Lecture 7 & 8: Hemoglobin & Myoglobin

Question 1

Hemoglobin and Myoglobin

Answer 1

Proteins are evolutionarily related; share sequence homology
Sequence Homology: how related the aa are to other proteins; similar sequence homology = similar structure
First proteins crystallized to determine structure by X-Ray crystallography
Both contain the heme prosthetic group
Prosthetic groups are permanently associated with a protein and contribute to its function
Heme holds Fe atom; Fe does not directly bind to aa

Question 2

Myoglobin

Answer 2

Stores oxygen in skeletal muscle
Single polypeptide chain; tertiary chain; monomer
Mb: 153 aa, 17,000 MW
Heme group: not in central cavity
When oxygen binds to heme, cavity is collapsed

Question 3

Hemoglobin

Answer 3

Transports oxygen, carbon dioxide, H+
Important role in pH balance
  CO2 with water forms carbonic acid and bicarbonate
  Acts as pH sensor
Tetramer
   2 alpha subunits of 141 residues
   2 beta subunits of 146 residues
Quaternary structure

Question 4

Why Heme For Oxygen Binding?

Answer 4

Oxygen is poorly soluble in water
Diffusion through tissue ineffective
Transition metals have a strong tendency to bind oxygen
Very reactive in free form, especially iron
Iron must be sequestered to render it less reactive

Question 5

Important Structural Features of Heme

Answer 5

Protoporphyrin Ring: hydrophobic, planar
Nitrogens prevent Fe2+ and Fe3+ through their electron donating capacity (Fe2+ binds oxygen reversibly, Fe3+ cannot); keeps Fe2+
Heme is sequestered within protein’s structure-prevents full transfer of electrons to give irreversible oxidation; key to understanding binding/release of O2
Free Heme does not bind O2 reversibly
Hydrophobic pockets

Question 6

Hemoglobin Function

Answer 6

Hemoglobin must bind oxygen in lungs and release it in capillaries
When the oxygen binds to Fe in heme of one Hb subunit, Fe is darn into the plane of the porphyrin ring –> Disrupts key nonco. interactions in that subunit which causes a change in conformation

Question 7

Disruption of Noncovalent Interactions on Binding Oxygen

Answer 7

As oxygen binds to Fe, F-helix moves
Loss of interactions between F & H helices
1. Salt bridge between Asp94 and His146 is broken
2. Salt bridge between Lys40 and His146 is disrupted
3. H bond between Val98 and Tyr145 also disrupted

Question 8

Binding of oxygen induces a 15 degree shift along the interface

Answer 8

No hole in the oxygenated state
Structure changes in such a way to make structure more compacted
Size of the central cavity change on binding oxygen

Question 9

Hemoglobin Shows Allosteric Behavior

Answer 9

Characteristics of proteins with allosteric behavior
Quaternary structure
Cooperativity leads to change in conformation
Communicated across the structure
Sigmoidal binding curve or kinetics

Question 10

Oxygen binding curves

Answer 10

Mb: hyperbolic
Hb: sigmoidal
Lower p50= tighter bind between Fe and Oxygen; less willing to give up oxygen

Question 11

How does the oxygen dissociation curve relate to delivery of oxygen in the body?

Answer 11

1.

Question 12

Role of 2,3-BPG in oxygen binding to Hb

Answer 12

No 2,3 BPG = a curve like Mb; hold onto oxygen; no cooperativity

Question 13

What type of noncovalent interactions stabilize 2,3 BPG binding in the central cavity?

Answer 13

Many negative charges in cavity of 2,3 BPG due to phosphate; stabilized by positive charged amino acids = electrostatic noncovalent interactions
Bound only in t state; central cavity would not close when O bound
2,3 BPG would not fit; when O binds 2,3 BPG comes off so central cavity can close

Question 14

Fetal Hb Differs from Adult Hb

Answer 14

2 alpha chains and 2 gamma chains
Serine substitutes for His143 in fetal Hb
Causes more preference in r state
Higher affinity because 2,3 BPG is not bound as tightly

Question 15

Hemoglobin as a pH sensor

Answer 15

Bohr Effect:

Regulation of oxygen binding to Hb by H+ and CO2

Question 16

How does H+ (pH) and CO2 affect O2 binding to Hb?

Answer 16

Hb carries 2 end products of respiration
Reaction is spontaneous but dissolved CO2 reacts slowly with H2O to form H2CO3
Rate is increased by carbonic anhydrase

Question 17

Hb carries 2 end products of respiration

Answer 17

Binding of O2 to Hb influenced by pH and [CO2]

Question 18

Bohr Effect: Chemistry of H+ Binding

Answer 18

Protons react with histidine side chains
His146 in B-subunit = important
Protonation promotes release of O22
Acid-base properities of Hb
Reformation of salt bridges in tissues upon release of O2
Same salt bridges that were disrupted on binding O2

Question 19

Bohr Effect: Chemistry of [CO2] binding

Answer 19

At low pH and high [CO2] in peripheral tissues, Hb affinity for O2 decreases
CO2 binds to a-amino group at amino terminus of each glob in chain:
Amino terminus becomes an anion
Reacts with Arg141 of a-chain; forms a salt bridge
Deoxygenated (T) state stabilized and promotes release of O2

Question 20

Hemoglobin as a pH sensor

Answer 20

Carbonic acid (H2CO3): major metabolic acid produced in body; major buffer - dissolved CO2 in body fluids is in equilibrium with air in lungs

1. Lots of dissolved CO2 exchange with air in lungs allows carbonic acid buffer system to have higher pKa
2. Increase of CO2 –> Decrease in pH
3. Hyperventilation: Extra buffering capacity; CO2 release in beginning can prevent muscle cramps