Oxygen, myoglobin and haemoglobin

Question 1

At which levels can metabolic regulation occur at?

Answer 1

The genetic level - multiple copies (alleles) of a particular gene
The transcriptional level - repressors and activators determine the levels of mRNA
The translational level - translational repressors and activators are known, mRNA degradation and regulatory RNA
The protein level - hormones/proteases
Regulation directly by interactions of small molecular weight ligands with proteins

Question 2

What is used in the regulation of O2?

Answer 2

Myoglobin and haemoglobin - bind oxygen, which is used to generate energy

Organisms either use simple or assisted diffusion to acquire the gaseous oxygen from the atmosphere
O2 carriers are then needed - either heme or non-heme based
Levels of saturated haemoglobin if healthy is 98% but if unhealthy around 92%
It is moved within proteins as bubbles of gaseous oxygen would stick in our capillaries, resulting in an embolism

Question 3

Describe myoglobin?

Answer 3

This is a monomeric protein, that resides in vertebrate muscles
153 residues - 8 a helices

The haem group (within a hydrophobic pocket) is a porphyrin derivative containing 4 pyrrole groups linked by methene bridges

The haem group contains Fe2+ in the middle, coordinated by 4 porphyrin N atoms as well as an interaction with histidine
O2 can act as a 6th ligand to the Fe atom

Question 4

What is significant about the Fe2+ interacting with the O2?

Answer 4

Normally Fe reacting with oxygen = red colour
Fe(II) + O2 = Fe(III) - a ferric ion - this doesn’t happen within a haem group due to surrounding ligands
It creates a special chemical environment where Fe remains as Fe2+ - the myoglobin reaction is reversible

Question 5

What can also bind to heme groups, other than O2?

Answer 5

CO, NO, and H2S

They bind with much higher affinity than O2 = very toxic

Question 6

What is myoglobin’s role?

Answer 6

It binds to O2 to facilitate its diffusion into cells, as it increases the effective solubility of O2 in muscle cells
It is known as O2 storage in aquatic mammals e.g. whales

Question 7

Describe the myoglobin binding curve?

Answer 7

Mb +O2 MbO2
Dissociation constant K = [Mb][O2] / [MbO2]

Myoglobin has a hyperbolic curve - this type of curve occurs when ligands interact independently with their binding sites
As myoglobin is mainly in one place (not moving around the bloodstream) it binds O2 better

Question 8

Why is transfer of O2 possible between myoglobin and haemoglobin?

Answer 8

Haemoglobin appears to sense the physiological conditions of pO2 and react appropriately = different binding curve
If the binding curve of the two were the same it wouldn’t work - there wouldn’t be an ability to transfer the oxygen (therefore the different affinities are essential)

Question 9

Describe haemoglobin?

Answer 9

This is a 2 alpha and 2 beta tetramer (similar to Mb)
In vertebrates, contained within erythrocytes (red blood cells) 6-9 μm
Within the genome there are some sections there for haemoglobin but not myoglobin
Haemoglobin has two forms the deoxy-form and the oxy-form

Question 10

What are some non-haem group O2 transporting molecules?

Answer 10

Haemrythrin - in marine worms, 13 kDa, intracellular and contains 2 Fe atoms with His and acidic residues as ligands

Haemcyanin - extracellular, transport O2 in mollusks and arthropods

Question 11

What happens when oxygen binds to haemoglobin?

Answer 11

This will trigger a conformational change
When O2 binds, there are electronic rearrangements that change Fe2+ from a high to low spin state - due to distributions of the electrons within the orbitals
This changes the volume occupied by the Fe

Deoxy-state (T-state)
The porphyrin ring (surrounding ligands to the Fe2+) isn’t flat = pyramidal doming as Fe-N bonds are too long
The Fe2+ is slightly displaced from being central

Oxy-state (R-state)
Due to electronic rearrangement and change in volume occupation, the Fe2+ drops into the centre of the ring - therefore the porphyrin becomes flat - as the Fe-N bonds shorten by 0.1 Å

Question 12

What happens as a result of the O2 binding?

Answer 12

Th Fe movement is 1/6 of an angstrom
As the Fe2+ is attached to a histidine (part of an alpha helical subunit), the movement of the Fe2+ results in a lever effect
This results in a significant orientation change within the alpha helix, which will in turn propagate through the whole molecule
The predominantley hydrophobic interactions between a and b subunits are altered as one ab dimer is rotated 15°

Question 13

What is the co-operative effect of proteins?

