08. Cellular oxygenation

Question 1

What is heme?

What is the structure of heme?

Answer 1

Heme is a prosthetic group that permanently binds to heme proteins.

It is made up of a porphyrin ring, with a central iron itom (Fe2+).

Question 2

In the structure of heme, the central iron atom is able to form 6 coordinate bonds. What atoms or groups are involved in these coordinate bonds?

Answer 2

In the 4 coordinate bonds in the equatorial plane, iron atom is binded to 4 N atoms belonging to the porphyrin ring.

axial coordinate bonds
The 5th coordinate bond (on top) : binds to Histidine F8 residue, which is bonded to F helix of hemeproteins.

The 6th coordinate bond (bottom): ligands such as O2, CO bind to iron atom, and there is the presence of Histidine E7 residue below the ligand

Question 3

What is the structure of hemoglobin and how many oxygen molecules can it bind?

Answer 3

It is a tetramer with 4 subunits (2α 2β). It consists of α1β1 and α2β2 dimers.

Since each subunit has a heme prosthetic group, each hemoglobin molecule can bind to 4 oxygen molecules.

Question 4

What is the function of hemoglobin?

Answer 4

Transport oxygen from lungs to tissues (and CO2 from tissues to lungs)

Question 5

What special structure in the body carries hemoglobin? List the features of this structure in helping in oxygen delivery. [2 main features]

Answer 5

Red blood cells

1. lacks a lot of organelles, including mitochondria
   - RBC does not consume oxygen it contains inside itself (respire through anaerbic glycolysis) + lack of organelles allow it to pack more hemoglobin and deliver more oxygen
2. Biconcave disc shape
   - High surface area to volume ratio
   - enhances flexibility of RBC to squeeze through narrow capillaries.

Question 6

What is the structure of myoglobin and how many oxygen molecules can it bind?

Answer 6

It is a monomer with 1 globin polypeptide chain with 1 heme group within it.

• 1 heme group = bind 1 oxygen molecule

Question 7

Where is myoglobin usually found and what is its function?

Answer 7

It is typically found in muscle fibres and provides localised oxygen reserves for time of intense respiration.

Myoglobin is usually found in muscles that are used extensively and it provides a fast source of oxygen to muscle tissues during periods of intense activity so that aerobic respiration can take place

Question 8

Sketch the oxygen dissociation curves for hemoglobin and myoglobin, including the axes. What does the shape of the graph indicate about hemoglobin?

Answer 8

Refer to notes.

But myoglobin has Michaelis-Menten kinetics, while Hemoglobin shows a sigmoidal curve

The sigmoidal shape of the graph for hemoglobin indicates cooperative binding

Question 9

Heme can bind oxygen without proteins such as myoglobin. However, the protein part is still important. List 2 reasons why.

Answer 9

1. The protein portion, such as myoglobin, protects the heme iron atom from oxidation.

• When reacting with oxygen, fe2+ gets oxidized into Fe3+, while O2 becomes a superoxide ion (O2)-
• Histidine E7 residue stabilizes the superoxide ion so that superoxide does not leave iron in the Fe3+ state (then fe3+ will nvr revert back to +2) and prevents the release of superoxide ions (harmful reactive species)

2. Myoglobin decreases the affinity of heme to ligands such as CO which binds irreversibly. This is due to the presence of His E7 residue from the protein, which poses steric hindrance and forces CO to tilt away from its preferred perpendicular arrangement, reducing affinity of heme for CO.

Question 10

What is the function of the His F8 residue?

Answer 10

It is to fix the heme group to hemoglobin F helix.

• Fe2+ from heme group is bonded to His F8, which is bonded to F helix of hemoglobin.

—- Fe2+ —– His F8 —– F-helix

Question 11

What is the function of the His E7 residue?

Answer 11

To make it more exclusive for O2, to expel / exclude CO by posing steric hindrance, reducing affinity of CO binding.

Question 12

Describe what happens before and after O2 binds to the 6th coordination site of heme in myoglobin, stating the conformational changes to the porphyrin ring/other components.

Answer 12

Before O2 binds

• The His F8 residue pulls the iron atom of heme away from the plane of the porphyrin ring, forming a dome shape.

After O2 binds

• as O2 binds, it pulls down iron atom to the same plane as the porphyrin ring.
• because iron atom is pulled down, proximal His E8 residue and F helix also gets pulled down.
• This induces a conformational change in the F helix of hemoglobin/myoglobin.

Question 13

In cooperative binding, what is the T state and R state and how is the iron atom positioned?

Answer 13

Tense state : not favorable for O2 binding because Fe2+ located above porphyrin ring, and the porphyrin ring repels O2 (steric hindrance)

Relaxed-state : Fe2+ lowered to porphyrin plane, O2 binding (in subsequent subunits) is enhanced.

Question 14

State the process of cooperative binding in hemoglobin.

