Synthesis of Heme

Heme is the prosthetic group of hemoglobin, myoglobin, & cytochromes. Heme is an asymmetric molecule. E.g., note the positions of methyl side chains around the ring system.

N

N

N

N

CH3 HC

CH3

S CH2

CH3

CH S CH2

CH3

CH2

CH2

COO−

CH3

H3C

CH2CH2−OOC

protein

protein

Fe

Heme c

The heme ring system is synthesized from glycine & succinyl-CoA.

Using isotopic tracers, it was initially found that N & C atoms of heme are derived from glycine and acetate.

It was later determined that the labeled acetate enters Krebs Cycle as acetyl-CoA, and the labeled carbon becomes incorporated into succinyl-CoA, the more immediate precursor of heme.

Heme synthesis begins with condensation of glycine & succinyl-CoA, with decarboxylation, to form δ-aminolevulinic acid (ALA).

−OOC CH2 CH2 C S-CoA

O

+ −OOC CH2 NH3+

−OOC CH2 CH2 C

O

CH2 NH3+

CO2CoA-SH

H+succinyl-CoA glycine

δ-aminolevulinate (ALA)

δ-Aminolevulinic Acid Synthase

Pyridoxal phosphate (PLP) serves as coenzyme for δ-Aminolevulinate Synthase (ALA Synthase), an enzyme evolutionarily related to transaminases. Condensation with succinyl-CoA takes place while the amino group of glycine is in Schiff base linkage to the PLP aldehyde. CoA & the glycine carboxyl are lost following the condensation.

Pyridoxal phosphate (PLP)

NH

CO

P

O−O

O

OH

CH3

CH O

−

+

H2

NH

CO

P

O−O

O

O−

CH3

HC−

+

H2

N

H2C

H+

COO−

glycine-PLP Schiff base (aldimine)

ALA Synthase is the committed step of the heme synthesis pathway, & is usually rate-limiting for the overall pathway.

Regulation occurs through control of gene transcription.

Heme functions as a feedback inhibitor, repressing transcription of the ALA Synthase gene in most cells.

A variant of ALA Synthase expressed only in developing erythrocytes is regulated instead by availability of iron in the form of iron-sulfur clusters.

PBG Synthase (Porphobilinogen Synthase), also called ALA Dehydratase, catalyzes condensation of two molecules of δ-aminolevulinate to form the pyrrole ring of porphobilinogen (PBG).

C

CNH

CH

C

CH2

COO−

CH2

CH2

COO−

H2C

NH3+

CH2

CH2

COO−

C

CH2

O

NH3+

CH2

CH2

COO−

C

CH2

O

NH3+

+

porphobilinogen (PBG)

2 δ-aminolevulinate (ALA)

2 H2O

PBG Synthase

As each of the two δ-aminolevulinate (ALA) substrates binds at the active site, its keto group initially reacts with the side-chain amino group of one of the two lysine residues to form a Schiff base. These Schiff base linkages promote the C-C and C-N condensation reactions that follow, assisted by the metal ion that coordinates to the ALA amino groups.

The reaction mechanism involves 2 Lys residues & a bound cation at the active site. Cation in mammalian enzyme is Zn++.

Lys−EnzymeCH2

CH2CH2

NH+C

H2C

CH2

H2C

NH2

HOOC

ALA in Schiff base linkage to active site lysine

a bacterial PBG Synthase with a substrate analog in Schiff base linkage at each of 2 ALA binding sites.

a yeast PBG Synthase crystallized with ALA substrate, having at its active site an intermediate resembling PBG in Schiff base linkage to one lysine side-chain.

Lys−EnzymeCH2

CH2CH2

NH+C

H2C

CH2

H2C

NH2

HOOC

ALA in Schiff base linkage to active site lysine

A proposed reaction mechanism is based on crystal structures of:

NH

CH2

COO−

CH2

CH2

COO−

H2C

NH3+

Porphobilinogen (PBG)

The Zn++ binding sites in the homo-octomeric mammalian Porphobilinogen Synthase, which include cysteine S ligands, can also bind Pb++ (lead). Inhibition of Porphobilinogen Synthase by Pb++ results in elevated blood ALA, as impaired heme synthesis leads to de-repression of transcription of the ALA Synthase gene. High ALA is thought to cause some of the neurological effects of lead poisoning, although Pb++ also may directly affect the nervous system. ALA is toxic to the brain, perhaps due to: • Similar ALA & neurotransmitter GABA

(γ-aminobutyric acid) structures. • ALA autoxidation generates reactive

oxygen species (oxygen radicals).