Answer 13

Due to a large number of intermolecular reactions
The small change in binding of oxygen (allosterically) results in large changes elsewhere
The change in conformation of the F helix is magnified over large distances (like a lever) resulting in many subtle changes at the interfaces between subunits
During the deoxy state the side chains of amino acids interact between alpha and beta subunits
When oxygen binds, these interactions are ripped apart

Haemoglobin’s oxygen binding curve has an S or sigmoid shape

Question 14

What does a sigmodial curve allow?

Answer 14

It shows a disgnostic of a cooperative interaction between binding sites
When O2 binds to haemoglobin, it increases the O2 affinity in the other subunits

Question 15

What does a Hill plot show?

Answer 15

Myoglobin has a straight line - slope = 1

Haemoglobin, has a straight line at first and at the end (giving lower and upper asymptotes) - slope = 3

Question 16

What information can we determine from the slopes of a Hill Plot?

Answer 16

Positive co-operativity - slope > 1, as a substrate binds it becomes easier for more substrate to bind
e.g. haemoglobin

Non co-operativity - slope = 1
e.g. myoglobin

Negative co-operativity - slope < 1, when something binds it becomes more difficult for more to bind

Question 17

What enhances O2 transport?

Answer 17

The Bohr shift:
Due to conformational changes to haemoglobin during O2 binding - this decreases the pK’s of some groups
Decreased pK = more acidic and more likely to give up protons
Haemoglobin, releases 0.6 protons per O2 binding
This increases the pH - stimulating haemoglobin to bind more O2

Question 18

How does the bohr shift help in affinity and roles in physiology?

Answer 18

CO2 + H2O H+ + HCO3- (slow reaction)
CO2 produced in respiration is removed using carbon anhydrase to form carbonic acid (H2CO3)

Deoxyhaemoglobin has an affinity for protons, therefore the protons released by the acid are taken up by the deoxyhaemoglobin before being transferred back

Question 19

What other than pH can affect O2 binding within haemoglobin?

Answer 19

The BPG effect
D-2,3-bisphosphoglycerate is an allosteric effector
It helps promote the deoxy (T) state as it binds tightly to T and weakly to R
Therefore it decreases haemoglobin’s O2 affinity

This is useful in adaptation to high altitudes
Fetal haemoglobin has low BPG affinity

Question 20

What are allosteric molecules?

Answer 20

Allosteric molecules - they are small molecules that bind to non-active sites that lead to regulation in activity

Small molecule ligands are called effectors
They can be positive or negative in terms of the molecules they regulate
Most often they act by inducing macromolecular conformational change
These changes could be Induced fit or displacement of conformational equilibria

Question 21

What is the symmetry model of allosterism?

Answer 21

1. An allosteric protein is an oligomer of symmetrically related subunits
2. Each oligomer can exist in two conformational states (R/T), in equilibirum
3. The ligand can bind to a subunit in either conformation
4. The molecular symmetry of the protein is conserved during the conformational change

Question 22

What is the sequential model of allosterism?

Answer 22

A ligand binding induces a conformational change in the subunit it binds, and cooperative interactions arise through the influence of those conformational changes on neighboring subunits
The conformational changes occur sequentially as more ligand-binding sites are occupied

Question 23

Which model is correct for haemoglobin?

Answer 23

It exhibits features of both models

Question 24

What disease is linked to haemoglobin?

Answer 24

Sickle Cell Anaemia:
This is caused by the alteration of a single amino acid Glutamic acid for Valine at the 6th position of each beta chain
The sickled red blood cells are oo large to fit through the capillaries, and therefore getting oxygen to extremities are very difficult - the cells can burst
SCA protects against malaria

Heterozygous - erythrocytes = shorter life
Homozygous - fatal

Question 25

What can treat sickle cell anaemia?

Answer 25

Hydroxyurea increases the fraction of cells containing fetal haemoglobin
As this haemoglobin contains gamma chains as opposed to the defective beta chains

Question 26

Describe a ligand induced affect on a virus?

Answer 26

Hepatitis B Virus has infected 2 billion people
There is now a vaccine but it is nor curative
HBV is a dsDNA virus but assembles a nucleocapsid around a pre-genomic RNA copy
There is a sequence-specific RNA-CP interaction, regulated by conformational change, promotes assembly
This blocks the RNA-CP contact and therefore a novel drug target
This is due to an allosteric mechanism