Lecture 6: Reversible Binding of a Protein to a Ligand: Oxygen-Binding Proteins Part II

Chapter 5.1

What type of cells transport oxygen?

erythrocytes (red blood cells)

What are erythrocytes formed from?

hemocytoblasts (precursor stem cells)

True or False: Arterial blood is 96% saturated with O₂, and the peripheral blood is 64% saturated with O₂.

true

True or False: Hemoglobin subunits are structurally similar to myoglobin, but myoglobin doesn’t have a quarternary structure.

true

What is the structure of hemoglobin?

• tetrameric protein with 4 heme groups

  • 2 alpha chains with 141 residues each

  • 2 Beta chains with 146 residues each

True or False: Hemoglobin and myoglobin have different primary structures but similar tertiary structure. Their primary structure is 70% identical

true

What are the interactions present in hemoglobin?

• hydrophobic effect (the main force of the quaternary structure)

• hydrogen bonds

• ion pairs (salt bridges)

How many amino acids are involved with a₁B₁ and a₂B₂?

over 30 amino acids

How many amino acids are involved with a₁B₂ and a₂ and B₁?

19 residues

What are the two conformations of hemoglobin?

R state and T state

What is the R state?

O₂ has a higher affinity for hemoglobin

What is the T state?

hemoglobin is more stable when O₂ is absent and is the predominant conformation of deoxyhemoglobin

What stabilizes the T state?

the number of ion pairs/salt bridges, which many of them lie at the a₁B₂ and a₂B₁ interface

What happens when O₂ binds to hemoglobin in the T state?

the R state undergoes a conformational change

Describe the conformational change that happens when O₂ binds to hemoglobin in the T state.

• aB subunit pairs slide past each other and rotate

• the pocket between the B subunits narrow

• some of the ion pairs that stabilize the T state beak and some new pairs are formed

When O₂ binds to hemoglobin in the T state (changing to the R state), what conformational changes are occurring near the heme?

the iron pulls His towards itself, and the alpha helix pulls closer to the heme group

What does the T state look like?

iron protrudes to the left side

What does the R state look like?

the iron is more centered and no longer protrudes to the right

What is the term for how hemoglobin binds oxygen?

hemoglobin binds oxygen cooperatively

True or False: Hemoglobin is good at binding and releeasing oxygen.

true

What is the shape of the hemoglobin binding curve?

a hybrid sigmoid binding curve for oxygen

What is an allosteric protein?

the binding of a ligand to one site affects the binding properties of another site on the same protein

What are modulators?

ligands that bind to an allosteric protein to induce a conformational change

What does homotropic mean?

the normal ligand and modular are identical

What is an example of a homotropic relationship?

oxygen to hemoglobin

What does heterotropic man?

the modulator is a molecule other than the normal ligand

What does cooperative binding mean?

the binding of a ligand to one subunit changes the conformation of the whole tetramer

True or False: CO has a 250-fold greater affinity for hemoglobin than does O₂.

true

What happens when CO binds to 1 or more heme sites?

the CO stabilizes the heme in the R state—increasing the affinity of the remaining unoccupied subunits of oxygen

What is the Hill equation?

log (Y/(1 - Y)) = n log [L] - log K_d

What is the Hill plot?

the plot of log [Y / (1 - Y)] versus log [L]

What is the Hill coefficient (n_H)?

the slope of the hill plot

What does it mean if n_H = 1?

ligand binding is not cooperative

What does it mean if n_H is more than 1?

there’s positive cooperativity, and oxygen binds to heme groups—influencing other oxygens to bind as well

What does it mean if n_H is less than 1?

there’s negative cooperativity

What is the concerted/MWC model?

the binding of an oxygen molecule shifts the equilibrium between the R and T state, with all four oxygen molecules binding to the four heme groups for the R state to be fully occupied

What is the sequential model?

hemoglobin exists in several states, with 3 intermediate states separating the T-state from the R-state

Which end-products of cellular respiration does hemoglobin carry?

H⁺ and CO₂

What does carbonic anhydrase do?

catalyzes the hydration of CO₂ to bicarbonate

What favors the T state, and what does it do to oxygen?

the binding of H⁺ and CO₂ to hemoglobin lowers the affinity of oxygen for hemoglobin

What does the Bohr effect describe?

describes the effect of pH and [CO₂] on the binding and release of O₂ by hemoglobin

What is the relationship between a slightly higher pH and O₂ affinity?

higher pH increases O₂ affinity

When [O₂] is high, what does hemoglobin do?

hemoglobin binds O₂ and releases H⁺

When [O₂] is low, what does hemoglobin do?

hemoglobin releases O₂ and binds H⁺

True or False: The pH is low in peripheral tissues—encouraging hemoglobin to release oxygen brought from the lungs.

true

Which part of hemoglobin does CO₂ bind to?

the NTD end

True or False: CO₂ binding to hemoglobin is inversely related to the binding of O₂—contributing to the Bohr effect by producing H⁺.

true

What regulates oxygen binding to hemoglobin?

2,3-biphosphoglycerate

What type of modulation is 2,3-biphosphoglycerate (BPG)?

heterotopic allosteric modulation

Where does BPG bind, and what does it do?

binds to a site distant from the oxygen-binding site—reducing the affinity of hemoglobin for oxygen (negative modulator)

When does BPG increase?

increases at high altitudes

What is hypoxia?

lowered oxygenation of peripheral tissues, which causes BPG to increase

Where does BPG bind to deoxyhemoglobin, and what does it stabilize?

BPG binds to a positively-charged cavity between the B subunits in the T state—stabilizing the T state and not allowing the T state to switch to the R state

What type of hemoglobin does a fetus synthesize?

synthesizes a₂y₂ hemoglobin

Why is fetus hemoglobin different from adult hemoglobin?

• fetus hemoglobin has a lower affinity for BPG than normal adult hemoglobin

• fetus hemoglobin has a higher affinity for O₂ than normal adult hemoglobin

True or False: Y-chains are 70% similar to adult B-chains.

true

What is important to know about sickle cell anemia?

• a homozygous condition

• single amino acid substitution in the B chains (Glu⁶ changes to Val⁶)—creating a hydrophobic patch

• sickle cells are less efficient in transporting O₂

• deoxygenated sickle hemoglobin becomes insoluble and forms polymers that aggregate

What is a potential cure for sickle cell anemia?

reactivating the gene for fetal hemoglobin, which doesn’t contain B-chains