Heme Synthesis

Heme, a porphyrin, is a co-factor in haemoglobin, myoglobin, cytochrome, catalase, heme peroxidase, and endothelial nitric oxide synthase. It has a complex structure with four pyrrole rings with a ferrous iron in the centre that allows it to carry oxygen. The synthesis of heme takes place from glycine and succinyl CoA in eight steps and is extensively studied. Mutations in genes encoding for enzymes involved in heme synthesis result in porphyrias.

Steps in Heme synthesis

About 85% of the heme is synthesised in the developing erythroid cells and almost all the remaining is synthesised in the liver. The control of synthesis differs in erythroid and non-erythroid cells reflecting the exceedingly high heme requirement of the former for haemoglobin synthesis. Heme synthesis takes place in the mitochondria as well as cytosol. The first step, formation of δ-aminolevulenic acid, takes place in the mitochondrial matrix. The next few steps take place in the cytosol. The heme precursor, corpoprophyrinogen III, returns to the mitochondria, is converted to protoporphyrin IX and iron incorporated. The steps in heme synthesis are as follows

  1. Synthesis of δ-aminoleuvelinic acid: Synthesis of δ-aminoleuvelinic acid (ALA) from glycine and succnyl CoA catalysed by ALA synthase (ALAS) is the first step in the synthesis of heme. This is a rate limiting step. ALA synthase is encoded by two genes ALAS1 (OMIM 125290) and ALAS2 (OMIM 301300). ALAS2 codes for the erythroid ALAS and ALAS1 for the non-erythroid (housekeeping) ALAS. The gene ALAS1 is located on chromosome 3p21.1. The product has 12 exons and undergoes is alternate splicing to yield two distinct forms, isoform 1 (640 amino acids) and isoform 2 (657 amino acids). The erythroid specific gene (ALAS2) on X chromosome at Xp11.21. It has 12 exons and also undergo alternate splicing to yield two forms, isoform b (587 amino acids), isoform c (574 amino acids). ALAS is synthesised in the cytosol and transported to the mitochondria. It has a short half life. Heme synthesis is consoled by regulating levels and activity of ALAS (discussed below).
  2. Synthesis of prophobilinogen: ALA moves to the cytosol and is dimerised to prophobilinogen by the action of prophobilinogen synthase (ALA dehydratase). The enzyme is a homo-octomer (made of eight similar units) and needs zinc. The gene (gene ALAD, OMIM 125270) encoding the enzyme is located at 9q32. It has 15 exons. Four isoforms from alternate splicing 361 amino acid, 344 amino acid, 321 amino acid and 304 amino acid are known.
  3. Synthesis of hydroxymethylbilane: Prophobilinogen is converted to hydroxymethylbilane by the action of hydroxymethylbilane synthase. This enzyme is also known as propohbilinogen deaminase. The gene (HMBS OMIM 609806) is located at 11q23.3, has 15 exons. Four alternately spliced forms with 361, 344, 321 and 304 amino acids are known.
  4. Synthesis of uroporphyrinogen: Hydroxymethylbilane is converted to enzymatically to uroporphyrinogen III as well as non-enzymatically to uroporphyrinogen I. The enzymatic conversion is catalysed by the enzyme uroporphyrinogen III synthase. Uroporphyrinogen III synthatase is encoded by a gene (UROS, OMIM, 606938) on 10q25.2-q26 that has 16 exons and encodes for a 265 amino acid protein.
  5. Synthesis of corpoporphyrinogen III: Uroporphyrinogen III is decrboxylated to corpoporphyrinogen III by uroporphyrinogen decarboxylase. The gene (UROD, OMIM 613521) for thes enzyme is at 1p34. It has 10 exons and encodes for a protein 367 amino acid long. This is the last step in the cytosol.
  6. Synthesis of protoporphrinogern IX: Coproporphyrinogen III is converted to propoporphyrinogen IX by a reaction catalysed by corpoporphyrinogen oxidase  in the mitochondria in an oxygen dependent reaction. The gene for corpoporphyrinogen oxidase (COPX, OMIM 612732) is at 3q11.2-q12.1 8 exons. The product has 454 amino acids.
  7. Synthesis of protoporphyrin IX: Propoporphyrin is the final product of the pathway into which iron is incorporated. Protoporphyrin IX is synthesised by the action of protoporphyrinogen oxidase. The gene (PPOX, OMIM 600923) for this enzyme is located at 1q22  and has14 exon. It encodes for a 477 amino acid enzyme.
  8. Synthesis of heme: Ferrochelatse (protoporphyrin ferrochelatase) catalysed the incorporation of iron into protoporphyrin IX. The gene (FECH, OMIM 612386) for ferrochelatse is located at 18q21.31 and has 11 exons. It encodes for a 477 amino acid enzyme.

