Unit 4 Question Bank
⚠ Note: lower affinity means P₅₀ increases (Harper's p.57). TMU Slide 20 says “decreases P₅₀”, which is an error — the two halves of that sentence contradict each other. Answer in terms of affinity and you are safe either way.Harper's ch.6, p.57 — corrects TMU Lecture 4 Slide 20
Some proteins consist of polypeptide alone; others, such as myoglobin and haemoglobin, require a prosthetic group — in their case haem, held in a hydrophobic pocket by non-covalent bonding.TMU Lecture 4 Slide 34
The β-position substituents are methyl, vinyl and propionate. The iron forms six coordination bonds: four to the pyrrole nitrogens, a fifth to the proximal histidine F8, and a sixth reserved for oxygen or left unoccupied. Its network of conjugated double bonds absorbs visible light and colours haem deep red.Harper's ch.6, p.52 · TMU Lecture 4 Slides 4, 35
It permits haemoglobin to maximise both the quantity of O₂ loaded at the pO₂ of the lungs and the quantity released at the pO₂ of peripheral tissues, and is the reason its dissociation curve is sigmoid rather than hyperbolic. Cooperative interactions are an exclusive property of multimeric proteins, and are critically important to aerobic life.Harper's ch.6, p.55
A high P₅₀ indicates LOW affinity, and therefore better release of oxygen at the tissues. In all instances P₅₀ exceeds the pO₂ of the peripheral tissues. HbA: 26 mm Hg. HbF: 20 mm Hg — the lower fetal value allowing HbF to extract oxygen from maternal HbA across the placenta.Harper's ch.6, p.55
The transition is triggered when O₂ binding pulls the haem iron into the plane of the ring, moving His F8, rupturing the salt bridges and rotating one α/β pair through 15°. The same terms describe the low- and high-affinity conformations of allosteric enzymes.Harper's ch.6, pp.55–56
In peripheral tissues: CO₂ combines with water to form carbonic acid, which dissociates into protons and bicarbonate. Deoxyhaemoglobin binds one proton for every two O₂ released, acting as a buffer; the resulting lower pH stabilises the T state and enhances O₂ delivery.
In the lungs: uptake of oxygen releases protons, which combine with bicarbonate to form carbonic acid; carbonic anhydrase dehydrates this to CO₂, which is exhaled. Oxygen binding therefore drives CO₂ exhalation.
The protons arise from rupture of salt bridges involving β-chain His 146. The effect depends on cooperativity and is therefore absent in myoglobin.Harper's ch.6, p.56
Of the over 1100 known mutations affecting human haemoglobins, almost all are extremely rare and benign and present no clinical abnormality; the term is reserved for those that impair function. More than 7% of the world's population carry a haemoglobin disorder.Harper's ch.6, p.57
Normally the enzyme methaemoglobin reductase reduces Fe³⁺ back to Fe²⁺. Methaemoglobinaemia arises from oxidation by agents such as sulfonamides, from hereditary haemoglobin M (in which His F8 is replaced by tyrosine, whose phenolate anion stabilises Fe³⁺), or from reduced methaemoglobin reductase activity.Harper's ch.6, pp.57–58
The central statement
Myoglobin and haemoglobin have different primary structures but nearly identical secondary and tertiary structures. They differ fundamentally in quaternary structure — and every difference in their biological function follows from that one structural difference.
Myoglobin — structure
- Primary: a single polypeptide of 153 aminoacyl residues, MW about 17 000.
- Secondary: about 75% of residues lie in eight right-handed α-helices of 7–20 residues, named A–H from the amino terminal.
- Tertiary: a compact, roughly spherical molecule of 4.5 × 3.5 × 2.5 nm, with a polar surface and an interior of non-polar residues — with two exceptions, His E7 and His F8, which lie close to the haem iron and function in oxygen binding.
- Quaternary: none — myoglobin is monomeric.
Haemoglobin — structure
- A tetramer of two subunit types: α₂β₂ in HbA. Subunits are held together by non-covalent forces, importantly salt bridges.
