Proteins in Action: Oxygen Transport and Storage by Haemoglobin and Myoglobin

Question 1

Describe the structure of Myoglobin (muscle globin)

Answer 1

Primary: ~150 AA’s
Secondary: 8 Alpha helices (A-H) and connecting loops
Tertiary: Globin fold; has hydrophobic pocket. Haem binds in pocket and also interacts with a residue called HisF8
Quaternary: Monomer, it is a single polypeptide chain

Question 2

Describe the bonds and structure of haem

Answer 2

Haem is a prosthetic group, or cofactor.
- Includes four pyrrole rings linked together in a plane
- Has six coordinate bonds - four to nitrogen atoms of the haem, one to a nitrogen atom of HisF8 and one to O2
- Molecular orbitals give haem its red colot
- Binding of oxygen to the Fe2+ is a reversible reaction

Question 3

How is oxygen binding to globins measured?

Answer 3

Spectroscopy of globins measures oxygen binding by quantifying dissolved molecules.
- Beer-Lambert law converts from absorbance to concentration
- Different wavelengths are absorbed more or less efficiently
- Shape of spectrum differs with colour and with chemical nature of solute

Haem has a visible absorbance (and therefore colour) that differs between bright red oxyhaemoglobin and dull red deoxyhaemoglobin.
- Can measure how much oxygen is bound to the globins by seeing how much light they absorb.

Question 4

Describe oxygen binding and haem structure

Answer 4

• HisF8 binds to Fe2+ out-of-plane
• Oxygen brings Fe2+ into plane
• There is an additional His on opposite side of the Haem which distorts gas (O2) binding, which allows dissociation (because it is not coordinated as tightly)

Question 5

Describe how Haemoglobin’s structure underlies its function

Answer 5

Overall structure is highly similar but it is evolved to be a tetramer that has two alpha subunits and two beta subunits. These associate non-covalently.
Each global contains one haem and each can bind one O2 molecule. So each tetramer can bind 1, 2, 3 or 4 O2 molecules (not all of the spaces have to be filled)

Question 6

What are the common mechanisms of Myoglobin and Haemoglobin binding?

Answer 6

• Haem Fe2+ is attached to globin protein by co-ordinate linkage to HisF8
• Another His (from helix E) on opposite side of haem distorts binding of gas molecules to 6th co-ordination position on haem Fe2+.
• This reduces the binding affinity of oxygen, making it easier to release oxygen to the muscle cell.

Question 7

What are the differences between how Haemoglobin and Myoglobin bind oxygen?

Answer 7

Myoglobin:
- Oxygen storage in tissues
- Needs to gold oxygen until it is required

Haemoglobin:
- Must acquire oxygen in lungs and deliver to tissues
- Must bind oxygen more weakly, to release at ‘correct’ time.

Question 8

Describe how the differences in binding curve reflect biological function

Answer 8

Myoglobin:
- Hyperbolic curve
- Saturated with oxygen at very low oxygen concentrations
- Only release O2 to the cell when cellular O2 levels are low

Haemoglobin:
- Sigmoidal curve
- In the tissues (where there is low O2 pressure), it will give up O2
- Only becomes saturates with O2 when its at very high partial pressure of oxygen.

Lungs have very high partial pressure and resting muscle has a much lower partial pressure

Question 9

How does the local environment of haemoglobin control the affinity of oxygen for it?

Answer 9

Allostery and co-operativity

Allostery: binding to additional sites. can also affect haemoglobin binding to oxygen
Co-operativty: will require an oligomer (a tetramer in haemoglobin)

Note: allosteric proteins can be monomers ro oligomers (‘Allo’ just means something changing away from the active site)

Question 10

Describe co-operativity in practice

Answer 10

• Co-operative behaviour = sinusoidal binding curve
• Likelihood of binding influenced by other subunits
• Two states with different affinities for substate (in haemoglobin we call these tense T and relaxed R)
  
  • T-state has low oxygen affinity (shown by the lower part of the curve)
  • R-state has higher oxygen affinity (shown by the upper part of the curve)
• Once oxygen binds, the speed of oxygen binding increases because it gets into a more relaxed state

Question 11

Describe how Co-operative enzymes diverge from Michaelis-Menten kinetics

Answer 11

• Many highly-regulated proteins are co-operative [enzymes as well as haemoglobin]
• V vs. [S] plot is a sigmoid, NOT a hyperbola
• Good on/off switches

Question 12

Describe the traits that both haemoglobin and myoglobin have

Answer 12

• Oxygen binds to iron of haem
• shift from dull to bright red allowing monitoring of O2 binding
• Oxygen-bound form has subtle change in haem (O2 in plane)

Question 13

Describe the differing traits between haemoglobin and myoglobin

Answer 13

• Store in tissue vs. transport molecule
• monomer vs tetramer
• tighter, hyperbolic binding vs. weaker sigmoidal binding curve - co-operativity of haemoglobin

Question 14

Co-operativity vs. Allostery

Answer 14

Co-operativity:
- Requires multiple interacting sub-units
- Co-operativity generates a sigmoidal binding curve
- Shifts binding affinity (and the steep part of the biding curve) to a physiologically relevant concentration (O2 in haemoglobin)

Allostery:
- Can occur in monomeric or multiple subunit proteins/enzymes
- Involves regulators or post-translational modifications away from the active site
- Can be linked with co-operativity