Haemoglobin Structure, Synthesis, and Breakdown

Structure of Haemoglobin

• Haemoglobin structure is organized into four hierarchical levels:

  1. Primary Structure: The sequence of amino acids in the polypeptide chains, which dictates the protein's overall shape and function.

  2. Secondary Structure: Refers to the local folding of the polypeptide chains into structures such as alpha-helices and beta-pleated sheets stabilized by hydrogen bonds.

  3. Tertiary Structure: This level describes the three-dimensional structure of a single polypeptide chain, including how the secondary structures fold and the spatial relationship of the different segments.

  4. Quaternary Structure: The assembly of multiple polypeptide subunits (the tetramers) into a functional complex, such as adult haemoglobin.

Haemoglobin as a Tetramer

• Haemoglobin is a tetrameric protein composed of two pairs of unlike chains (alpha and beta).

• Adult haemoglobin (HbA) composition:

  • 97% consists of HbA: α2β2, which is the most prevalent form in adults.

  • 1-3% is composed of HbA2: α2δ2, which is present in small amounts.

  • ~1% is HbF (fetal haemoglobin): α2γ2, which has a higher affinity for oxygen than adult haemoglobin, allowing effective oxygen transfer from mother to fetus.

Primary Structure

• The primary structure of haemoglobin consists of 574 amino acids distributed across its four chains:

  • The alpha chains (2 subunits) each have 141 amino acids.

  • The beta, gamma, and delta chains (2 subunits each for beta and delta, 2 for gamma) each have 146 amino acids, with the variation in amino acid sequences contributing to the different properties of the chains.

Alpha Globin Gene Location

• The alpha chain genes are located on chromosome 16.

• Gene sequence: 5' - ζ - α2 - α1 - 3'.

• There is a duplication of the alpha-globin genes which is significant for haemoglobin synthesis throughout development (embryonic to adult).

Beta Globin Gene Location

• The beta chain gene is located on chromosome 11.

• Gene sequence: 5' - ε - γ - 3' G - γA - δ - β, which indicates the order of gene expression from embryonic development to adulthood.

• The sequences encode for different proteins with varying functions in oxygen transport and regulation of blood pH levels.

Foetal Haemoglobins

• Fetal haemoglobins consist of several chains:

  • Zeta (ζ) chain (embryonic)

  • Alpha (α) chain

  • Gamma (γ) chain

  • Epsilon (ε) chain

  • Beta (β) chain

  • Delta (δ) chain

• During embryonic development, the key forms include:

  • Gower 1: ζ2ε2

  • Portland 1: ζ2γ2

  • Gower 2: α2ε2

• Hb F (fetal haemoglobin) is α2γ2, facilitating oxygen transfer across the placenta.

Secondary Structure

• The secondary structure features alpha-helices which are stabilized by hydrogen bonds between backbone amide and carbonyl groups.

• Each of the globin chains contains 8 helical segments designated as A through H, contributing to the compact and functional form of haemoglobin.

Alpha Helix Details

• Approximately 80% of the amino acids in the chains adopt a right-handed spiral configuration characteristic of alpha-helices.

• It has been determined that an alpha-helix averages about 3.6 amino acids per turn, crucial for the stability and functionality of the structure.

Non-Helical Segments

• The helical segments are interconnected by non-helical segments (designated AB, BC, CD, etc.), which can impact the overall flexibility and function of the protein.

• Certain residues, such as proline, act as helix breakers and introduce kinks in the chains, affecting the overall shape and interactions of haemoglobin molecules, as noted in the Hb Sabine F variant where β91 Leu → Pro.

Tertiary Structure

• In tertiary structure, non-helical segments bend back on themselves, contributing to a globular shape.

• Polar amino acids are primarily located on the exterior to interact with the aqueous environment, while non-polar amino acids are tucked away in the interior, minimizing hydrophobic interactions with the surrounding medium.

Haem Location

• Haem groups fit into non-polar pockets formed by the globin chains and are held in place by hydrogen bonds and hydrophobic interactions from surrounding amino acids.

• Each haem group contains iron (Fe2+) which is hexavalent, allowing it to bind oxygen molecules reversibly.

Haem Structure

• The haem structure features a central iron (Fe) core bound to nitrogen atoms and histidine residues, along with other groups such as methyl CH3, vinyl CH=CH2, and proprionate C2H4COO^-, which play a role in the stability and solubility of the haem group.

Haem Stability

• The presence of the haem group imparts additional stability to the tertiary structure of haemoglobin.

• Loss of the haem group can lead to unfolding of the chain and significant loss of helical content, resulting in potential loss of function.

Quaternary Structure

• The quaternary structure of haemoglobin can be represented as:

  • α1β2

  • α2β2 (the typical adult form)

  • α1β1 is quite rigid.

  • α1β2 exhibits flexibility, essential for functional interactions in oxygen binding and release.

Quaternary Structure Interactions

• Notable interactions in the quaternary structure include:

  • For α1β1: involves interactions among 34 amino acids

  • For α1β2: involves interactions among 19 amino acids.

