#08 Hemoglobin

Question 1

Importance of Heme

Answer 1

• Heme is required for oxygen binding because amino acids don’t bind to oxygen and the diffusion rates for oxygen are very limited.

Question 2

Blood Color

Answer 2

• The heme group gives muscle and blood their distinctive red color.

Question 3

Protoporphyrin

Answer 3

• The heme group consists of an organic component and a central iron atom. The organic component, called protoporphyrin, is made up of four pyrrole rings linked by methine bridges to form a tetrapyrrole ring. Four methyl groups, two vinyl groups, and two propionate side chains are attached.

Question 4

Coordinations of Heme Group In Both States

Answer 4

• In deoxy state of heme group, iron kind of hangs out of the plane of the ring. It’s coordinated by the four nitrogens that make up the protoporphyrin ring. But it also has two other coordinations, one proximal histidine, other distal histidine. Histidine is not free, it’s bonded to the helix. When oxygen binds to iron, the distal histidine forms a hydrogen bond with it to stabilize oxygen. This causes oxygen to be fixed at an angle, which is actually due to the fact that there is an imidazole group that forces oxygen to bind at an angle rather than perpendicular.

Question 5

Will Oxygen Bind to Fe+3?

Answer 5

• Oxygen will not bind to iron in +3 state.

Question 6

Importance of Binding Oxygen At An Angle in Heme

Answer 6

• The fact that oxygen binds to iron at an angle has an important physiological function. Carbon monoxide can bind to hemoglobin much better than oxygen, so have this sixth coordination site with imidazole and histidine pretty much reduces the extent of carbon monoxide binding to iron.

Question 7

Position of Iron When Binded To Oxygen

Answer 7

• When oxygen binds to iron, iron gets pulled into the plane of the protoporphyrin ring, and its radius gets a little smaller to fit into ring of ring (due to rearrangement of electrons within iron). Also drags the proximal histidine which is a part of the helix. Causes change in structure.

Question 8

Distal Histidine Importance

Answer 8

• Distal histidine prevents the oxidation of the heme iron to the ferric iron (Fe+3), which cannot bind oxygen, and also reduces ability of carbon monoxide to bind to the heme.

Question 9

Noncovalent Interactions in Deoxy State of Hemoglobin

Answer 9

• In deoxy state of hemoglobin, you have three noncovalent interactions. Two types, three total. One involves Asp94 and His146. It is an electrostatic interaction, or salt bridge. This composes the F helix. For the H helix, you’ll have the same His146 binding to Lys40 as a salt bridge as well. At the end of the H helix, you have Val98. The carbonyl group of it can hydrogen bond with the hydroxyl group of tyrosine.
○ The numbers tell you where in the sequence the amino acid is found.

Question 10

Chaneg in Structure from Deoxy to Oxy

Answer 10

• When oxygen binds, iron will move into the plane of the ring. At this point, all three noncovalent interactions of the deoxy state are broken. That’s the change in the structure that occurs. This will translate eventually to the entire hemoglobin molecule.

Question 11

Central Cavity

Answer 11

• According to crystallography, you get two different defraction patterns in hemoglobin whether oxygen is bound or not. In deoxy state, you have a small “donut hole” in center of molecule, while it is gone in oxy state. This is called the central cavity, and it gets smaller as oxygen binds. There is also a 15 degree rotation between both conformations.

Question 12

Hemoglobin As a Dimer?

Answer 12

• Hemoglobin behaves like a dimer. The Beta 1 and Alpha 1 subunit function together, and the beta 2 and alpha 2 subunit function together. This interaction is called the interface. What goes on is translated throughout entire structure.

Question 13

Hemoglobin Has Allosteric Behavior

Answer 13

• The fact that hemoglobin changes structure between its deoxy and oxy forms is the basis for why it shows allosteric behavior. Hemoglobin has two different conformations. Allosteric behavior in general is only possible with quaternary structure, because you need another polypeptide to interact with.

Question 14

Hemoglobin & Cooperativity

Answer 14

○ Quaternary structure leads to cooperativity. The subunits by virtue of their interaction are actually communicating with one another, which results in change in conformation. Binding of oxygen to one subunit will make it easier for the same to happen to other three subunits. This breakage of the first noncovalent interaction loosens the structure up for the others to do the same. In case of hemoglobin it’s positive cooperativity as when oxygen binds to one subunit, makes it easier for other subunits to bind.

Question 15

Sigmoidal Binding Curve

Answer 15

• Sigmoidal Binding Curve (S-shaped) shows proof for cooperative behavior. The graph has fractional saturation as its Y value, and partial pressure of oxygen in the tissue as the X value. Allosteric behavior allows hemoglobin to have cooperativity.
○ At a high partial pressure, like 100 torr, both myoglobin and hemoglobin are able to bind to oxygen fully.
○ At rest, partial pressure of tissue is around 40 torr. At this pressure, myoglobin has only given up about 5% of oxygen, while hemoglobin is 25%. This difference only increases as you go lower in partial pressure, as hemoglobin becomes more willing to deliver oxygen to tissue while myoglobin continues to only release a little.
○ Sigmoid shape of oxygen saturation curve of hemoglobin indicates cooperativity between subunits in binding oxygen.

Question 16

Why Myoglobin Has No Allosteric Function

Answer 16

• Myoglobin cannot function same as hemoglobin due to tertiary structure, no cooperativity, and no allosteric function.

Question 17

T-State vs. R-State

Answer 17

• T-State represents deoxyeganated state, while R-State represents oxygenated state.

