Hemoglobin: Structure, Oxygen Transport & Hemoglobinopathies

PART 1 - Heme Proteins: Why They Matter

• Cyanide kills by blocking cytochrome oxidase (Complex IV of the electron transport chain) - it poisons cellular respiration directly.
• Carbon monoxide (CO) kills by binding to hemoglobin with extremely high affinity, preventing O2 delivery to tissues.

PART 2 - The Heme Group

Fe²⁺ Coordination - 6 bonds in total:

Why free heme can't carry O₂ safely:

PART 3 - Structural Changes on Oxygenation

• Deoxyhemoglobin: the iron lies 0.06 nm outside the plane of the porphyrin ring, pulled toward the proximal His F8 (slightly domed ring).
• Oxyhemoglobin: the iron moves to 0.021 nm from the plane - almost perfectly in-plane.

PART 4 - Hemoglobin as the Ideal Respiratory Pigment

1. Large O₂ carrying capacity - each molecule carries 4 O₂ (one per heme)
2. High solubility - can reach concentrations of ~33 g/dL inside red cells without precipitating
3. Appropriate P50 - loads at lung pO₂ (~100 mmHg), releases at tissue pO₂ (~20-40 mmHg)
4. Powerful buffer - hemoglobin is one of the most important blood buffers

PART 5 - Structure of Hemoglobin

Globin chain types:

Two conformational states:

Major hemoglobin types:

PART 6 - Oxygen Saturation Curves: Myoglobin vs. Hemoglobin

Myoglobin (single chain):

• Hyperbolic saturation curve
• P50 = 1 mmHg - saturates almost completely at very low pO₂
• Acts as an O₂ storage protein in muscle

Hemoglobin (tetramer):

• Sigmoidal (S-shaped) curve - the signature of cooperativity
• HbF: P50 = 20 mmHg
• HbA: P50 = 26 mmHg

Key physiological pO₂ values:

PART 7 - Cooperativity

The mechanism:

• Hemoglobin is an allosteric protein - binding of O₂ at one subunit changes the structure and O₂ affinity of the remaining subunits.
• The first O₂ binds with low affinity (T state). But once it binds, it helps flip the subunit toward R state, making the next O₂ bind more easily, and so on.
• This positive cooperative binding produces the sigmoidal curve.

The KNF Sequential Model (slide 22):

Haldane Effect (slide 21):

PART 8 - Allosteric Modulators (The Big Four)

1. pH - The Bohr Effect (slides 24-26)

• At the lungs: pH = 7.4 → hemoglobin binds O₂ readily (R state)
• At the tissues: pH = 7.2 (due to CO₂ + H₂O → H⁺ + HCO₃⁻) → hemoglobin binds H⁺ → shifts to T state → releases O₂ to tissues
• Deoxyhemoglobin binds 1 proton for every 2 O₂ molecules released
• H⁺ binding stabilizes the T state, lowering O₂ affinity
• The curve shifts right as pH drops (increased P50 = more O₂ released)

2. Carbon Dioxide - Hemoglobin Carbamates (slide 27)

Hb-NH₂ + CO₂ → Hb-NH-COO⁻ + H⁺

3. 2,3-Bisphosphoglycerate (2,3-BPG) - slides 29-32

• Synthesized in RBCs via the Rapaport-Leubering cycle (a side-branch of glycolysis)
• When tissue pO₂ is low → more 2,3-BPG is made
• 2,3-BPG is a negatively charged molecule that fits into the central cavity of deoxy-hemoglobin, cross-linking the two β subunits via positively charged residues
• Binds much more tightly to deoxy-HbA (T state) → stabilizes T state → O₂ is released
• Shifts the ODC to the right (raises P50)

Why HbF has higher O₂ affinity than HbA:

High altitude adaptation (slide 32):

4. Temperature (slide 35)

• A 20°C increase in temperature raises P50 by 15% → promotes O₂ release
• At 37°C, P50 = 26 mmHg; raising temperature from 20°C → 37°C causes 88% increase in P50
• Fever: shifts ODC right → more O₂ delivered (meets increased metabolic demand)
• Hypothermia: shifts ODC left → less O₂ released (can contribute to tissue hypoxia)

PART 9 - Clinical Applications of the Oxygen Dissociation Curve (Slide 36)

PART 10 - Carbon Monoxide Poisoning (Slides 39-41)

