Lecture 7 - Proteins in action

Question 1

Problem of oxygen availability in animals

Answer 1

Concentration of oxygen in saline solution is limited to approx 0.2 mmol/L, while the concentration of haemoglobin is approximately 5 mmol/L

Highly active tissue e.g. exercising muscle or the brain, is limited by the availability of oxygen

There is strong evolutionary pressure for efficient oxygen delivery

Question 2

Haemoglobin

Answer 2

Has 4 subunits - 2 alpha and 2 beta
Each subunit has a globing (protein part) and a haem/heme (non-protein part)
Haemoglobin binding to oxygen is tightest in the lungs and releases oxygen into the tissues easier

Haemoglobin collects and binds oxygen and transports it around the body

Held together by non-covalent interactions between side chains - displays cooperatively which describes the phenomenon when one subunit affects the other
Evolved to have a weaker binding affinity to oxygen (transport) compared to myoglobin (high affinity, storage)

Question 3

Myoglobin

Answer 3

Myoglobin, a protein found in the muscle cells of animals. It functions as an oxygen-storage unit, providing oxygen to the working muscles.

Myoglobin binds and stores oxygen for use in the muscles in bursts of high requirement

Primary structure - Approx 150 amino acids
Secondary structure - 8 alpha helices A-H and connecting loops (arranged into the globin fold)
Tertiary structure - globing fold with a hydrophobic pocket
Haem binds to His F8 (8th amino acid in helix F) in globing protein
Quaternary structure - monomeric (a single polypeptide chain)
The globin fold provides a hydrophobic pocket (Val E11 and Phe CD1) to bind a haem group

Question 4

What are the two major components of a myoglobin or haemoglobin molecule?

Answer 4

The haem group and the globin (the polypeptide chains of the molecule)

Question 5

What secondary structure dominants the globin protein?

Answer 5

Alpha helices with connecting loops

Question 6

Active tissues and oxygen

Answer 6

Active tissues can use more oxygen than blood can deliver. The haem protein myoglobin meets this challenge by storing oxygen in muscles against bursts of high requirement.

Human muscles have 0.5-0.7 mmol/L myoglobin, enough for about 7 seconds of intense activity. After this store is exhausted the tissue depends on oxygen delivery from the lungs.
Whale muscles have up to 3 mmol/L, perhaps helping with deep dives.

Question 7

Heme

Answer 7

Is a prosthetic group or cofactor
4 pyrrole rings linked together (a protoporphyrin) in a plan
6 coordinate bonds - Four to nitrogen atoms of the haem, one to a nitrogen atom of histidine F8 from the globin and one to O2
Electronic molecular orbitals of protoporphyrin give a red colour
Binding of oxygen to the Fe2+ is a reversible interaction

RIng of hydrogen and nitrogen

Histidine (N) donor from the 8th AA in F helix (HisF8). His E7 weakens binding of oxygen. HisE7 allows for weak binding so that it is reversible.

Question 8

Describe the structure of the haem group

Answer 8

Contains a central Fe, with four pyrrole rings linked together (a protoporphyrin) in a plane

Question 9

Fe2+has six coordination bond sites, what binds to each of these sites?

Answer 9

Four to nitrogen atoms of the haem, one to a nitrogen atom of histidine F8 from the globin and one to O2

Question 10

What is the role of HisE7 in myoglobin?

Answer 10

Located on opposite side of His F8 and reduces binding affinity of oxygen to myoglobin, making it easier to release oxygen to the muscle cells

Question 11

Spectroscopy and absorbance

Answer 11

Higher concentration = less transmitted light = higher absorbance
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.
Protein is colourless (but has UV absorbance).
Haem has visible absorbance (and therefore colour) that differs between bright red oxyhaemoglobin (HbO2) dull red deoxyhaemoglobin (Hb).

Question 12

Myoglobin - haem interaction with oxygen

Answer 12

Haem Fe2+ is attached to globin protein by co-ordinate linkage to His F8. Another His in helix E (His E7) is located on opposite side of haem and
distorts binding of gas molecules to 6 th co-ordination position on haem Fe2+
This reduces the binding affinity of oxygen to myoglobin, making it easier to release oxygen to the muscle cell.

Question 13

Deoxyhaemoglobin

Answer 13

The form resulting from when oxyhemoglobin loses its oxygen

Has a dished haem

Question 14

What will weaken oxygen binding?

Answer 14

Anything that keeps helix F away will weaken oxygen binding

Question 15

Oxyhaemoglobin

Answer 15

Oxygen flattens the haem, and pulls histidine F8 and helix F towards the binding site

Question 16

Oxygen changes haemoglobin’s shape …

Answer 16

Compared to deoxyhaemoglobin, O2 binding to oxyhaemoglobin moves the Fe2+ into the plane of the haem, draws His F8 down, and repositions helix F.
Shifts in the orientation of protein secondary elements, such as helix F moving relative to helix C, are called ‘conformational changes’.

Anything that keeps the heme in the dished form (deoxy) is going to weaken oxygen binding. Anything that keeps the heme flat in the form it likes to be in when binding oxygen will make oxygen binding strong, so it will bind oxygen more strongly and this is how we tune the binding in our lungs versus out muscles to either promote binding or release oxygen

Question 17

Mechanism of oxygen binding in haemoglobin

Answer 17

1. Low PO2, oxygen has low saturation and all subunits in low-affinity T-state
2. PO2 increases (i.e. in lungs) —> 1 O2 molecule binds to one sub unit, conformational change from T to R state.
3. Non-covalent interactions cause the other three subunits to also change shape in response to initial subunits change —> all at high affinity R state
4. Rapid binding of O2 Reverse is also true
   Change from T to R can be sequential (one at a time) but mostly concerted (all at once) [realistically a mix]

Allows for better loading in the lungs and unloading in the tissues - better transporter. Haemoglobin’s tetrameter structure allows for its function as a transporter… as we increase the partial pressure of oxygen, go more into R state and therefore increase in binding of oxygen.

