Protein Function

Question 1

What are the properties of myoglobin?

Answer 1

1. One subunit that binds one heme group
2. Found in muscle tissue of vertebrates
3. Used to determine first X-ray crystal structure
4. Made of 8 helices connected to loops

Question 2

What are prosthetic groups?

Answer 2

Small, organic molecules that are permanently attached to a protein

Question 3

What prosthetic group do myoglobin and hemoglobin bind?

Answer 3

Heme group

Question 4

What are the properties of a heme group?

Answer 4

1. Central Fe(II) atom
2. Conjugated system
3. Iron has 6 coordination positions
   3a. 4 positions coordinated by nitrogen in pyrrole groups
   3b. 1 position with His residue
   3c. Final position coordinated with oxygen

Question 5

Why does oxygen bind reversibly in living organisms (mammals)?

Answer 5

Heme and protein (myoglobin or hemglobin)

Question 6

Where does the heme group sit in myoglobin?

Answer 6

Deep hydrophobic cleft

Question 7

How does myoglobin interact with heme group?

Answer 7

His F8 coordinates Fe (proximal)
His E7 coordinates oxygen at 120 degree angle (distal)

Question 8

What is the function of myoglobin?

Answer 8

1. Improves oxygen solubility to facilitate diffusion in muscle (heme binds oxygen but very hydrophobic and oxygen in not very soluble in aqueous solution)
2. Prevents heme oxidation (physically separates heme groups from each other prevention transfer of electrons)
3. Prevents CO binding (distal His blocks, CO binds free heme 25,000x more than O2)

Question 9

Fractional saturation

Answer 9

Describes how much oxygen is bound to myoglobin
Y= pO2/(Kd +pO2)

Question 10

Dissociation constant (Kd)

Answer 10

[Mb] [O2] / [MbO2]

Question 11

What is the shape of myoglobin’s fractional saturation equation?

Answer 11

Hyperbola

Question 12

How is myoglobin’s oxygen binding described?

Answer 12

Independent, only 1 O2 binds 1 heme group at a time, no influence on binding

Question 13

What is p50 and how does it explain oxygen affinity?

Answer 13

1. Kd=pO2
2. Pressure when 50% of Mb sites are bound with O2
3. High p50= low affinity for oxygen
4. Low p50 means high affinity for oxygen

Question 14

What is myoglobin’s p50?

Answer 14

2.8 torr

Question 15

What are the properties of hemoglobin?

Answer 15

1. Tetramer (dimer of dimers)
2. Has 2 conformations (R and T)
3. Found in red blood cells of vertebrates (96% of dry content)
4. Sophisticated system used to transport oxygen from respiratory system to organs and tissues
5. One of the first proteins given a physiological function

Question 16

What is the structure of hemoglobin?

Answer 16

1. Two alpha-beta subunits
2. Dimer of dimers
3. Alpha-beta units are related by 180 degree rotation (2-fold symmetry)
4. Each subunit binds 1 heme group

Question 17

How are alpha and beta subunits of hemoglobin related to each other and myoglobin?

Answer 17

1. All have the globin fold (conserved based in important conserved residues: proximal His, distal His, and heme-binding residues
2. Sequences only 18% identical at amino acid level (sequences diverge faster than structures)

Question 18

What are the structural changes in hemoglobin when it changes from T state (deoxy) to R state (oxy)?

Answer 18

1. One alpha-beta subunit rotates 15 degrees with respectto symmetry mate (quanterary structure)
2. Tightens central channel by bringing beta subunits closer
3. Changes contacts between A1-B2 and A2-B1
4. All 4 subunits exist in one state at the same time
5. Change driven by oxygen binding

Question 19

What is the fractional saturation equation for hemoglobin?

Answer 19

Y= (pO2)^n / (p50)^n + (pO2)^n
n= Hill coefficient

Question 20

What is the Hill coefficient?

Answer 20

An estimate of cooperativity (n<1= negative, n=1 independent, n>1=positive)
Based on slope of Hill plot (y axis: log (YO2/1-YO2), x axis: log pO2)
Hb=3
Mb=1

Question 21

What is the p50 of hemoglobin?

Answer 21

26 torr

Question 22

What is the shape of hemoglobin’s fractional saturation curve?

Answer 22

Sigmoidal

Question 23

What is the structural basis of hemoglobin’s conformation change (T—R)

Answer 23

Allosteric effect
1. Oxygen pull irons into optimal position in heme plane (porphyrin rings go from domed to flat)
2. Iron pulls proximal His F8
3. His F8 pulls helix F to avoid steric clash with heme group
4. Helix tilts and translated 1 angstrom across heme plane
5. Helix translation causes register shift (moves up a turn) that changes A1-B2 and A2-B1 interactions (C helix in alpha and FG region in beta)
6. Ion pairs break at N terminus of alpha subunits and C terminal His of beta subunits (powered by Fe(II)-O2 bond b/c energetically unfavorable) as basic residues are deprotonated
7. Causes p50 and Kd to decrease (higher affinity)

Question 24

What is the purpose of the Bohr effect?

Answer 24

Lower the affinity of hemoglobin for O2 and promote release
Causes P50 increase
Promotes R-T state transition

Question 25

What are the 2 parts of the Bohr effect?

Answer 25

A. Protons
1. C terminal His (beta) and N-terminus (alpha) pick up H+ to stabilize charge
2. pK increases
3. lower pH drives oxygen dissociation (T state)

B. CO2
1. CO2 exists as bicarbonate in blood
2.Carbonic anhydrase catalyzes CO2 to HCO3- and releases protons
3. Hemoglobin uptake of protons facilitates this formation (pushes equilibrium right)
4. Proton uptake to promote bicarbonate formation favors T state

C. CO2
1. CO2 can modify amino groups to form carbamate
2. Releases protons to lower pH
3. Causes Hb to uptake protons and favor T state