- Each subunit closely resembles myoglobin: rich in α-helix, highly compact, hydrophilic residues outside and hydrophobic inside, the hydrophobic interior forming a pocket that binds one haem by non-covalent bonding. The α subunit has seven rather than eight helical regions.
- Four haem groups, four oxygen molecules — one per subunit.
The prosthetic group
Haem is a cyclic tetrapyrrole of four pyrroles linked by methyne bridges, with central Fe²⁺. The iron forms six coordination bonds: four to the pyrrole nitrogens, a fifth to the proximal histidine F8, and a sixth reserved for oxygen. The distal histidine E7 lies on the opposite face and hinders the binding of carbon monoxide.
Structure determines function
| Myoglobin | Haemoglobin | |
|---|---|---|
| Location | Red skeletal muscle | Erythrocytes |
| Function | Oxygen storage | Oxygen transport, plus CO₂ and protons |
| Cooperativity | None — monomeric | Present — a multimeric property |
| O₂ curve | Hyperbolic | Sigmoid |
| Bohr effect | Absent | Present |
The mechanism linking the two. In deoxymyoglobin the iron lies about 0.03 nm outside the plane of the haem. On oxygenation it moves into the plane, and because it is bonded to His F8 the histidine and its attached residues are pulled with it. In monomeric myoglobin this motion has no further consequence. In the haemoglobin tetramer it ruptures salt bridges between the carboxyl-terminal residues of all four subunits, so that one α/β pair rotates 15° relative to the other, converting the molecule from the low-affinity T (taut) state to the high-affinity R (relaxed) state. Because subsequent binding events require the rupture of fewer salt bridges, the affinity of the remaining haems rises — cooperativity, and hence the sigmoid curve.
Why this matters physiologically. A sigmoid curve is steep across the range between lung (pO₂ 100 mm Hg) and tissue (40, or 20 mm Hg in active muscle), so haemoglobin loads almost fully in the lung and unloads a large fraction in the tissues. Myoglobin's hyperbolic curve leaves it nearly saturated at tissue pO₂, releasing oxygen only when muscle pO₂ falls to about 5 mm Hg — exactly the behaviour required of a reserve store.
The principle
Sickle cell anaemia is the classic molecular disease: a change in a single amino acid of the primary structure produces, through a chain of structural consequences, a clinical illness. The gene, the protein, the cell and the patient can be connected in one continuous argument.
1 · The mutation
In haemoglobin S, the non-polar valine replaces the polar surface residue glutamate at position 6 of the β subunit:
HbA Val-His-Leu-Thr-Pro-Glu-Glu-Lys
HbS Val-His-Leu-Thr-Pro-Val-Glu-Lys
A single base change in the β-globin gene; a single residue changed in a chain of 146.
2 · The consequence for the protein surface
Substituting a hydrophobic residue for a charged one at a surface position generates a hydrophobic “sticky patch” on the surface of the β subunit. This patch is present in both oxyHbS and deoxyHbS — it is a permanent feature of the mutant protein.
3 · The complementary patch
Both HbA and HbS possess a complementary sticky patch elsewhere on their surface. Critically, this complementary patch is exposed only in the deoxygenated T state.
4 · Polymerisation
Consequently, at low pO₂, the sticky patch of one deoxyHbS molecule binds the complementary patch of the next, and deoxyHbS polymerises into long, insoluble, twisted helical fibres. In the oxygenated R state the complementary patch is hidden and no polymer forms — which is why the disease is episodic and precipitated by hypoxic stress such as high altitude, infection or exertion.
5 · The cell and the patient
The fibres distort the erythrocyte into the characteristic sickle shape, converting a normally flexible cell into a stiff one. Such cells are vulnerable to lysis in the interstices of the splenic sinusoids, producing the haemolytic anaemia, and cause multiple secondary clinical effects through vascular occlusion.
Why heterozygotes are protected
Binding of deoxyHbA terminates fibre polymerisation, because HbA possesses only the complementary patch and lacks the second sticky patch needed to bind a further haemoglobin molecule. HbA therefore acts as a chain terminator. The same reasoning underlies an emerging treatment — inducing HbF expression to inhibit HbS polymerisation — alongside stem cell transplantation and, in future, gene therapy.
