What are the properties of myoglobin?
- One subunit that binds one heme group
- Found in muscle tissue of vertebrates
- Used to determine first X-ray crystal structure
- Made of 8 helices connected to loops
What are prosthetic groups?
Small, organic molecules that are permanently attached to a protein
What prosthetic group do myoglobin and hemoglobin bind?
Heme group
What are the properties of a heme group?
- Central Fe(II) atom
- Conjugated system
- 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
Why does oxygen bind reversibly in living organisms (mammals)?
Heme and protein (myoglobin or hemglobin)
Where does the heme group sit in myoglobin?
Deep hydrophobic cleft
How does myoglobin interact with heme group?
His F8 coordinates Fe (proximal)
His E7 coordinates oxygen at 120 degree angle (distal)
What is the function of myoglobin?
- Improves oxygen solubility to facilitate diffusion in muscle (heme binds oxygen but very hydrophobic and oxygen in not very soluble in aqueous solution)
- Prevents heme oxidation (physically separates heme groups from each other prevention transfer of electrons)
- Prevents CO binding (distal His blocks, CO binds free heme 25,000x more than O2)
Fractional saturation
Describes how much oxygen is bound to myoglobin
Y= pO2/(Kd +pO2)
Dissociation constant (Kd)
[Mb] [O2] / [MbO2]
What is the shape of myoglobin’s fractional saturation equation?
Hyperbola
How is myoglobin’s oxygen binding described?
Independent, only 1 O2 binds 1 heme group at a time, no influence on binding
What is p50 and how does it explain oxygen affinity?
- Kd=pO2
- Pressure when 50% of Mb sites are bound with O2
- High p50= low affinity for oxygen
- Low p50 means high affinity for oxygen
What is myoglobin’s p50?
2.8 torr
What are the properties of hemoglobin?
- Tetramer (dimer of dimers)
- Has 2 conformations (R and T)
- Found in red blood cells of vertebrates (96% of dry content)
- Sophisticated system used to transport oxygen from respiratory system to organs and tissues
- One of the first proteins given a physiological function
What is the structure of hemoglobin?
- Two alpha-beta subunits
- Dimer of dimers
- Alpha-beta units are related by 180 degree rotation (2-fold symmetry)
- Each subunit binds 1 heme group
How are alpha and beta subunits of hemoglobin related to each other and myoglobin?
- All have the globin fold (conserved based in important conserved residues: proximal His, distal His, and heme-binding residues
- Sequences only 18% identical at amino acid level (sequences diverge faster than structures)
What are the structural changes in hemoglobin when it changes from T state (deoxy) to R state (oxy)?
- One alpha-beta subunit rotates 15 degrees with respectto symmetry mate (quanterary structure)
- Tightens central channel by bringing beta subunits closer
- Changes contacts between A1-B2 and A2-B1
- All 4 subunits exist in one state at the same time
- Change driven by oxygen binding
What is the fractional saturation equation for hemoglobin?
Y= (pO2)^n / (p50)^n + (pO2)^n
n= Hill coefficient
What is the Hill coefficient?
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
What is the p50 of hemoglobin?
26 torr
What is the shape of hemoglobin’s fractional saturation curve?
Sigmoidal
What is the structural basis of hemoglobin’s conformation change (T—R)
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)
What is the purpose of the Bohr effect?
Lower the affinity of hemoglobin for O2 and promote release
Causes P50 increase
Promotes R-T state transition
