Hemoglobin and Heme Proteins

Haemoglobin Overview

• Learning Objectives: Molecular features of haem and globins, pathways of haem biosynthesis/degradation, diseases associated with haem and globin metabolism, the metabolism of bilirubin and the main types of jaundice
• Haemoglobin contains both a haem group and a globin chain. This makes up a heme group and a globin chain

Haem-containing Proteins

• Various proteins use a prosthetic haem group:
• Hemoglobin transports oxygen in red blood cells
• Myoglobin stores oxygen in muscle cells
• Cytochromes are involved in electron transport chains.
• Catalase breaks down hydrogen peroxide
• Peroxidases catalyze oxidation reactions
• Tryptophan pyrrolase is an enzyme involved in tryptophan metabolism.
• Haem proteins are continuously synthesized and degraded, with 6 to 7 g of hemoglobin produced daily

Haem Structure

• It is an oxygen-binding prosthetic group that enables the transport of oxygen
• Consists of a porphyrin ring, specifically protoporphyrin IX, chelated with ferrous iron (Fe2+)
• Protoporphyrin IX has four pyrrole rings linked by methene bridges
• The porphyrin ring contains double bonds, which contribute to blood's color.
• Includes eight side chains and porphyrins that vary in their nature on the four pyrrole rings
• Uroporphyrin transforms into acetate & propionate groups
• Coproporphyrin changes to methyl & propionate groups
• Protoporphyrin IX (and haem) becomes vinyl, methyl & propionate groups
• The structure is hydrophobic and planar
• Iron(Fe) it contains is the most important part of the haem group
• Haem iron in hemoglobin and myoglobin is always in the ferrous (Fe2+) state.
• Fe2+ is inserted in the center and Co-ordinately linked to the four N-atoms of the pyrrole rings
• Is complexed with imidazole N-atom of a histidine residue in the protein (globin) chain
• Hb binds four oxygen molecules, while Mb binds one oxygen molecule.

Biosynthesis of Haem

• Liver and bone marrow (erythroblasts) are the major synthesis sites
• In bone marrow haem production equals globin synthesis.
• Cytochrome P450 synthesis in the liver depends on haem pool balance, iron availability, and the body’s needs.
• It happens in the mitochondria and others in the cytoplasm
• Red blood cells cannot make haem because they lack mitochondria
• δ-aminolevulinic acid (ALA) is formed:
  • This is the rate-limiting step
  • It is catalyzed by ALA synthase (mitochondrial) and requires pyridoxal phosphate
  • Condensation of glycine with succinyl CoA
  • Excess haem is oxidised to hemin decreasing the activity of ALA synthase and decreasing synthesis (mRNA synthesis)
• Glucose also negatively regulates this and synthesis increases in ALA synthesis.
• The enzyme aminolevulinic acid dehydrase condenses two ALA molecules to create porphobilinogen (PBG)
  • Requires Zinc.
  • Formation of very sensitive to lead and other heavy metals
  • Lead poisoning causes increased ALA and anemia
• Four porphobilinogens form hydroxymethylbilane which then becomes uroporphyrinogen III
• Two cytoplasmic enzymes are involved
  • Hydroxymethylbilane synthase (PBG deaminase)
  • Uroporphyrinogen III synthase
• Acetate groups on uroporphyrinogen III are decarboxylated to coproporphyrinogen III by UPP decarboxylase
• Coproporphyrinogen III is transported back into mitochondria
• Converted to protoporphyrinogen IX (decarboxylation of 2 propionate groups – CPP oxidase) then oxidised to protoporphyrin IX (PPP oxidase)
• Haem forms by incorporation of iron

Haem Biosynthesis Regulation

• Free haem inhibits pre-existing ALA synthase activity
• Free haem diminishes ALAS transport from cytoplasm to mitochondria after synthesis.
• It also represses that production of 8-ALA synthase by regulating gene transcription and stimulates globin synthesis to ensure that levels of free haem remain low.
• ALA synthase inhibition and globin synthesis stimulation are the most critical parts in balancing hemoglobin production.

