3 - Protein function III - Myoglobin

Question 1

Hypoxia

Answer 1

A deficiency of oxygen at tissue
The brain is the most sensitive of our tissues to any deficiency in oxygen.
Hypoxia can cause:-
– Unconsciousness in 15 sec
– Irreversible brain damage in 2 min
– Cell death in 4-5 min

Question 2

How to measure Oxygen abundance

Answer 2

The way that is usually done is by reporting a partial pressure – this is usually measured in Torr or mm of Hg
Oxygen abundance at sea-level is about 21% of the air and that is about 155.5 Torr.
Atmospheric pressure is around 760 Torr.

Question 3

Myoglobin

Answer 3

First Protein Structure
• Myoglobin was purified from whale blood by Max Perutz in 1958.
• Showed a 75% α-helical with 155 amino acids and ~ 17 kDa.

Question 4

Myoglobin – Key features

Answer 4

• About 75% of the main chain is folded into RH α-helices, referred to as A, B, C,….H,
• Helical segments are generally terminated by Proline residues, a cyclic aa with a locked conformational angle.

Question 5

Myoglobin - Structure

Answer 5

• The interior of the molecule consists almost entirely of non-polar residues.
• A haem group is located in a crevice in the molecule, where it is surrounded by non-polar residues except for two histidines shown in green.

Question 6

Problems with transportingoxygen

Answer 6

– O2 has only limited solubility (1 X 10-4 M) in water.
– O2 will diffuse only very slowly in tissues.
– Binding of O2 to a free heme group is irreversible.

Question 7

How does Myoglobin overcome problems with transporting oxygen?

Answer 7

Enclosure the heme group in a protein to allow reversible binding and overcome the solubility problem and this acts as the transporter.

Question 8

The Haem group

Answer 8

• The non-polypeptide Haem group confers oxygen-binding capacity on myoglobin and haemoglobin (and it gives them their colour).
• The haem group contains an iron atom (Fe2) and an organic part, protoporphyin IX, that occurs in myoglobin and haemoglobin.
• The Fe2 in haem binds to 4 N atoms in a plane and has 2 other coordination positions on either side of the haem plane – the 5th and 6th coordination positions.

Question 9

Structure of the Haem group

Answer 9

• The haem group contains an iron atom (Fe2) and an organic part, protoporphyin IX, that occurs in both myoglobin and haemoglobin.
• This structure is flat and interactions with the Fe2 occur in front of, and behind, the plane of the screen in the 5th and 6th coordination positions.
• Binding of O2 alters heme electronic structure causing changes in colour.
• Hence the bright scarlet colour of oxygenated blood in arteries and the dark purple color of deoxygenated blood in veins

Question 10

Resodue

Answer 10

When two or more amino acids combine to form a peptide, the elements of water are removed, and what remains of each amino acid is called an amino-acid residue.

Question 11

Two histidine residues in helices E and F are:

Answer 11

Two histidine residues in helices E and F are:
• DISTAL His (E7)
• PROXIMAL His (F8)

Question 12

The iron atom of the haem

Answer 12

• The iron atom of the haem is directly bonded to F8 (proximal), which occupies the 5th coordination position.
• The iron atom is 0.3 Å out of the plane of the porphyrin on this side of this histidine.
• The iron atom of the haem is NOT directly bonded to E7

Question 13

which coordination position is occupied by Histidine E7

Answer 13

The 6th coordination position is occupied by the distal histidine at E7.

Question 14

Histidine E7 roles

Answer 14

• It reduces the affinity of binding carbon monoxide for the site, which nevertheless remains higher than that foroxygen.
• It sterically blocks the formation of myoglobin associates in which haem – O2 – haem sandwiches would form.

Question 15

Why does CO need to be stopped binding to the haem? How is this done?

Answer 15

• CO binds tightly; linear.
• O2 binds less tightly, bent structure.
• Distal His forces bent binding of both, weakens CO binding.

Question 16

Equilibrium of oxygen binding

Answer 16

Mb + O2 <=>MbO2

Question 17

Kd of myoglobin

Answer 17

Mb + O2 <=> MbO2

KD = [Mb] [O2] / [MbO2]

KD=2.75 mm Hg
In this case KD and p50 are the same.

Question 18

How does myoglobin release oxygen in lower concentrations?

Answer 18

Just like any equilibrium we can force it to one side or the other of the equation by changing the conditions.
If the complex arrives at a place with low O2 concentration then that forces the equilibrium to the left to release O2 from the complex.

Question 19

Le Châtelier’s principle

Answer 19

If we have a mixture of A and B which combine to form AB, then if the concentration of B is reduced, some of the AB mixture will dissociate to release free A and B.
In our discussions A is the molecules which carry B, which is oxygen
A + B ⇔ AB

Question 20

Myoglobin Function in Humans

Answer 20

Myoglobin inside muscle cells facilitates the diffusion of oxygen by binding oxygen when O2 concentration is high (at the interface with the extracellular fluid) and releasing it where O2 concentration is low (at the mitochondria).
In humans myoglobin facilitates the diffusion of oxygen.