Answer 14

• When O2 binds to a subunit, it pulls Fe2+ and f helix towards porphyrin ring, causing subunit to convert from T to R state.
• O2 bound subunit transmits its conformational change to 1 neighboring subunit and increases their binding affinity.
• As more oxygen is bound to the tetramer, more molecular strain accumulates in the O2‐bound subunits, making subsequent binding of oxygen more likely.

Question 15

[Ions/molecules that reduce binding of O2 to hemoglobin]

What is the name of the effect that protons (H+) have on oxygen binding capabilities of hemoglobin?

Answer 15

Bohr effect.

Question 16

What does the Bohr effect state, and how is it represented in a graph of % saturation against partial pressure of O2?

Answer 16

Bohr effect states that hemoglobin’s affinity for O2 decreases in response to changes in pH / carbon dioxide concentration.

• as pH decreases, the oxygen dissociation curve shifts to the right.

Question 17

[Ions/molecules that reduce binding of O2 to hemoglobin]

How do protons decrease the affinity of O2 binding to hemoglobin?

Answer 17

With higher [H+], pH decreases.

In the T-state, the basic side chain of a His 146 residue is protonated, and forms ionic interactions with negatively charged carboxy group of an Asp 94 residue. In R-state, His 146 residue is deprotonated and thus uncharged side chain of His 146 does not form interactions with C terminus of Asp 94.

• when pH lowers, position of equilibrium tends to favor the T-state where His 146 basic side chain is protonated and forms the ionic salt bridge w C terminus of asp 94. Since T state is favored, it is less favorable for O2 binding, and heme’s affinity for O2 decreases. thus at lower pH, more oxygen is released from hemoglobin to cells.

Question 18

[Ions/molecules that reduce binding of O2 to hemoglobin]

How does CO2 act as an allosteric effector to reduce binding of O2 to hemoglobin?

Answer 18

Co2 binds to N terminus of αβ dimer to form the carbamate ion, with a free COO- group.

The COO- group of the dimer can react with the N terminus of another αβ dimer, forming an ionic bridge.

This induces the T state and reduce binding of O2 to hemoglobin

Question 19

[Ions/molecules that reduce binding of O2 to hemoglobin]
When CO2 is present, the conformational change that occurs during the T to R transition takes place primarily in the positions between individual subunits. True or False?

Answer 19

False, the T to R transition takes place primarily in the positions of the two dimers relative to one another (α1β1 dimer and α2β2 dimer)

Question 20

What is the full name fr 2,3-BPG?

Answer 20

2,3- biphosphoglycerate

(it is an intermediate of the glycolytic pathway)

Question 21

[Ions/molecules that reduce binding of O2 to hemoglobin]

How does 2,3-BPG reduce binding of O2 to hemoglobin?

Answer 21

2,3-BPG binds to the positively charged cavity (space) formed by β1 and β2 subunits. This stabilises the T state, reducing affinity of hemoglobin for O2.

Question 22

Role of 2,3-BPG in high-altitude adaptation

How does the oxygen dissociation curve for normal levels of BPG and high levels of BPG look like? What does this imply?

Answer 22

When there is high BPG, the oxygen dissociation curve shifts to the right.

This means at the same partial pressure of oxygen, the fractional saturation of hemoglobin with oxygen is lower when BPG levels are higher as compared to normal.

(at higher altitude, there is less oxygen in the air, so o2 affinity to hemoglobin reduces to supply more o2 to cells)

Question 23

What is the difference between fetal and adult hemoglobin? How does this affect oxygen binding to hemoglobin and what does this signify?

Answer 23

HbF (fetal hemoglobin) consists of α2γ2 subunits (2α, 2γ) and HbA (adult hemoglobin) consists of α2 β2.

• In γ subunit, residue is serine instead of His, thus there is a lack of positive charges in the γ cavity
• This mean that 2,3-BPG binds weaker to fetal Hb, and thus fetal Hb has higher affinity to O2 as compared to HbA (R state favoured in fetus)→ ensuring transfer of O2 from maternal circulation across placenta to RBC of fetus

Question 24

Sickle cell anemia is a genetic disorder caused by?

Answer 24

A single (amino acid) residue substitution in the** β globin chain. In *normal hemoglobin, residue is *glutamic acid but in sickled cell, residue is valine

Question 25

How does this mutation affect the hemoglobin and cause the red blood cell to form a sickle shape?

Answer 25

[refer to notes for more detail]

After O2 has been delivered, when hemoglobin exists as deoxyhemoglobin, β1 subunit has a hydrophobic pocket while β2 has a valine residue protruding out.

1. The mutant Val side chain in a HbS tetramer fits into a hydrophobic pocket on surface of a β subunit in another HbS tetramer
2. Hydrophobic pocket accommodates Val binding only in T state (deoxyhemoglobin)
3. Hydrophobic interactions between mutant val from one β subunit and hydrophobic pocket of neighbouring hemoglobin initiates aggregation of deoxyhemoglobin (forming rod shapes)
4. The aggregation of deoxyhemoglobin in rod-like shapes causes the sickling of cells.
5. HbS fibres dissolve instantaneously on oxygenation, thus sickle cells often have a short life span as compared to normal RBC