Porphyria's Overview

• Occur because of partial deficiency of haem synthesizing enzymes (other than ALA synthase) and all porphyrins are autosomal dominant, except congenital erythropoietic porphyria, which is recessive.
• Has caused accumulation and increased excretion of porphyrins and porphyrin precursors in the urine (each unique)
• Can be hepatic or erythropoietic, which are either acute or chronic.
• Accumulation of tetrapyrrole intermediates causes photosensitivity which leads to:
  • Formation of superoxide radicals
  • Skin blisters, itches (pruritis) and may darken, grow hair (hypertrichosis)

Erythropoietic vs Hepatic

• Hepatic porphyria primarily affects the nervous system
  • Results in abdominal pain, vomiting, acute neuropathy, muscle weakness, seizures, and mental disturbance, constipation, cardiac arrhythmia and tachycardia.
• Erythropoietic mainly affects the skin, causing photosensitivity (photodermatitis), blisters and necrosis of the skin and gums as well as increased hair growth ie on the forehead

Haem Degradation Overview

• Most haem comes from RBCs (85%) and originates with turnover of cytochrome P450 and immature erythrocytes
• The RBCs lifespan is 120 days and they get degraded by reticuloendothelial (RE) systems
• The degraded byproduct is Bilirubin
  • The RE cells microsomal haem oxygenase & biliverdin the reductase as well as this needs NADPH, O2.
  • Hydroxylates methenyl bridge, Fe+2 oxidised to Fe+3 where the second reaction cleaves ring and CO is released with Fe+3

Bilirubin Metabolism

• Bilirubin is hydrophobic- so transported by albumin.
• Some anionic drugs displace bilirubin, causing CNS damage in infants (salicylates)
• Bilirubin binds to intracellular proteins in the liver (ligandin) and Conjugation to 2 glucuronate molecules increases solubility
• Uses Bilirubin glucuronyltransferase where UDP-glucuronic acid is the donor and conjugates also bind albumin, but much weaker.
• Bilirubin is actively transported into bile and requires energy, being susceptible to liver disease and unconjugated bilirubin is not excreted
• Gut bacteria hydrolyse bilirubin diglucuronide and reduce it to urobilinogen where some urobilinogen is reabsorbed back into the blood

Urobilinogen Metabolism

• Bacteria in the intestine oxidize urobilinogen to stercobilin, giving stool its brown color.
• Urobilinogen follows in the enterohepatic cycle (liver to re-excretion in bile)
• Some that remains is converted to yellow urobilin in kidney to give urine its color

Jaundice/Icterus

• Hyperbilirubinemia is the cause (>50µM/3mg/dL) where Bilirubin deposits in skin, nail beds, and sclera of eyes and is a symptom, not a disease
• Direct Bilirubin is conjugated with glucuronic acid
• Indirect Bilirubin is unconjugated and insoluble in water
• Indirect bilirubin can cross into the brain (especially in infants)
• Bilirubin in the basal ganglia → irreversible brain damage → kernicterus à leading to hypotonia → atonia → death and then survivors have permanent neurological impairment if levels are >25mg/dL.
• Kidneys excrete direct Conjugated bilirubin

Jaundice Types

• Prehepatic (haemolytic) jaundice happens from excess bilirubin production that overwhelms the livers ability to conjugate it, due to of RBC hemolysis
  • Could stem from autoimmune disease, haemolytic disease of the newborn or structurally abnormal RBCs, with high levels of unconjugated bilirubin
• Intrahepatic (hepatocellular) jaundice where uptake, conjugation, or secretion, is impaired due to dysfunction in the liver itself and is accompanies by liver damage markers
• Posthepatic (obstructive) jaundice is due to biliary tree obstruction by conjugated bilirubin, which gives plasma a pale colour to feces. A complete obstruction has absent urobilin in the urine
• Neonatal jaundice is common. UDPGT is the enzyme that conjugates biliruben and is low in premature babies and newborn infants and they can get Kernicterus with high levels of unconjugated bilirubin. They apply blue white light in phototherapy to break it down.
• Genetic disorders of bilirubin metabolism:
  • Crigler-Najjar syndrome where the UDP glucuronyltransferase is extremely defective leading to profound jaundice
  • Gilbert's syndrome which has reduced UDP glucuronyltransferase leading to jaundice in cases of illness
  • Dubin-Johnson syndrome which has abnormal transport of conjugated bilirubin into the biliary system

Haemoglobin Types

• Major adult (HbA) composed of α2β2 (97% of adult Hb)
• Minor adult (HbA₂) made of α2δ2 (2-3% of adult Hb)
• HbA1c has a2ẞ2-glucose (3-9% of adult Hb)
• Hb Gower 1 is ζ composed of2 ε2 (Embryonic Hb)
• Foetal (HbF) has α2 Y2 (Major Hb in 2nd and 3rd trimesters of pregnancy)
• The 2,3-BPG forms a salt bond with lysine and histidine in the B-chain of HbA and HbF binds 2,3-BPG weakly for easier O2 transfer to the foetus.