• Free heme binds CO 25,000× more strongly than O₂
• Inside hemoglobin, the distal histidine (His E7) sterically blocks CO from binding at its preferred 90° angle - forces it to bind at a less favored angle → reduces CO affinity to 200× that of O₂ (still very dangerous)
• CO binding stabilizes the R state - paradoxically this makes the remaining heme groups hold on to O₂ even more tightly (shifts ODC left) → tissues get no O₂
• Changes the sigmoidal O₂ saturation curve to hyperbolic
• CO crosses the placenta freely → HbF has higher CO affinity than HbA → fetuses are at particularly high risk

PART 11 - Hemoglobinopathies

A. Sickle Cell Disease - HbS (Slides 45-50)

• This creates a hydrophobic "sticky patch" on the surface of the β subunit
• At low pO₂, deoxyHbS polymerizes: the sticky patch on one molecule inserts into a complementary hydrophobic pocket on a neighboring deoxyHbS molecule (at residues Ala 70, Phe, Leu 88)
• Long, insoluble fibers form → distort RBCs into a sickle (crescent) shape
• Sickled cells are rigid → cannot squeeze through the spleen's sinusoids → hemolysis
• Sickled cells occlude small vessels → vaso-occlusive crises (pain, organ damage, stroke)

• Autosomal recessive
• Homozygous (HbSS) = sickle cell disease - severe
• Heterozygous (HbAS) = sickle cell trait - usually asymptomatic (1/12 African Americans)
• Reading assignment on slide 50: heterozygotes are protected against malaria - the HbS trait makes RBCs less hospitable to Plasmodium and causes infected cells to sickle under the relatively low pO₂ of the spleen → parasite destroyed

B. Methemoglobinemia (Slides 51-53)

• Drugs/chemicals: sulfonamides, nitrites, dapsone
• Reduced activity of NADH methemoglobin reductase
• Genetic mutations: HbM Iwate (proximal His F8 → Tyr) and HbM Boston (distal His E7 → Tyr) - tyrosine stabilizes Fe³⁺ directly

C. Thalassemia (Slides 54-56)

α-Thalassemia (chromosome 16, 2 gene copies per chromosome = 4 total)

• Loss of α-gene copies → excess unpaired γ chains (in fetus) or β chains (in adults)
• Excess γ chains form Hb Bart's (γ₄)
• Excess β chains form HbH (β₄)
• Both γ₄ and β₄ are stable and soluble but have hyperbolic O₂ curves (no cooperativity, no heme-heme interaction) → useless for tissue O₂ delivery
• Loss of all 4 α genes = hydrops fetalis (no α chains = no HbF = incompatible with life)

β-Thalassemia (chromosome 11, 1 gene copy per chromosome = 2 total)

• Reduced/absent β chain synthesis → excess α chains accumulate
• Excess α chains are unstable - they cannot form a stable tetramer and lyse red cell precursors in the bone marrow (ineffective erythropoiesis)
• HbF (α₂γ₂) accumulates as a compensatory mechanism
• Thalassemia minor (heterozygous): mild microcytic anemia
• Thalassemia major (Cooley's anemia, homozygous): severe anemia, requires regular transfusions, bone marrow transplant is definitive treatment

D. G6PD Deficiency (Slides 57-58)

• X-linked recessive (predominantly affects males)
• G6PD catalyzes the first step of the pentose phosphate pathway, producing NADPH
• In RBCs, NADPH → reduced glutathione (GSH) is the ONLY defense against oxidative stress
• Without G6PD, RBCs cannot protect themselves from reactive oxygen species → oxidative hemolysis
• Triggered by: oxidant drugs (primaquine, dapsone, sulfonamides), infections, fava beans
• Results in: hemolytic anemia (episodic, often self-limiting)

E. Pyruvate Kinase (PK) Deficiency (Slides 59-60)

• Second most common enzyme-deficient hemolytic anemia (after G6PD deficiency)
• PK catalyzes the last step of glycolysis: phosphoenolpyruvate → pyruvate + ATP
• RBCs have no mitochondria - they depend entirely on glycolysis for ATP
• Without PK, RBCs cannot make enough ATP → loss of K⁺ homeostasis → cell dehydration and membrane failure → hemolysis
• Also causes splenomegaly (spleen engorged with destroyed RBCs)
• Peripheral blood smear shows echinocytes (spiky cells)

Summary Table - Hemoglobinopathies at a Glance

ODC Shift Summary