Heme Synthesis

Molecular Biochemistry II

Synthesis of Heme

Contents of this page:
Heme
Synthesis of d-aminolevulinate & porphobilinogen
Formation & modification of the tetrapyrrole ring system
Porphyrias
Regulation of iron absorption and transport

Heme is the prosthetic group of hemoglobin, myoglobin, and the cytochromes. The heme of cytochrome c is shown at right. (For the slightly different structure of heme a, see the notes on electron transfer.) Heme is an asymmetric molecule. (Note the positions of the methyl side chains around the ring system.)

The heme ring system is synthesized from glycine and 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 first enters Krebs Cycle as acetyl-CoA, and the labeled carbon becomes incorporated into succinyl-CoA, which is the more immediate precursor of heme.

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

Pyridoxal phosphate (PLP) serves as coenzyme for d-Aminolevulinate Synthase (ALA Synthase), an enzyme is evolutionarily related to transaminases.

Condensation with succinyl-CoA takes place while the amino group of glycine is in Schiff base linkage to the aldehyde of PLP. Coenzyme A and the carboxyl of glycine are lost following the condensation reaction. Diagram p. 1015.

d-Aminolevulinate Synthase (ALA Synthase) is the committed step of the heme synthesis pathway, and 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 gene for d-Aminolevulinate Synthase 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 d-aminolevulinic acid (ALA) to form porphobilinogen (PBG).

The reaction mechanism involves two lysine residues and a bound cation at the active site. The bound cation in the mammalian enzyme is Zn⁺⁺.

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.

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 synthesis of heme results in de-repression of transcription of the gene for ALA Synthase.

High ALA is thought to cause some of the neurological effects of lead poisoning, although Pb⁺⁺ also may directly affect the nervous system. ALA (d-aminolevulinate) is toxic to the brain. This may be due in part to the fact that ALA is somewhat similar in structure to the neurotransmitter GABA (g-aminobutyric acid). In addition, autoxidation of ALA generates reactive oxygen species (oxygen radicals).

Current views of the reaction mechanism are based on solved crystal structures of:

a bacterial PBG Synthase with a substrate analog in Schiff base linkage at each of two binding sites for ALA.
a yeast PBG Synthase crystallized in presence of the ALA substrate and having at its active site an intermediate resembling the product PBG still in Schiff base linkage to one lysine side-chain.

Explore these structures at right.

PBG Synthase - ALA

PBG Synthase - PBG

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

The porphyrin ring is formed by condensation of four molecules of porphobilinogen.

Porphobilinogen Deaminase catalyzes successive condensations of PBG, initiated in each case by elimination of the amino group. Diagram p. 1018.

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

The enzyme itself catalyzes formation of this prosthetic group.

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

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

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

Explore at right the structure of the enzyme Porphobilinogen Deaminase, with its covalently linked prosthetic group dipyrromethane.

PBG Deaminase

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

Uroporphyrinogen III Synthase catalyzes ring closure, and flipping over one of the pyrroles, to yield an asymmetric tetrapyrrole. Note the distribution of acetyl and propionyl side chains in the diagram above.

This rearrangement is thought to proceed via a spiro intermediate, as depicted at right and in the animation below.

The active site of Uroporphyrinogen III Synthase is located 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 is the precursor for synthesis of vitamin B₁₂, chlorophyll, and heme, in organisms that produce these compounds.

Conversion of uroporphyrinogen III to protoporphyrin IX (above) occurs in several steps, as presented in the animation below.

These steps include:

decarboxylation of all 4 acetyl side chains, converting them to methyl groups (catalyzed by Uroporphyrinogen Decarboxylase).
oxidative decarboxylation of 2 of the 4 propionyl side chains, converting them to vinyl groups (catalyzed by Coproporphyrinogen Oxidase)
oxidation adds more double bonds (catalyzed by Protoporphyrinogen Oxidase), yielding protoporphyrin IX.

Fe⁺⁺ is added to protoporphyrin IX via Ferrochelatase. This enzyme in mammals is homodimeric and contains two [2Fe-2S] iron-sulfur clusters.

A conserved active site histidine, 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 and other tissues.

There is relatively steady production of pathway enzymes in erythrocyte-forming cells, limited only by iron availability.
Expression of pathway enzymes in other tissues, is more variable and subject to feedback inhibition by heme.

Porphyrias are genetic diseases resulting in decreased activity of one of the enzymes involved in heme synthesis (e.g., PBG Synthase, Porphobilinogen Deaminase, etc...). Symptoms vary depending on the enzyme, the severity of the deficiency and 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. Treatment of acute attacks is by injection of hemin (a form of heme). The heme, in addition to supplying needs, would repress transcription of ALA Synthase in non-erythroid tissues.

Photosensitivity is another common symptom of porphyrias. Skin damage may result from exposure to light. This is attributable to elevated levels of light-absorbing pathway intermediates and their degradation products.
For more information on these diseases, search the OMIM (Online Mendelian Inheritance in Man) website with the keyword porphyria.

Explore at right an animation summarizing the process of heme synthesis.

of heme synthesis

Regulation of iron absorption and transport

Iron for use in synthesis of heme, iron-sulfur centers and other non-heme iron proteins is obtained from the diet and via release of recycled iron from macrophages of the reticuloendothelial system that ingest old and damaged erythrocytes (red blood cells). There is no mechanism for iron excretion. Iron is significantly lost from the body only by bleeding, including menstruation in females, with small losses, e.g., from sloughing of cells of skin and 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.

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, as well as 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 and storage. Genes mutated in these disorders include those for the transferrin receptor, a protein HFE that interacts with the transferrin receptor, hepcidin and 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.

For more information on these diseases, search the OMIM (Online Mendelian Inheritance in Man) website with the keyword hemochromatosis.