Lecture 6

Question 1

Oxygen transport overview?

Answer 1

If you try to hold your breath, it is hard to do. This is due to physiological adaptations that allow us to hold our breath for a very short time. This is because breathing allows us to take in oxygen and release CO2.

Question 2

Why breathe?

Answer 2

We need to get oxygen to tissues in a way that’s very effective and allows those tissues to get oxygen even though they’re not in contact with the outside.

Question 3

How do we breathe?

Answer 3

We already know the physiological/biological way - but in biochemistry we want to observe this phenomenon at the molecular level.

Question 4

Myoglobin?

Answer 4

Myoglobin is a monomeric O2 carrier in muscle cells. This protein consists of eight alpha-helices (A-H) and it has a heme group.
- It is a small but globular protein that has tertiary structure folded specifically with a hydrophobic core.
- “Myo” refers to muscles - and is typically found in red muscle.

Question 5

How does myoglobin carry O2?

Answer 5

Myoglobin is able to carry oxygen to to its heme prosthetic group.

Question 6

What forms heme?

Answer 6

A poryphorin ring coordinates with Fe to form heme (distinct poryphorin with iron at the centre. The jobs to carry and transport iron depending on its location - the iron then carries oxygen).

Question 7

Apoprotein?

Answer 7

Without prosthetic group.

Question 8

Holoprotein?

Answer 8

With prosthetic group.

Question 9

Apoprotein vs. Holoprotein?

Answer 9

Same protein, just one has prosthetic group and the other does not.

• proteins without one at all are not considered apoproteins.

Question 10

What does the heme group do?

Answer 10

The heme group coordinates to oxygen and myoglobin.

Question 11

Organization of heme?

Answer 11

The way that the heme is organized in the myoglobin is with 2 histidine residues that arevvery closet the heme and play a role in stabalizing the heme and the oxygen. The proximal histidine coordinates iron in heme group and allows everything to stay in place and are very important. When heme binds oxygen (O2) there is an H bond between the distal oxygen and distal histidine (stabilizes the heme and oxygen). The proximal histidine steps behind and coordinates the iron. The distal histidine can hydrogen bond with he oxygen when its bound.

Question 12

Proximal His?

Answer 12

Fe coordinates to the O2 and the proximal histidine (His in helix F8)

Question 13

Quantitative treatment of O2 binding?

Answer 13

The binding of O2 (ligand of myoglobin) to myoglobin (Mb) can be expressed as a dissociation:

MbO2 <-> Mb + O2

(follow slide 6 for actual problem)

Question 14

P50 meaning?

Answer 14

[O2] is usually expressed as a partial pressure (units of mmHg or torr).
P50 is the oxygen pressure when myoglobin is 50% bound. It
is inversely proportional to the affinity of myoglobin for O2. This means we will have a hyperbolic curve (goes up and flattens out at the top), which tells us about how myoglobin binds oxygen and it makes a difference in terms of how oxygen gets from the blood to the tissues.

Question 15

Myoglobin O2 Saturation Curve?

Answer 15

We want to find the P50, when 50% of myoglobin has O2 bound to it. We compare the percentage of mooglobin bound to O2 with the partial pressure of oxygen.

Y = 0.5 when pO2 equals P50.

• The P50 for the association of myoglobin with O2 is ~2.5 torr pO2
• This means that half of the myoglobin has bound O2 at 2.5 torr.

Question 16

The Kd?

Answer 16

The Kd is informative - The stronger the protein binds to its ligand, the lower the P50 value and need less ligand.

ON SLIDE 8

• Blue line: lower P50 and stronger binding
• Pink line: higher P50 and weaker binding

Question 17

What is Y equal to?

Answer 17

Y = pO2 / P50 + PO2

Question 18

Myoglobin works best?

Answer 18

Myoglobin can effectively store and release intracellular oxygen!

Question 19

Intracellular PO@?

Answer 19

Intracellular P02 is ~65%

Question 20

pO2 in Capillaries?

Answer 20

~90%

Question 21

Delta Y?