The wider lesson
Compare HbS with haemoglobin M, in which His F8 is replaced by tyrosine. There the substituted residue is bonded to the haem iron itself: the phenolate anion stabilises Fe³⁺ and oxygen binding is abolished. Two single-residue substitutions, two entirely different mechanisms of disease — because what matters is not only which amino acid changes but where it sits in the folded structure.
The problem both solve
A transport protein must do two contradictory things: bind oxygen tightly in the lung and release it readily in the tissues. Cooperativity provides part of the answer. The rest comes from ligands that shift the T⇌R equilibrium according to local conditions — and the general rule is that anything stabilising the T state lowers affinity and improves delivery.
The Bohr effect
Definition: the reciprocal coupling of proton and oxygen binding by haemoglobin.
In the peripheral tissues. CO₂ generated by respiration combines with water to form carbonic acid — a reaction catalysed in the erythrocyte by carbonic anhydrase — which dissociates into bicarbonate and protons. Deoxyhaemoglobin binds one proton for every two O₂ molecules released, contributing significantly to the buffering capacity of blood. The resulting lower pH, aided by carbamate formation, stabilises the T state and thereby enhances oxygen delivery.
In the lungs. The process reverses. As O₂ binds to deoxyhaemoglobin, protons are released; they combine with bicarbonate to form carbonic acid, and dehydration of H₂CO₃ by carbonic anhydrase yields CO₂, which is exhaled. The binding of oxygen thus drives the exhalation of CO₂.
The molecular origin. The protons arise from the rupture of salt bridges when O₂ binds to T-state haemoglobin — specifically those involving β-chain His 146. On release of oxygen the T structure and its salt bridges re-form, which increases the pKa of His 146 so that it binds protons once more.
Note: the Bohr effect depends on cooperative interactions between the haems of the tetramer, so the monomeric structure of myoglobin precludes it.
Carbon dioxide transport
Haemoglobin also carries CO₂ directly, as carbamates formed with the amino-terminal nitrogens of the chains, accounting for about 15% of the CO₂ in venous blood. Carbamate formation changes the charge on the amino terminals from positive to negative, favouring salt bridge formation between α and β chains — so it, too, stabilises T. Most of the remaining CO₂ travels as bicarbonate.
2,3-bisphosphoglycerate
Synthesis. A low pO₂ in peripheral tissues promotes the synthesis of BPG in erythrocytes from the glycolytic intermediate 1,3-bisphosphoglycerate.
Binding. The tetramer binds one molecule of BPG in the central cavity formed by its four subunits. The space between the H helices of the β chains lining that cavity is wide enough to accommodate BPG only when haemoglobin is in the T state.
Mechanism. BPG forms salt bridges with three positively charged groups on each β chain — the terminal amino group via Val NA1, and Lys EF6 and His H21. These are additional salt bridges that must be broken before conversion to the R state. BPG therefore stabilises deoxygenated T-state haemoglobin and lowers oxygen affinity.
Two consequences worth stating.
- Fetal haemoglobin. Residue H21 of the γ subunit is serine rather than histidine. Serine cannot form a salt bridge, so BPG binds more weakly to HbF; the T state is less stabilised, and HbF therefore has a higher oxygen affinity (P₅₀ 20 versus 26 mm Hg) — enabling it to extract oxygen from maternal HbA across the placenta.
- Adaptation to altitude. Prolonged exposure raises erythrocyte number, haemoglobin concentration and BPG synthesis. Elevated BPG lowers the affinity of HbA for oxygen, enhancing release at the peripheral tissues — a trade of loading efficiency for unloading efficiency, which is the correct trade when ambient oxygen is scarce.
The unifying statement
Protons, carbon dioxide, chloride and BPG all stabilise the T state, and the higher their concentration the more oxygen must bind to trigger the transition to R. All four are signals that a tissue is metabolically active — and all four act by the same means, preserving or adding the salt bridges that hold the taut state together.