Control of heme sythesis

The rate limiting enzyme of heme synthesis is the synthesis of ALA. ALA synthase has a short half life. Heme synthesis is regulated  by controlling the levels and activity of ALA synthase.

  1. Inhibition of ALA synthase: ALA synthase is subject to feedback inhibition by heme and and it’s oxidation product hemin. ALA synthase is synthesised in the cytosol and transported to the mitochondrial matrix. In addition to being an inhibitor of ALA synthase hemin also inhibits the metochondrial transport of the enzyme.
  2. Promotion of ALA synthase activity: Cellular iron and factors promoting erythroid differentiation increase the synthesis of ALAS-2, the enzyme responsible ALA synthesis in erythroid cells. Erythroid specific factors like GATA-1 promote the transcription of the ALAS-2 gene. Untranslated portions of the ALAS-2 mRNA have iron responsive elements (IRE) that promote translation. The activity of ASLS in iron deficient cells is low.


Porphyrias are inherited diseases resulting from a mutation of genes involved in heme synthesis. With one exception, X-linked porphyria that results from a gain of function mutation of ALAS synthase 2, porphyrias result from a partial deficiency of the enzymes involved in heme synthesis. Enzyme deficiency results in accumulation of substrates for the reaction catalysed by the enzyme encoded by the gene. Symptoms of porphyrias may be intermittant and/or chronic. The symptoms are diverse and include skin changes, photosensitivity, abdominal pain, muscle weakness, CNS disturbance, seizures, hyponatremia, discolouration of urine. Enzyme deficiencies associated with porphyrias as as follows:

  1. ALA synthatase 2: Gains of function mutation in X linked protoporphyria
  2. ALA dehydratase: ALA dehydrate deficient porphyria (ADP). Lead displaces zinc from binding sites inhibiting the function of the  with enzyme. In patients with tyrosinaemia type 1 Succinylacetone (4,6-dioxoheptanoic acid) accumulates in tyrosinaemia type I. It is structurally similar to ALA and a potent inhibitor of ALA dehydratase.
  3. PBG Deaminase deficiency results in acute intermittent porphyria
  4. Uroporphyrin III synthatase deficiency results in congenital erythrocytic porphyria
  5. Uroporphyrin decarboxylase deficiency results in porphyria cutanea tarde. All patients with porphyria cutanea trade do not have a mutation. Only type II has gene mutations. Types I and III are due to mulifactorial effects on the gene.
  6. Coproporphyrin III oxidase deficiency results in hereditary coproporphyria
  7. Protoporphyrin oxidase results in varigate porphyria
  8. Ferrochalase results in erythropoietic porphyria

Further Reading

Porphyrin and Heme Metabolism
Erythroid Heme Biosynthesis and Its Disorders (doi:  10.1101/cshperspect.a011676)


Pathophysiology of Anaemia

Low haemoglobin decreases the oxygen carrying capacity of the blood. This is offset by the following mechanisms.

  1. Increased cardiac output: Tissue hypoxia causes peripheral vasodialatation and decreased haemoglobin decreases blood viscosity. Together these reduce peripheral resistance allowing a higher cardiac output without increasing blood pressure.  Increased cardiac output increases tissue oxygen delivery but decreases the patient’s  exercise tolerance. Increase in cardiac output is seen at haemoglobin values less than 7g/dL.
  2. Redistribution of cardiac output: Blood flow from non-critical areas like the skin is diverted to critical organs like heart and brain.
    The Figure shows three oxyhaemoglobin dissociation curves. The blue curve is the physiological curve. This may be shift to the right (red) or left (green). When the curve shifts to the right the oxygen affinity of haemoglobin falls. Haemoglobin is more desaturated ar a given P02. The opposite happens when curve shifts to the left. The three arrows show the change in oxygen saturation between arterial and venous blood at venous and arterial PO2
  3. Increased Oxygen Extraction: Anaemic patients have increased tissue oxygen extraction from the blood. This is not seen in organs like the heart and brain that already have high oxygen extraction.
  4. Changes in oxygen affinity of haemoglobin: Patients with chronic anaemia have increased of 2,3 diphosphoglycerate (2,3-DPG). 2,3-DPG shifts oxyhaemoglobin dissociation curve to the right and increases oxygen delivery. The figure above shows three curves. The blue curve is the curve of normal haemoglobin the red shows shift to left and the green shift to right. Shift to left increases the oxygen delivery.
  5. Isovoluaemic haemodilution: Patinets with anaemia have increased plasma volume. This allows maintaining a normal cardiac output.