Answer 21

The delta Y difference is large so myoglobin ca easily pick up O2 from capillaries.

Question 22

A new type of myoglobin gene has been discovered in pokémon that has a P50 of 10 mmHg. This is significantly higher than human myoglobin, which has a P50 of 2.5
mmHg. This means that:

A. Pokémon myoglobin has a higher affinity for O2 than human myoglobin.
B. Pokémon myoglobin can only bind more oxygen than human myoglobin at high oxygen
pressures.
C. Pokémon myoglobin binds more oxygen than human myoglobin at low oxygen pressures.
D. Human myoglobin binds more oxygen than pokémon myoglobin at all oxygen pressures.
E. None of the above

Answer 22

The answer is B: Human myoglobin binds more oxygen than Pokemon myoglobin at all oxygen pressures.

• Human myoglobin has a lower P50 than the pokémon one.
  This means that the human myglobin binds O2 more effectively
  (tightly).

Question 23

What is hemoglobin used for?

Answer 23

Hemoglobin is used for oxygen transport.

Question 24

Hemoglobin structure?

Answer 24

Hemoglobin has 4 subunits, each in identical pairs (alpha and beta subunits). Each subunit corresponds very closely to myoglobin (with similar structure).

Question 25

Hemoglobin overview - structure, function?

Answer 25

Hemoglobin is a heterotetrameric protein with two identical alpha subunits and two identical beta subunits that transports O2 from lungs to tissues. Each submit has a heme prosthetic group. - The area around heme is the most conserved.

Question 26

Oxygen binding to hemoglobin is...?

Answer 26

Oxygen binding to hemoglobin is sigmoidal. - There is some kind of cooperation going on- which is part of the reason it has 4 subunits: has to be able to change oxygen binding strength based on its location. This allows it to bind and let go in different places very effectively.

Question 27

Sigmoidal behaviour indicates?

Answer 27

Sigmoidal behavior indicates cooperative interactions due to protein allostery (regions of proteins communicating with each other)

Question 28

Tissues vs. Lungs PO2?

Answer 28

Tissues: lower pressure - makes difference for protein function. Hemoglobin can let go of O2 in its tissues more readily than myoglobin. Lungs: much higher pressure - almost 100% of what it can reach in solution. Hemoglobin will not readily let go of the O2 in the lungs- instead taking it up.

Question 29

Hb transportation meaning?

Answer 29

Hb can transport (ΔY) much more O2 than myoglobin (Mb) because it has a greater variation in O2 binding over the range of pO2

Question 30

What does O2 binding cause in hemoglobin?

Answer 30

O2 binding causes a structural change in hemoglobin. - When O2 is bound by Fe2+, that O2 coordinates with a His located in helix E of hemoglobin. This coordination with His E7 brings the Fe2+ into the plane of the heme ring. - When Fe2+ has no O2 bound, it is coordinated by the His in helix F (F8), which pulls it slightly out of the plane of the heme. Essentially, proximal histidine coordinates the iron thats in the middle of the heme. Also have a distal histidine that can interact with bound O2.

Question 31

Deoxy vs. Oxy?

Answer 31

Deoxy (tense) has no iron interactions. H8 pulls molecules down. Oxy (relaxed) interacts with the iron, which is in line with the heme.

Question 32

Hemoglobin has how many states?

Answer 32

Hemoglobin exists in two states with different O2 affinity.

Question 33

What are the two states of hemoglobin and why do they have them?

Answer 33

There is the deoxygenated (Tense) state with low O2 affinity and the oxygenated (Relaxed) state with high O2 affinity. - Need to be able to go into a different state to let us go of O2 at the lungs.

Question 34

What happens once the oxygen is released from the hemoglobin?

Answer 34

Once an oxygen is released from the hemoglobin in the capillaries of the tissues, there is a change in confirmation of the one subunit that lost an oxygen. Salt bridges are able to reform between the subunits because they had a conformation change - leading other units to also release oxygen really quickly.

Question 35

Deoxygenated Tense (T) state?

