David Hart

Dec 12, 2006

Heme

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)•

The Heme Pocket in Hemoglobin

Heme

• One ferrous (Fe2+) 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 H2O2

(catalase and peroxidase)• Nitric Oxide Synthesis• Regulation of cellular processes• Effector of apoptosis

N

NH N

HN

B

CD

Porphyrin: Cyclic molecule formed by linkage of four pyrrole rings through methenyl bridges

A

Porphyrin Side Chains

• M = Methyl (-CH3)

• V = Vinyl (-CH=CH2)

• P = Propionyl (-CH2-CH2-COO-)

• A = Acetyl (-CH2-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

CH2

CH2

COSCoACH2 NH2

COOH

SUCCINYL CoA

GLYCINE

All Carbon and Nitrogen atoms provided by 2 building blocks:

COOH

CH2

CH2

COSCoACH2 NH2

COOH

SUCCINYL CoA

GLYCINE isDecarboxylated

IN MITOCHONDRIA

AMINOLEVULINIC ACID SYNTHASE

- CO2

COOH

CH2

CH2

C=OCH2

NH2

Condense to form: AMINOLEVULINIC ACID (ALA)

MOVES OUT OF THE MITOCHONDRION

COOH

CH2

CH2

C=OCH2

NH2

COOH

CH2

CH2

C=OCH2

NH2

2 Molecules dehydrated byALA DEHYDRATASE

-2 H2O

COOH

CH2

CH2

CC

NH

COOH

CH2

C

CCH2

NH2

To form Porphobilinogen (PBG)

COOH

CH2

CH2

NH

COOH

CH2

CH2

NH2

Porphobilinogen (PBG)

AcetateCH2COO-

PropionateCH2CH2COO-

Porphobilinogen (PBG)

NH

CH2

NH2

A P

NH

CH 2

NH 2

A

PN

H

CH

2

NH2

A

P

NH

CH2

NH2

A

P

NH

CH2

NH2

A

P

Hydroxymethylbilane synthase& Uroporphyrinogen III synthase

• Four PBG molecules condense• Ring closure • Isomerization

NH

NH HN

HN

A B

CD

A

A

P

A

P

P

P

A

Uroporphyrinogen III

NH

NH HN

HNHOOC-H2C-

HOOC-H2C-

-CH2-CH2-COOH

-CH2-COOH

CH2

CH2

COOH

CH2

CH2

COOH

COOHCH2

CH2

COOHCH2

Uroporphyrinogen III

Series of decarboxylations & oxidations• Porphyrinogens:

– Chemically reduced– Colorless intermediates

• Porphyrins:– Intensely colored– Fluorescent

• Uroporphyrinogen III• Coproporphyrinogen IIIMoves back into Mitochondrion• Protoporphyrinogen IX• Protoporphyrin IX

NH

N HN

NH3C-

H3C-

-CH=CH2

-CH3

CH2

CH2

COOH

CH2

CH2

COOH

CH3

Protoporphyrin IX

CH=CH2

HEMEFe2+ chelated by Protoporphyrin IX

Assisted by Ferrochelatase

CH3-

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

CH2

CH2

COSCoACH2 NH2

COOH

SUCCINYL CoA

GLYCINE

IN MITOCHONDRIA

AMINOLEVULINIC ACID SYNTHASERATE-CONTROLLING STEP IN

HEPATIC HEME SYNTHESIS

COOH

CH2

CH2

C=OCH2

NH2

ALA

BonkovskyASH Education BookDecember 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 Anemiasimprove with Pyridoxine (B6)

COOH

CH2

CH2

C=OCH2

NH2

COOH

CH2

CH2

C=OCH2

NH2

-2 H2O

ALA moves out of the mitochondrion

ALA DEHYDRATASEInhibited by Heavy Metal: LEAD POISONING

PBG

NH

CH2

NH2

A P

Lead Poisoning

Lead PoisoningALAD and FerrochelataseAre particularly sensitive

to Lead inhibition

Lead Poisoning

Fe + PPIX

Ferrochelatase

Heme

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

Iron35% of Earth’s mass

nasa

Fe3+

Heme

Fe2+

Fe2+

BloodApicalDuodenal Enterocyte

MitochondrialHemeSynthesis

HepatocyteMacrophageErythroid Cell

diFe3+

Transferrin

GUTContents

NEJM June 2004

Fe2+

Fe2+

Blood

Macrophage

RBC

HemoglobinHaptoglobin

HemeHemopexin

Syed, Hemoglobin 2006

?

http://walz.med.harvard.edu

Hentze, Muckenthaler & AndrewsCell, Vol 117, 285-297, April 30, 2004

Hepcidin

Hepcidin: 25 Amino AcidsJ Med Genet 2004

Nem

eth

et

al,

Sci

ence

, D

ec 2

004

Beutler,ScienceDec 2004

Hentze, Muckenthaler & AndrewsCell, Vol 117, 285-297, 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

NormalLiver

www.med.niigata-u.ac.j

Granular, Dark Reddish BrownSurface of Liver in Hemochromatosis

http://eduserv.hscer.washington.edu

Iron Accumulation in Chronic Disease

Ring SideroblastPrussian Blue stains Iron

In Mitochondria

www.uchsc.edu

Iron Deficiency Anemia

Hypochromic,Microcytic

http://eduserv.hscer.washington.edu

Normal Red Blood Cells

www.lsuagcenter.co

Spinach:Non-HemeIron LessReadilyAbsorbed

OxalatesPhytatesTanninsFiberCalcium

www.mcgil.com/food/pics

Heme Iron is More Readily Absorbed

www.agnet.org/library

Iron Deficient Spinach“Chlorosis”

www.geoimagery.com

Harvesting Latex

www.sentientkinetics.com

Geophagia

www.awesomedrinks.com

Pagophagia

Solemnity Scale:0 = No smiles/hour

5 = “wreathed”In smiles

www.drmhijazy.com

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

AcuteHepatic

PBG and ALA Accumulate in Urine

PBG in Urine: Diagnostic ScreenUrine darkens with exposure

NOT photosensitiveNeuro-visceral attacks

Precipitated by Drugs, EtOHwhich induce cytochrome P450

Hydroxymethylbilane Synthase

Lead Poisoning

ALA-D Porphyria