HEME

By
Dr. A. Kwena
• 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.
 Synthesis
• 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.
• 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).
• 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. 
• 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
• 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 rearran
gement is thought to proceed via a
spiro intermediate
, as depicted at right and in the animation belo
w.
• The active site of Uroporphyrinogen III
Synthase
is located in a cleft between two domains of t
he enzyme. The structural flexibility inherent i
n 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 i
n 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 c
onduct released protons away, as Fe
++ binds from the other side of the porphyrin

ring, to yield heme.

 Regulation
• 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
• 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.
Regulation of Iron transport and absorption

• 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.
Iron transportation
• 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.
 Catabolism
• Heme oxygenase or haem oxygenase (HO) is
an enzyme that catalyzes the degradation of
heme.
• This produces biliverdin, iron, and
carbon monoxide.[1]
• Heme oxygenase cleaves the heme ring at the
alpha-methene bridge to form either biliverdin
or, if the heme is still attached to a globin,
verdoglobin.
• Biliverdin is subsequently converted to
bilirubin by biliverdin reductase.]
• The reaction occurs as follows:
• Heme b + 3O2 + 3½NADPH + 3½H+ + 7e- →
biliverdin + Fe2+ + CO + 3½NADP+ + 3H2O
Biliverdin to Bilirubin
• This reaction can occur in virtually every cell;
the classic example is the formation of a bruise
, which goes through different colors as it
gradually heals: red heme to green biliverdin
to yellow bilirubin.
• Under normal physiological conditions, the
activity of heme oxygenase is highest in the
spleen, where old erythrocytes are
sequestrated and destroyed.
 Isoforms of the Enzyme
• There are three known isoforms of heme oxygenase.
• Heme oxygenase 1 (HO-1) is an inducible isoform in
response to stress such as oxidative stress, hypoxia,
heavy metals, cytokines, etc.
• Heme oxygenase 2 (HO-2) is a constitutive isoform
which is expressed under homeostatic conditions. Both
HO-1 and HO-2 are ubiquitously expressed and
catalytically active.
• A third heme oxygenase (HO-3) is not catalytically
active, but is thought to work in oxygen sensing.
• Biliverdin is a green tetrapyrrolic bile pigment,
and is a product of heme catabolism.
• It is the pigment responsible for a greenish
color sometimes seen in bruises.
 Catabolism cont’d
• Biliverdin results from the breakdown of the
heme moiety of hemoglobin in erythrocytes.
• Macrophages break down senescent
erythrocytes and break the heme down into
biliverdin, which normally rapidly reduces to
free bilirubin.
• Biliverdin is seen briefly in some bruises as a
green color. Its breakdown into bilirubin in
bruises, leads to a yellowish color.
• Haemoglobin (red)

• Biliverdin (green)

• Bilirubin (Yellow)
 Biliverdin in Disease
• Biliverdin has been found in excess in the
blood of humans suffering from hepatic
diseases.
• Jaundice is caused by the accumulation of
biliverdin or bilirubin (or both) in the
circulatory system and tissues.
• Jaundiced skin and sclera (whites of the eyes)
are characteristic of liver failure.
 Potential Test Question:

• 1.a. Write out the reaction leading to

formation of d-aminolevulinate (ALA). Include
names and structures of substrates and
product of the reaction (mechanism not
required). What vitamin-derived cofactor
participates in the reaction?
   
• b. Write out the reaction catalyzed by
Porphobilinogen Synthase (PBG Synthase),
giving names and structures of reactants and
product. How are the substrates of the PBG
Synthase reaction initially held at the active
site? Explain the effect of Pb++ on the PBG
Synthase reaction. How are effects of lead
poisoning on the brain explained?
   
• c. Summarize and explain the effect of genetic
deficiency of PBG Synthase on synthesis of
ALA.