CH2

CH2

COO−

C

CH2

O

NH3+

CH2

CH2

COO−

CH2

NH3+

ALA GABA

NH

pyrrole

The porphyrin ring is formed by condensation of 4 molecules of porphobilinogen. Porphobilinogen Deaminase catalyzes successive PBG condensations, initiated in each case by elimination of the amino group.

NH

CH2

COO−

CH2

CH2

COO−

H2C

NH3+

Porphobilinogen (PBG)

Porphobilinogen (PBG) is the first pathway intermediate that includes a pyrrole ring.

Porphobilinogen Deaminase has a dipyrromethane prosthetic group, linked at the active site via a cysteine S.

The enzyme itself catalyzes formation of this prosthetic group.

NH

CH2

COO-

CH2

CH2

COO-

SEnz NH

CH2

COO-

CH2

CH2

COO-

dipyrromethane

PBG units are added to the dipyrromethane until a linear hexapyrrole has been formed.

NH

CH2

CH2

COO-

CH2

COO-

HN

CH2

COO-

CH2 CH2

HN

CH2

CH2

COO-

CH2 COO-NH

CH2

COO-

CH2

CH2

COO-

COO-

NH

CH2

COO-

CH2

CH2

COO-

SEnz NH

CH2

COO-

CH2

CH2

COO-

Porphobilinogen Deaminase is organized in 3 domains. Predicted interdomain flexibility may accommodate the growing polypyrrole in the active site cleft.

HO

NH

CH2

CH2

COO-

CH2-OOC

HN

CH2

COO-

CH2 CH2

HN

CH2

CH2

COO-

CH2 COO-NH

CH2

COO-

CH2

CH2

COO-

COO-

hydroxymethylbilane

Hydrolysis of the link to the enzyme's dipyrromethane releases the tetrapyrrole hydroxymethylbilane.

Uroporphyrinogen III Synthase converts the linear tetrapyrrole hydroxymethylbilane to the macrocyclic uroporphyrinogen III.

HO

NH

CH2

CH2

COO-

CH2-OOC

HN

CH2

COO-

CH2 CH2

HN

CH2

CH2

COO-

CH2 COO-C NH

C

CH2

COO-

CH2

CH2

COO-

COO-

NH

CH2

CH2

COO-

CH2-OOC

HN

CH2

COO-

CH2 CH2

HN

CH2

CH2

COO-

CH2 COO-

COO-

C NH

C

CH2

CH2

COO-

CH2-OOC

Uroporphyrinogen III Synthase

hydroxy- uroporphyrinogen methylbilane III

NH

CH2

CH2

COO-

CH2-OOC

HN

CH2

COO-

CH2 CH2

HN

CH2

CH2

COO-

CH2 COO-

COO-

CN

HC

CH2

COO-CH2

CH2

COO-

postulated intermediate

Uroporphyrinogen III Synthase catalyzes ring closure & flipping over of one pyrrole to yield an asymmetric tetrapyrrole.

This rearrangement is thought to proceed via a spiro intermediate.

Note the distribution of acetyl & propionyl side chains, as flipping over of one pyrrole yields an asymmetric tetrapyrrole.

HO

NH

CH2

CH2

COO-

CH2-OOC

HN

CH2

COO-

CH2 CH2

HN

CH2

CH2

COO-

CH2 COO-C NH

C

CH2

COO-

CH2

CH2

COO-

COO-

NH

CH2

CH2

COO-

CH2-OOC

HN

CH2

COO-

CH2 CH2

HN

CH2

CH2

COO-

CH2 COO-

COO-

C NH

C

CH2

CH2

COO-

CH2-OOC

Uroporphyrinogen III Synthase

hydroxy- uroporphyrinogen methylbilane III

Uroporphyrinogen III is the precursor for synthesis of vitamin B12, chlorophyll, and heme, in organisms that produce these compounds. Conversion of uroporphyrinogen III to protoporphyrin IX occurs in several steps.

The active site of Uroporphyrinogen III Synthase is in a cleft between two domains of the enzyme. The structural flexibility inherent in this arrangement is proposed to be essential to catalysis.

All 4 acetyl side chains are decarboxylated to methyl groups (catalyzed by Uroporphyrinogen Decarboxylase)

Oxidative decarboxylation converts 2 of 4 propionyl side chains to vinyl groups (catalyzed by Coproporphyrinogen Oxidase)

Oxidation adds double bonds (Protoporphyrinogen Oxidase).

