Oxalates Phytates Tannins Fiber Calcium

Heme Iron is More Readily Absorbed

Iron Deficient Spinach “Chlorosis”

Harvesting Latex

Geophagia

Pagophagia

Solemnity Scale: 0 = No smiles/hour 5 = “wreathed” In smiles

Spoon Nails

Blue Sclera

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

Heme  porphuros (purple)

Heme Synthesis: Porphyrias 8 Enzymatic Reactions 7 Deficiencies: “Porphyrias” Most are Autosomal Dominant Hepatic or Erythroid depending on main site of synthesis / accumulation

Porphyrias Accumulation and excretion of porphyrins –Pattern depends on which enzyme affected Multiple alleles Acute and Chronic –Acute: Neurovisceral attacks Porphyrin accumulation: Photosensitivity –Formation of reactive oxygen species –Damage tissues, Release lysosomal enzymes

Very Rare Recessive Porphyria ALA-D Porphyria Lead Poisoning

Acute Hepatic PBG and  ALA Accumulate in Urine PBG in Urine: Diagnostic Screen Urine darkens with exposure NOT photosensitive Neuro-visceral attacks Precipitated by Drugs, EtOH which induce cytochrome P450 Hydroxymethylbilane Synthase Lead Poisoning ALA-D Porphyria