Question 18

2,3-Bisphosphoglycerate: What Does It Effect?

Answer 18

• The saturation curve of hemoglobin can be affected by binding of other molecules. One of them is called 2,3-Bisphosphoglycerate (2,3-BPG).

Question 19

Origin of 2,3-BPG

Answer 19

○ You get 2,3-BPG from glycolysis in RBCs, since RBCs have no mitochondria.

Question 20

Effect of 2,3-Bisphosphoglycerate on Saturation Curve of Hemoglobin

Answer 20

○ Without it in hemoglobin, the saturation curve looks just like myoglobin’s, while with the molecule makes hemoglobin more likely to release oxygen. Curve is more sigmoid in shape. 2,3-BPG decreases oxygen affinity by a sizable amount.

Question 21

How Does 2,3-BPG Make Its Effect?

Answer 21

How does 2,3-BPG decrease oxygen affinity of hemoglobin? Well looking at structure of it, it has two -2 phosphate groups and one -1 carboxyl group, -5 total. In the central cavity where the molecule will be held, there are electrostatic interactions due to the amino acids around it that stabilizes the deoxy state.

Question 22

Deoxy to Oxy WRT Noncovalent Interactions

Answer 22

To change to the oxy conformation, these bonds must be broken. This is why in oxy conformation the central cavity is much smaller.

Question 23

Fetal vs. Adult Hemoglobin

Answer 23

• The difference between a fetal and adult hemoglobin is a substitution. Instead of beta chains in fetal, there are gamma chains. In fetal hemoglobin, histidine is replaced by serine. Serine is uncharged. Since serine is uncharged, 2,3-BPG will not be able to bind to hemoglobin nearly as well because the nonpolar serine will try to push it away. Thus, the oxygen affinity will not be able to decrease, and the fetus can have a higher affinity for oxygen. This function is crucial because it depends on maternal circulation for oxygen, so it needs to be able to extract oxygen from the circulation, otherwise fetus will not get proper amount of oxygen.

Question 24

Hemoglobin At High Temperature

Answer 24

• If you suddenly go up to a higher altitude, your body will produce more 2,3-BPG because you need more oxygen release to get to the tissues.

Question 25

Hemoglobin As pH Sensor

Answer 25

• Hemoglobin is also a pH sensor because it contributes to homeostasis of body and responds to changes in blood pH.

Question 26

Bohr Effect

Answer 26

• 2,3-BPG is not the only allosteric regulator of hemoglobin activity. Actively metabolizing tissues, those in most need of oxygen, release signal molecules that further reduce affinity of hemoglobin for oxygen. They are hydrogen ion and carbon dioxide. The regulation of oxygen binding by hydrogen ions and carbon dioxide is called the Bohr Effect.

Question 27

Bohr Effect Equilibrium

Answer 27

○ CO2 is much better at diffusing through walls then oxygen is. The partial pressure between the tissue and outside though also very much favors release of CO2 into bloodstream. CO2 combines with water to become carbonic acid, which dissociates into bicarbonate ion and H+. This equilibrium is spontaneous, although there is an enzyme in red blood cells called carbonic anhydrase that increases rate of this reaction.

Question 28

pH Effect of Oxygen Affinity

Answer 28

○ Small changes in the pH, like 7.4 to 7.2, results in over 10% increase in dropping of oxygen. This tells us that tissues need more oxygen when pH drops, and that is a signal for hemoglobin to release oxygen.

Question 29

CO2 Effect on Oxygen Affinity

Answer 29

○ Tissues also produce carbon dioxide. Adding CO2 to blood will also have an effect of decreasing the oxygen affinity of hemoglobin. So CO2 is another signal for release of oxygen.

Question 30

Importance of His146 and Asp94 Salt Bridge

Answer 30

○ When the salt bridge between His146 and Asp94 is broken in the lungs, when they get to the capillaries, His146 becomes protonated and can now reestablish the salt bridge that was broken when oxygen is bound.
§ The pKa for His146 when Hb is oxygenated is 6.6. A little higher then strictly histidine pKa due to neighboring side chains. In deoxy state, pKa is 8.2. In oxy, acts as acid, in deoxy, acts as base. So, this is where hemoglobin is a pH sensor, as histidine can donate and accept protons depending on partial pressure of oxygen. It will absorb some of the protons in circulation and become protonated, and then go to lungs to give up proton. So where proton binds is to histidine.

Question 31

Where CO2 Binds To In T-State

Answer 31

○ CO2 is going to bind to free amino terminus of each chain, forming a carbamate ion, negatively charged. Binding of CO2 also causes release of an additional proton. This ion is going to form a salt bridge with arginine of an alpha-chain, because when you break a salt bridge, another one forms. Ultimately contributes to stabilization of T state.

Question 32

CO2 Transport in Blood

Answer 32

• Tissues produce CO2, CO2 diffuses enters RBC, converted to carbonic acid, then bicarbonate and proton. Bicarbonate exits RBC, and is going to be major transport form of CO2. Then once at the lungs, bicarbonate ion will reenter the RBC, form carbonic acid, and then dissociate into CO2, and at the point be transported into alveoli.

Question 33

Hypothalamus

Answer 33

• Hypothalamus is very sensitive to changes in pH. It’ll adjust rate of breathing according to pH that exists in blood. Blood pH cannot change very much, very strict.

Question 34

Carbon Monoxide

Answer 34

• Carbon monoxide binds to hemoglobin much more tightly and prevents binding of oxygen. Its symptoms mimic the flu, cannot be smelled, and is invisible.