Question 18

T state

Answer 18

Oxygen not bound
Taut = deoxyhaemoglobin
Low affinity - doesn’t really want to bind oxygen

Question 19

R state

Answer 19

Oxygen is bound
Relaxed = oxyhemoglobin
High affinity - wants to bind oxygen but doesn’t want to let it go

Question 20

Myoglobin and haemoglobin both show…

Answer 20

allosteric control’ of oxygen affinity ….LActate decreases myoglobin’s affinity for oxygen but does not bind where oxygen binds. Lactate build up in muscle promotes oxygen release from myoglobin, increasing oxygen availability for respiration

Allosteric builds on ‘steric hinderance’, the impossibility of two atoms occupying the same space

Question 21

Myoglobin binds quite _____ to oxygen

Answer 21

Tightly

Myoglobin is O2 saturated at low pO2 only releasing O2 to muscle cells when the cellular pO2 is very low. This is shown in a ‘hyperbolic’ curve.
This suits its function as a “back-up” store of O2 in muscle cells.
Mb + O2 MbO2
The partial pressure of oxygen in lungs, or pO2, is ~100 Torr, and in resting muscle it is ~ 20 Torr.

Reversible oxygen binding but binds a lot stronger than haemoglobin

Question 22

Haemoglobin oxygen binding and release

Answer 22

The availability of O2 to cellular proteins depends on:
- The pO2 in the local environment
- The binding affinity of O2 to myoglobin or haemoglobin

Haemoglobin in red blood cells in the blood needs to be able to:
- Bind O2 in the vicinity of the lungs where the pO2 is ~100 Torr
- Release the O2 in the vicinity of peripheral tissues where the pO2 is ~20 Torr

Haemoglobin therefore evolved to bind O2 less tightly.

Question 23

Which one is a tetramer and which one is a monomer?

Answer 23

Haemoglobin is a tetramer (4 subunits) and myoglobin is a monomer (1 subunit)

Question 24

Haemoglobin evolved to be a …

Answer 24

Tetramer - 4 globing proteins associate together non-covalently
Each globing protein contains one haem and each can bind one oxygen

Question 25

Colour of oxy and deoxy (haemoglobin and myoglobin)

Answer 25

Dull red is deoxy and it shifts to bright red for oxy

Question 26

Different haemoglobin conformations

Answer 26

Hexangonal, plate shaped crystals AND anaerobic zone goes into needle-shaped crystals, aerobic zone and interface of air and protein solution

Question 27

Similarities between haemoglobin and myoglobin

Answer 27

Oxygen binds to iron of haem
Shift from dull to bright red allows the monitoring of oxygen binding
Affinity for oxygen is altered by molecules (e.g. lactate to myoglobin) binding elsewhere (allosteric control)

Question 28

Differences in haemoglobin and myoglobin

Answer 28

Tetramer vs monomer
Weaker, Sigmodial binding curve VS tighter, hyperbolic curve
Transport molecule vs store in tissue

Question 29

Hb vs Mb

Answer 29

Have very different amino acid sequence but are structurally very similar
Each subunit has 8 alpha helices (A-H), 1 heme per subunit, 1 oxygen per heme (in Hb since it is a tetramer there is 4 oxygen but that is still one per heme)

Question 30

Haem Fe

Answer 30

Can bind 6 ligands (6 coordination sites)
4 of the 6 attachment sites are occupied by N atoms
Can only bind oxygen in the Fe2+ state (reduced state)

Question 31

Pyrrole ring

Answer 31

a five-membered ring with the formula C4H4NH

Question 32

Porphyrin ring

Answer 32

A porphyrin is a large ring molecule consisting of 4 pyrroles, which are smaller rings made from 4 carbons and 1 nitrogen. These pyrrole molecules are connected together through a series of single and double bonds which forms the molecule into a large ring.

Question 33

Fe binds 6 ligands

Answer 33

4 N atoms
N atom from side chain of His F8 (also known as proximal histadine) (histadine, F helix (no. 6), 8th amino acid of this helix)
6th coordination site is available for oxygen

Question 34

His E7

Answer 34

Prevents oxidation of haem Fe2+ when oxygen binds
Ensures weakish binding of oxygen to ensure that binding is reversible
Distal His - prevents linear (strong) bond forming between oxygen atom and iron atom and it does so by sterically pushing on to the oxygen molecule when it binds. It plays a role in preventing the oxidation of the haem iron atom. All the other hydrophobic amino acids surrounding the haem group also prevent oxidation. Fe atom size shrinks when oxygen binds, moved into the plane of the heme and the His F8 will be pulled towards it and therefore this helix moves position as the F helix is pulled towards it

Question 35

Oxygen binding to Hb

Answer 35

Structural configuration of whole subunit and therefore the rest of the subunit - C helix moves the most as a result of the F helix moving

Question 36

What would happen if the bond was linear/vertical to the haem plane in Hb

Answer 36

Oxidation to Fe3+ and therefore unable to release oxygen

Question 37

Why does the conformation of haemoglobin change on O2 binding?

Answer 37

O2 binding moves the Fe into the plane of the haem, draws His F8 down, repositions helix F and the conformational changes move haemoglobin into the relaxed R-state