Detoxifying Haemoglobin

It is difficult to imagine that a molecule as essential to life as haemoglobin (hb) would need a detoxification mechanism. Anyone who has treated a patient with severe intravascular haemolysis knows the havoc cell-free haemoglobin can cause. Haemoglobin is small enough to be filtered by the glomerulus and causes renal failure due to pigment nephropathy. Haemoglobin depletes nitric oxide resulting in vasculopathy. Mechanisms to detoxify cell-free haemoglobin counter the oxidative and pro-inflammatory effects of haemoglobin.

Haemolysis releases cell-free haemoglobin. Haptoglobin (Hp), alpha-2 globulin, secreted by the liver is a haemoglobin scavenger. It rapidly binds cell-free haemoglobin in the plasma protecting the vessels and the kidneys from it’s deleterious effects. When the scavenging capacity of haptoglobin is overwhelmed cell-free haemoglobin appears in the plasma. It converts nitric oxide to biologically inactive nitrate and is itself converted to methemoglobin in the process. Degradation of cell-free haemoglobin results in the formation of heme and free iron which deplete nitric oxide by their oxidizing action. Hemopexin scavenges free heme. Free iron is taken up and transported by transferrin.

The haemoglobin-haptoglobin complex is taken by the CD163 receptor on the reticuloendothelial macrophage and the heme-hemopexin complex is taken up by the CD91 (low density lipoprotein-1, LRP1) receptor. The interactions of haemoglobin-haptoglobin by CD163 and heme-hemopexin by CD91 have an anti-oxidant and anti-inflammatory action by activation of heme oxygenase-1 and IL10. Haptoglobin and hemopexin are acute phase reactants. The body’s capacity the counter the effects of cell-free haemoglobin increases during acute inflammation. The expression of CD163 and CD91 is increased by corticosteroids which are also secreted as a part of response to acute inflammation.

Pigment nephropathy from precipitation of haemoglobin in renal tubules has long been recognized as a serious complication of massive intravascular haemolysis. The vascular effects of cell free-haemoglobin are evident at lesser haemolysis. There is evidence in rodent malaria model that heme, a degradation product of cell-free haemoglobin released during intravascular haemolysis, is involved in the pathogenesis of cerebral and non-cerebral malaria (Proc Natl Acad Sci U S A. 2009 September 15; 106(37): 15837–15842, Nat Med. 2007 Jun;13(6):703-10. Epub 2007 May 13). Heme has been implicated in the pathogenesis of severe sepsis in animal models (Sci Transl Med. 2010 Sep 29;2(51):51ra71). Cell-free haemoglobin has been implicated in the pathogenesis of pulmonary hypertension, leg ulceration, priapism, and cerebrovascular disease related to sickle cell anaemia (Blood Rev. 2007 January; 21(1): 37–47). The list of haemolytic anaemias where cell-free haemoglobin has a pathogenic role is increasing and now includes thalassemia, autoimmune haemolytic anaemia, paroxysmal nocturnal hemoglobinuria, unstable hemoglobinopathy, and hereditary membranopathies. Corticosteroids can increase the clearance of cell-free haemoglobin and its degradation products. They have been shown to benefit patients of sickle cell disease with acute chest syndrome and vaso-occlusive crisis. Thrombotic thrombocytopenic purpura, a disease characterized by intravascular haemolysis, is also treated with corticosteroids in addition to plasmapheresis. Plasmapheresis removes cell-free haemoglobin and corticosteroids enhance its clearance by macrophages.

Too much of a good thing is bad. Haemoglobin is safe when in erythrocytes. Outside erythrocytes it needs detoxification.