Molecular Biochemistry II

Synthesis of Heme

Copyright 1999-2008 by Joyce J. Diwan. All rights reserved.

CH3 CH3 HC S CH2 protein

N H3C N

CH3 Fe N CH3 N CH S CH2 protein

OOC

CH2 CH2

CH2 CH2 COO

CH3

Heme c

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.

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.

OOC

CH2

CH2

S-CoA + H+

OOC

CH2

NH3+

succinyl-CoA d-Aminolevulinic Acid Synthase

glycine
CO2 O C CH2 NH3+

CoA-SH

OOC

d-aminolevulinate (ALA)

CH2

CH2

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

O C OH

Pyridoxal phosphate (PLP) serves as coenzyme for d-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.

O P O

O O

H2 C

N CH3 H Pyridoxal phosphate (PLP)

H2C N+ O P O O N H CH3 O H2 C HC H O COO

CoA & the glycine carboxyl are lost following the condensation.

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.

COO CH2 CH2 C CH2 NH3

+

COO CH2 + CH2 C CH2 NH3

+

PBG Synthase
COO

COO CH2 CH2 C CH N H

2 H 2O
O H2C

CH2 C C

NH3+

2 d-aminolevulinate (ALA)

porphobilinogen (PBG)

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

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

HOOC CH2 H2C C H2C NH2 CH2 CH2 CH2 NH+ LysEnzyme

ALA in Schiff base linkage to active site lysine

As each of the two d-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.

HOOC CH2 H2C C H2C NH2 CH2 CH2 CH2 NH+ LysEnzyme
COO CH2

COO CH2 CH2

ALA in Schiff base linkage to active site lysine

H2C NH3+

N H

A proposed reaction mechanism is based on crystal structures of:

Porphobilinogen (PBG)

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.

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++ COO COO also may directly affect the nervous system. ALA is toxic to the brain, perhaps due to: Similar ALA & neurotransmitter GABA (g-aminobutyric acid) structures. ALA autoxidation generates reactive oxygen species (oxygen radicals).
CH2 CH2 C CH2 NH3+ O CH2 CH2 CH2 NH3+

ALA

GABA

COO COO CH2 CH2 CH2

N H

pyrrole

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

H2C NH3+

N H

Porphobilinogen (PBG)

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.

COOCOOCH2 CH2 CH2 COO CH2

-

COO CH2 CH2

Enz

N H

N H

dipyrromethane

PBG Deaminase

PDB 1PDA

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.

COO CH2 COO COO CH2 CH2 CH2 COO CH2 COO - COO -CH CH2 CH2 NH HN CH2 CH2 CH2 COO 2

COO CH2

Enz

N H

N H CH2

NH

HN CH2 COO -

CH2 CH2 COO -COO -

CH2 CH2 COO -

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

COO CH2 COO CH2

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

CH2

OOC

CH2 NH HO NH HN HN

CH2

CH2 COO -

CH2 CH2 CH2 CH2 COO -COO CH2 CH2 COO -

COO -

hydroxymethylbilane

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

hydroxymethylbilane

COO CH2 CH2 COO CH2

COO CH2 CH2 COO CH2

uroporphyrinogen III -

OOC

CH2 NH HO C NH C CH2 CH2 CH2 COO -COO CH2 CH2 COO HN HN

CH2

CH2 COO -

OOC CH2 NH HN

CH2

CH2 COO -

C CH2 COO -

NH C

HN CH2 COO -

OOC CH2

CH2

CH2 CH2 COO -

Uroporphyrinogen III Synthase

CH2 COO -

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

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

COO CH2 CH2 COO CH2

OOC

CH2 NH HN

CH2

CH2

COO -

N HC

HN CH2 COO -

This rearrangement is thought to proceed via a spiro intermediate.

CH2 CH2 COO CH2 COO CH2 CH2 COO -

postulated intermediate

hydroxymethylbilane

COO CH2 CH2 COO CH2

COO CH2 CH2 COO CH2

uroporphyrinogen III -

OOC

CH2 NH HO C NH C CH2 CH2 CH2 COO -COO CH2 CH2 COO HN HN

CH2

CH2 COO -

OOC CH2 NH HN

CH2

CH2 COO -

C CH2 COO
-

NH C

HN CH2 COO -

OOC CH2

CH2

CH2 CH2 COO -

Uroporphyrinogen III Synthase

CH2 COO -

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

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.
Uroporphyrinogen III PDB 1JR2 Synthase

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.

COOCH2 CH2

uroporphyrinogen III
COOCH2

protoporphyrin IX
CH2 CH CH3

OOC CH2 NH HN

CH2

CH2 COO-

H3C NH N

CH CH2

NH
-

HN CH2 COO
-

N H3C

HN CH3

OOC CH2

CH2 CH2 COO

-

CH2 CH2 COO

-

CH2 CH2 COO

-

CH2 CH2 COO

-

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).

CH2 CH

protoporphyrin IX
CH3

CH2 CH

heme
CH3

H3C NH N

CH CH2

H3C

CH CH2 N N

Fe++
CH3

2H+
H3C

Fe
N N CH3

N H3C

HN

CH2 CH2 COO-

CH2 CH2 COO-

Ferrochelatase

CH2 CH2 COO-

CH2 CH2 COO-

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.

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 d-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.

transferrin with bound Fe extracellular space transferrin receptor

receptor-mediated endocytosis

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.

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.