Answer 35

This state has low O2 affinity. Salt bridges (ionic interactions) between subunits in this form. In the T state, salt bridges between subunits favour a low affinity conformation

Question 36

Oxygenated Relaxed (R) state?

Answer 36

This state has high O2 affinity. Salt bridges (ionic interactions) broken in this form. In the R state, these ionic contacts are broken and the O2 affinity is increased. Salt bridges broken between adjacent subunits = relaxed. This state binds O2 much better and is less rigid.

Question 37

Hemoglobin O2 binding cooperativity?

Answer 37

Concerted model: O2 binding to one subunit in the R state encourages other subunits to adopt the R state (allostery). Hb is sigmoidal R is hyperbolic (high - lungs) T is hyperbolic (low - tissues)

Question 38

Allosteric effectors of hemoglobin?

Answer 38

These are things that let hemoglobin release O2 in the T state - changing the behaviour of a protein. - Bohr Effect - CO2 - BPG

Question 39

Bohr Effect?

Answer 39

Decreased pH protonates key histidine side chains at αβ interface (stabilizes T)

Question 40

CO2 allosteric effector?

Answer 40

CO2 (stabilizes T by contributing to [H+]: CO2 + H2O H2CO3 H2CO3 HCO3- + H+ HCO3- CO32- + H+ - 10-15% of CO2 is transported to the lungs this way. - Both H+ and CO2 reduce the affinity of hemoglobin for oxygen at the tissues.

Question 41

BPG?

Answer 41

BPG is a long-term allosteric effector. BPG (2,3-bisphosphoglycerate) binds (and stabilizes) only the deoxy (T) state of Hb in its positively charged center, reducing O2 affinity. - BPG is increased at high altitudes - BPG levels in blood can be adjusted to adapt to long-term changes.

Question 42

Bohr effect and proton transport?

Answer 42

H ions stabilize the T state. Hemoglobin, O2 transport, and CO2 transport are linked.

Question 43

In the presence of lowered pH and increased BPG: A. Nothing would happen because H+ and BPG would counteract each other. B. Hemoglobin would adopt the T state and have a lower affinity for oxygen. C. Hemoglobin would adopt the R state and have a lower affinity for oxygen. D. Hemoglobin would adopt the T state and have a higher affinity for oxygen. E. Hemoglobin would adopt the R state and have a higher affinity for oxygen.

Answer 43

B) Hemoglobin would adopt the T state and have a lower affinity for oxygen. - Both H+ and DPG stabilize the T state of hemoglobin, which has the lower O2 affinity.

Question 44

Global production during development?

Answer 44

Fetal hemoglobin contains ɣ (gamma) subunits instead of β subunits. The α2ɣ2 hemoglobin cannot bind BPG (giving it a higher affinity for O2). Appling et al. (2018) - The fetus produces a modified hemoglobin with a higher O2 affinity, allowing it to obtain O2 from the mother. - Gamma has a higher O2 affinity than alpha or beta subunits.

Question 45

Sickle Cell Disease Overview?

Answer 45

Sickle cell disease is due to a a single amino acid mutation in hemoglobin (glutamate to valine). The hydrophobic Val on the cell surface causes the hemoglobin to clump and this affects the shape of the cells. People who are homozygous for this mutation have sickle cell disease and those who are heterozygous can also be affected. However, this mutation can be protective against malaria. - This is why it has persisted: it is actually higher fitness to have this trait in areas with high risk for malaria.

Question 46

Molecular basis of sickle cell disease?

Answer 46

1. A point mutation in the DNA codes for structurally altered HbS. 2. In the deoxygenated state, HbS polymerizes into long, rope-like fibres. 3. Intracellular fibers of HbS distort the erythrocyte.

Question 47

Treatments?

Answer 47

Treatments include: Hydroxyurea (stimulates ɣ chain production and people will express their fetal hemoglobin) , bone marrow transplant (very invasive), gene therapy (repairing of gene through CRISPR). - Things are looking a lot better now with our ability to research the molecular root of SCD.