Globin Genetics

• Occur on 2 chromosomes.
• A-globin genes exists on chromosome 16 and it is 2 genes for a-globin chains
• 4 a-genes exist per diploid genome and are located as pairs 2.5kb apart
• Β-globin genes exist on the short arm of chromosome 11 having a single gene for the β-globin chain

Globin chains

• Polypeptide chains form the globin protein and a high amount of α-helical structure
• L-valine it contains exists at the amino terminus.
• Includes 2 histidine residues that are present amongst the nonpolar aa in the crevice of the molecule
  • Proximal (F8) binds with iron
  • Distal (E7) stabilizers binding with oxygen
• Types are:
  • a-globins, β-globins, 8-globins, γ -globins, & -globins and ζ-globins

Diseases of Globin Metabolism

• Are genetic disorders with structurally abnormal Hb
• Happens with insufficient production of Hb
  • Inherited as an autosomal recessive trait.
  • Arise due to the substitution of one amino acid for another, deletion of a portion of the amino acid sequence, abnormal hybridisation between two chains and elongation of the globin chain
• Abnormal synthesis: Hb Constant spring (rare) ter142a→ Gln causing Loss of termination codon

Haemoglobinopathy Classification

• Abnormal solubility has cellular crystallisation of oxygenated protein leading to Mild anaemia
• A decreased affinity for O2 has a heterodimer interface leading to Mild cyanosis
• Increased oxygen causes reduced binding which leads to mild polycythemia
• Altered haem loss leads to cyanosis

Sickle Cell Anemia

• It is an autosomal recessive genetic disorder that results from mutations in the genes coding for ẞ-chains and a amino acid substitution in β-chains.
• It is common in black populations (African origin)
• Causes Fibrous network which stiffens and distorts the sickle shaped RBCs because of capillary blocks leading to severe haemolytic disease, including pain in bones, chest and the abdomen
• Happens with impaired growth, susceptibility to infections and multiple organ damage
• Electrophoresis: HbA migrates faster (negative charge/phosphate groups) than HbS
• Its treated by strict avoidance of hypoxia and dehydration through hydroxyurea, as well as heterozygotes where they will be partial resistance against malaria

Methaemoglobinemia Definition

• When Fe2+ oxidised to Fe3+ due it is not being reduced.
• NADH methaemoglobin reductase reverses this reaction so if enzyme is lacking, it leads to blue baby syndrome
• Can cause "Chocolate cyanosis" with hypoxia and dyspnea
• Its treated by ascorbic acid (antioxidant) and methylene blue (electron donor) as well as having methaemoglobin reductase that Uses NADH as a reductant and congenital methaemoglobinemia deficiency

Overview of Thalassemias

• Are genetic disorders when there is an imbalance in the creation of a- and β- globin chains and occur due to single deletions, substitutions and they are classified by levels of globin synthesis.
• They are autosomal recessive defects happening in Africa, Mediterranean, Asia, India & Burma.
• Classified as:
  • a-thalassemia (deficiency of α chains) and β-thalassemia (deficiency of ẞ chains)
  • Thalassemia minor (heterozygous mild anaemia) or major (homozygous severe anaemia) and happens across any race

α-Thalassemias

• From a defect in one or all of the four genes and is directly correlated to how severe it is
• HbBart's (γ4) and HbH ẞ4) have an impact, deleting genes cause Non-deletion forms
• Defect in one or 2 is mild (1 or 2), a defect in 3 causes HbH (tetramer of β-chains) and HbBart's (γ4) and HbH β4) cause non-a-chains lead to hydrops foetalis

β-Thalassemia

• B-thalassemmia happens becasue some mutations and an increased Synthesis of 8 and y globins
• Has
  • B+-thalassemia with Low ß-globin chains and Bº-thalassemia (homozygous) causes no detectable ß-chains
  • Elevated levels of globin lead to RBC haemolysis with anaemia, facial deformities("chipmunk faces") due to the overactive red bone marrow
  • Has only genetic counselling that can be given, as well as Iron Overload needing blood transfusion and needing Desferrioxamine (iron chelator) and a possible bone marrow replacement