Quaternary Structure Stabilization

• The rigid forms α1β1 and α2β2 provide structural stability essential for consistent function, whereas the forms α1β2 and α2β1 are more flexible, allowing for necessary sliding and rotational movements during the oxygen binding process.

Functional Significance of Quaternary Structure

• The interaction of α1β2 chains is responsible for the sigmoid (S-shaped) dissociation curve of oxygen binding, allowing for improved oxygen uptake and release characteristics, particularly influenced by molecules like 2,3-diphosphoglycerate (2,3-DPG).

Synthesis and Breakdown of Haemoglobin

• Haemoglobin synthesis and breakdown include two critical processes: synthesis (involving pathways such as porphyria) and degradation (primarily extravascular).

Synthesis Pathway

• Glycine + Succinyl CoA are converted to d-Amino-levulinic acid (dALA) by the enzyme ALA synthetase.

• Two molecules of dALA are converted to Porphobilinogen by ALA dehydrogenase.

• Four molecules of Porphobilinogen are converted to Hydroxymethyl bilane by PB deaminase.

• Hydroxymethyl bilane is then transformed into Uroporphyrinogen III by Uroporphyrinogen synthase.

• Uroporphyrinogen III is subsequently converted to Coproporphyrinogen III by Uroporphyrinogen decarboxylase with the release of CO2.

• Following that, Coproporphyrinogen III is converted to Protoporphyrinogen IX by coproporphyrin oxidase.

• Protoporphyrinogen IX is finally converted to Protoporphyrin IX by protoporphyrin oxidase.

• Protoporphyrin IX is then converted to Heme by Ferrochelatase, facilitated by iron insertion.

• The interaction of Heme with globin chains results in the formation of functional Hemoglobin.

• The synthesis process involves both the mitochondrion and cytoplasm of cells in erythropoietic tissues.

Some Synthetic Steps and Accumulation points

• Various intermediates in the synthesis pathway include structural formulas for:

  • Porphobilinogen (linear tetrapyrrole)

  • Uroporphyrinogen I, which accumulates in congenital erythropoietic porphyria

  • Uroporphyrinogen III, which is typically involved in normal haem synthesis.

Further Synthesis Steps

• Additional intermediates include:

  • Coproporphyrinogen III

  • Protoporphyrin IX

  • Heme

  • Uroporphyrinogen III

Porphyria-Related Interferences

• Key substrates in haem synthesis that may lead to various porphyrias include:

  • Glycine and Succinyl CoA: associated with Plumboporphyria

  • Aminoleulinic Acid (ALA): indicative of Acute Intermittent Porphyria (AIP)

  • Porphobilinogen (PGB)

  • Hydroxymethylbilane

  • Uroporphyrinogen I: associated with Congenital Erythropoietic Porphyria (CEP)

  • Uroporphyrinogen III

  • Coproporphyrinogen I

  • Coproporphyrinogen III: associated with Hereditary Coproporphyria (HCP)

  • Protoporphyrinogen: associated with Variegate Porphyria (VP)

  • Protoporphyrin: Protoporphyria (PP)

  • Heme, with its conversion products also being implicated in disorders.

Symptoms of Porphyria

• Common clinical manifestations of porphyria include:

  • Sensitivity to sunlight

  • Darkened urine due to porphyrin accumulation

  • Skin blisters resulting from photosensitivity

  • Abnormal hair growth can also occur in some forms of the disorder.

Vampirism

• Porphyria has historical associations with vampirism, as individuals suffering from the condition often exhibited symptoms that could be misconstrued as vampiric characteristics, such as sensitivity to sunlight and the appearance of skin disorders.

Degradation of Haemoglobin

• The degradation of haemoglobin involves a complex series of steps within the reticuloendothelial system, followed by transport and circulation throughout the body to facilitate breakdown.

• Key steps include:

  1. Production and processing by macrophages, particularly in the spleen and bone marrow.

  2. Uptake into hepatocytes where heme is converted to Biliverdin by the enzyme heme oxygenase; this reaction also produces carbon monoxide (CO) and free iron (Fe).

  3. Biliverdin is then converted to Bilirubin by Biliverdin reductase.

  4. Conjugation with albumin occurs, forming conjugated Bilirubin which is then processed by the liver and stored in the gall bladder.

  5. Urobilinogen, a product of Bilirubin reduction in the intestines, can also undergo further conversion to Urobilin and stercobilin, leading to excretion in feces.

  6. Additional recycling processes include reabsorption in the circulation.

• The degradation products of haemoglobin predominantly originate from:

  • 80% from hemoglobin

  • 20% from hemoproteins including myoglobin and ineffective erythropoiesis.

Extracellular degradation of haemoglobin

• The extracellular degradation of haemoglobin occurs chiefly in macrophages involved in erythrocyte turnover within the spleen and bone marrow.

• Following degradation, heme is converted to Bilirubin, which is subject to conjugation in the liver.

• Overall metabolic end-products include urobilin and stercobilin, which reflect the proper breakdown and processing of bilirubin following haem degradation, emphasizing the significance of efficient hepatic and intestinal function in maintaining bilirubin homeostasis.