NH

CH

CH2

H3CN

CH3

CH CH2

HN

CH2

CH2

COO-

CH3

N

CH2

CH2

COO-

H3C

protoporphyrin IX

NH

CH2

CH2

COO-

CH2-OOC

HN

CH2

COO-

CH2 CH2

HN

CH2

CH2

COO-

CH2 COO-

COO-

NH

CH2

CH2

COO-

CH2-OOC

uroporphyrinogen III

Fe++ is added to protoporphyrin IX via Ferrocheletase, a homodimeric enzyme containing 2 iron-sulfur clusters. A conserved active site His, along with a chain of anionic residues, may conduct released protons away, as Fe++ binds from the other side of the porphyrin ring, to yield heme.

NH

CH

CH2

H3CN

CH3

CH CH2

HN

CH2

CH2

COO-

CH3

N

CH2

CH2

COO-

H3C

N

CH

CH2

H3CN

CH3

CH CH2

N

CH2

CH2

COO-

CH3

N

CH2

CH2

COO-

H3C

Fe

Ferrochelatase

protoporphyrin IX heme

2H+ Fe++

Regulation of transcription or post-translational processing of enzymes of the heme synthesis pathways differs between erythrocyte forming cells & other tissues. In erythrocyte-forming cells there is steady production

of pathway enzymes, limited only by iron availability. In other tissues expression of pathway enzymes is more

variable & subject to feedback inhibition by heme. Porphyrias are genetic diseases in which activity of one of the enzymes involved in heme synthesis is decreased (e.g., PBG Synthase, Porphobilinogen Deaminase, etc…). Symptoms vary depending on the enzyme the severity of the deficiency whether heme synthesis is affected primarily in liver or

in developing erythrocytes.

Occasional episodes of severe neurological symptoms are associated with some porphyrias. Permanent nerve damage and even death can result, if

not treated promptly. Elevated δ-aminolevulinic acid (ALA), arising from

de-repression of ALA Synthase gene transcription, is considered responsible for the neurological symptoms.

Photosensitivity is another common symptom. Skin damage may result from exposure to light. This is attributable to elevated levels of light-absorbing

pathway intermediates and their degradation products.

Question: How do you think episodes of acute neurological symptoms would be treated?

Treatment is by injection of hemin (a form of heme).

Why would this work?

The heme, in addition to supplying needs, would repress transcription of the gene for ALA Synthase, rate-limiting for the pathway and the source of excess ALA.

View an amination of the heme synthesis pathway.

Regulation of iron absorption & transport.

Iron for synthesis of heme, Fe-S centers & other non-heme iron proteins is obtained from: the diet release of recycled iron from macrophages of the

reticuloendothelial system that ingest old & damaged erythrocytes.

There is no mechanism for iron excretion. Iron is significantly lost only by bleeding, including

menstruation in females. Small losses occur from sloughing of cells of skin &

other epithelia.

Iron is transported in blood serum bound to the protein transferrin.

The plasma membrane transferrin receptor mediates uptake of the complex of iron with transferrin by cells via receptor mediated endocytosis.

transferrin with bound Fe

transferrin receptor

extracellular space

receptor-mediated endocytosis

Iron is stored within cells as a complex with the protein ferritin. The main storage site is liver.

The plasma membrane protein ferroportin mediates: release of absorbed iron from intestinal cells to

blood serum release of iron from hepatocytes (liver cells) and

macrophages.

Control of dietary iron absorption and serum iron levels involves regulation of ferroportin expression.

Transcription of the gene for the iron transporter ferroportin is responsive to iron.

Hepcidin, a regulatory peptide secreted by liver, induces degradation of ferroportin.

Hepcidin secretion increases when iron levels are high or in response to cytokines produced at sites of inflammation.

Degradation of ferroportin leads to decreased absorption of dietary iron and decreased serum iron.

Hepcidin is considered an antimicrobial peptide because by lowering serum iron it would limit bacterial growth.

Hereditary hemochromatosis is a family of genetic diseases characterized by excessive iron absorption, transport & storage. Genes mutated in these disorders include those for: transferrin receptor a protein HFE that interacts with transferrin receptor hepcidin hemojuvelin, an iron-sensing protein required for

transcription of the gene for hepcidin. E.g., impaired synthesis or activity of hepcidin leads to unrestrained ferroportin activity, with high dietary intake and high % saturation of serum transferrin with iron. Organs particularly affected by accumulation of excess iron include liver and heart.