Lecture 4 - Hemoglobin: Model of Protein Allostery

Question 1

What is Heme + its structure?

Answer 1

A cofactor bound to hemoglobin and myoglobin which is made of protophorphyrin 9 and an iron in its ferrous (Fe+2) reduced state.

Porphyrins have four pyrrole rings linked by methylene bridges

Question 2

What are coordination bonds of iron and what provides them?

Answer 2

Fe+2 has six coordination bonds. 4 are provided by nitrogens in the plane of the porphyrin ring system, and 2 are perpendicular to it.

5th coordination bond: via proximal His (HisF8)
6th coordination bond: via bound oxygen

Question 3

What favors the ferrous state over the ferric state overall?

Answer 3

The environment around is non-polar, and Fe+3 would be much worse than Fe+2 in this environment

Question 4

What are the two most important histidines of globins? Why?

Answer 4

HisF8 - proximal histidine - holds Ferrous 5th coordination bond

HisE7 - distal histidine - stabilizes oxygen on an angle, lowering affinity of CO binding by 100 fold since it’s triple bond prefers to bind linearly with iron (still 250x affinity, but better). Also prevents auto-oxidation of hemoglobin by H-bonding to it so the super-oxide anion does not move.

Question 5

What type of proteins are globins?

Answer 5

All-helix proteins: 8 alpha-helices A-H, separated by loops

Question 6

What are two reasons why you want to prevent the auto-oxidation of hemoglobin?

Answer 6

1. Superoxide radical promotes chain reactions leading to hydroxy radicals destroying cellular components
2. Heme iron must be in +2 state to bind oxygen. Leaving in the superoxide state would leave iron in ferric (+3) state

HisE7 distal histidine prevents superoxide from leaving via stabilization from hydrogen bonding

Question 7

What are methemoglobin / metmyoglobin and what keeps them in check?

Answer 7

Oxidized globin proteins -> where heme iron is oxidized to ferric (Fe+3) state. Turns beef brown. Delivery of oxygen to tissues becomes very serious, and can cause death at >70% methemoglobin saturation.

NADH-dependent reductases in muscles and RBCs keep these in check under normal conditions

Question 8

What is Kd vs Ka?

Answer 8

Ka = association constant = equilibrium constant of forward reaction of ligand binding

Kd = dissociation constant = equilibrium constant of reverse reaction of ligand binding.

Low Kd or high Ka mean the same thing -> very high affinity for substrate, or tendency of enzyme-substrate complex to want to stay together

Question 9

What is the formula for fraction of protein with bound ligand?

Answer 9

Theta = fraction of protein with bound ligand

Theta = ([L]) / ([L] + Kd)

Thus, Kd is the dissociation constant for binding, and is equal to the free ligand concentration at which the enzyme-substrate complex will be half saturated

Question 10

What is meant in comparison of rectangular hyperbola vs linear?

Answer 10

Rectangular hyperbola -> there is a specific affinity for ligand binding to active site. Dependent on Kd.

Linear -> amount of binding is dictated by nonspecific binding, and could never be saturated

Question 11

What is the formula for binding of O2 via pO2 curve?

Answer 11

Theta = (pO2) / (pO2 + P50)

P50 is the partial pressure of oxygen for which the receptor is half saturated.

Question 12

Why is myoglobin good for storage but lousy for delivery?

Answer 12

Its P50 is around 2.8 torr. Tissue capillaries have around 25-35 Torr, and lungs have around 100 Torr. The affinity is too high and it will be saturated with oxygen even in tissue capillaries where it needs to let go

Question 13

What makes up hemoglobin structure?

Answer 13

Still 8 alpha helicies per subunit, but it has two alpha and two beta globin subunits which make up a whole hemoglobin, with 4 globins in the quaternary structures.

Myoglobin, alpha-hemoglobin, and beta-hemoglobin subunits are all analogous.

Question 14

Why is hemoglobin’s oxygen binding curve sigmoidal shaped?

Answer 14

It exhibits positive cooperativity - a characteristic of oligomeric proteins with two or more identical polypeptide subunits which each bind a particular ligand

Question 15

What is a homotropic allosteric effect? What are its two types?

Answer 15

The effector and affected ligand are the same type of molecule (oxygen, carbon monoxide, etc) and the interaction is between duplicate structural domains in the protein (they bind analogous molocules, i.e. heme domain)

1. Positive cooperativity - first ligand increases affinity for second
2. Negative cooperativity - first ligand decreases affinity for second

Question 16

What is the hill coefficient?

Answer 16

n, which approximates the number of interacting sites in homotropic allostery.

In the formula

log (theta / 1 - theta) = nlog[L] - logKd

n = 1 indicates zero cooperativity
n > 1 indicates positive cooperativity
n < 1 indicates negative cooperativity

Question 17

What is the approximate hill coefficient value for hemoglobin in the p50 range?

Answer 17

n ~ 2.8. Outside of this range, when no sites are filled (low affinity) or 3 sites are filled (high affinity) n=1. Within this range, n is greater than 1 because hemoglobin affinity is rapidly increasing as an aggregate as multiple oxygens are binding and exhibiting positive cooperativity.

Question 18

What does the X intercept indicate in the hill equation?

Answer 18

X intercept = log Kd, shows relative affinity. Kd will be lower for higher affinity things -> the reason why hemoglobin’s high affinity x-intercept is lower farther back.

Question 19

What are the two states of hemoglobin and which is more stable in vitro?

Answer 19

T state and R state.

T state has a large hole in the middle, in the R state the alpha-beta pairs slide past eachother and rotate, narrowing the pocket between betta subunits.

T state is more stable due to ionic bonding between chains, especially between alpha1-beta2 and alpha2-beta1

Question 20

What leads the transition from T->R state of hemoglobin?

Answer 20

Local effects of O2 binding to the first hemoglobin subunit (positive cooperativity).

In the deoxy (Tense) state, iron is out of the heme plane. When oxygen binds, the iron is pulled into the heme plane to form that 6th coordination bond, pulling the proximal His (F8) to the right, changing the confirmation of that subunit to the relaxed state. This moves the entire F helix.

Question 21

What is the concerted vs the sequential model of oxygen binding?

Answer 21

Concerted = symmetry model, all or none. Binding of oxygens makes switching to R state more likely.

Sequential model = there are hybrid states of T/R hemoglobin, fully oxygenated Hb is the R state.

Likelihood is that reality is somewhere in between the two.

Question 22

Why is it good that hemoglobin is allosteric?

Answer 22

The steepest part of the sigmoidal binding curve is between 20-40 torr (20-30 exercise, 30-40 resting), which allows for the greatest differential fraction of bound ligand to be released. Large changes in bound ligand can meet metabolic needs better.

Question 23

What is heterotropic allostery?

Answer 23

An interaction where the effector and the affected ligands are different types of molecules and bind to different domains that are not structurally similar.

Affected ligand -> binds to activity site
Effector -> bding to regulatory site

Question 24

What is a negative vs positive allosteric affector?

Answer 24

Negative allosteric effector - an inhibitor or inactivator - regulatory effector ligand which has a negative effect on events at active site

Positive allosteric effector - activator regulatory effector ligand which has a positive effect on events at active site.