Chapter 7: Hemoglobin and Myoglobin pt.1 Flashcards
Myoglobin relating to 3D structure
1st protein whose 3D structure was determined (by x-ray crystallography)
John Kendrew
used sperm whale myoglobin to determines myoglobin structure
-Mb has O2 storage/transport protein
Myoglobin facts
-monomer
-8 alpha-helices labeled A through H
-globular
Hemoglobin facts
-tetramer of 4 Mb like homologous subunits
*2 Alpha-globin and 2 Beta-globin subunits
Max Perutz (and Kendrew)
solved Hb’s crystal structure
Heme
*KNOW STRUCTURE S.4
-protoporphyrin IX + Fe^2+ = Heme
-1 heme (prosthetic group) per subunit
*prosthetic group: non-amino acid portion of a protein
-allows oxygen to bind
O2 binding pocket in heme general
-oxygen binds to the Fe^2+ that is in between 4 N
-His F8 (proximal HIS)
-Val E11
-His E7 (distal HIS)
-Phe CD1
How to determine oxy and deoxy Hb
-they have different wavelengths so diff colors
-color changes due to presence or absence of oxygen
O2 binding pocket in heme: His F8
-proximal His (8th residue on F helix)
-heme held in place to each Hb subunit/Mb by coordinate covalent bond to His F8
**connects Heme to Nitrogen of His F8
-keeps oxygen on the same face
O2 binding pocket in heme: His E7
-distal His
-forces any ligand binding to Fe (II) to bind and a BENT angle
*bent angle allows for O2 to bind Fe(II) on the heme reversibly
O2 binding pocket in heme: Val E11 and Phe CD1
holds heme in place through hydrophobic interactions
CO posioning
CO and other small molecules have a very strong affinity for Fe^2+ in heme
-when it binds linearly CO is 20,000 X stronger than O2
what helps prevent CO poisoning
-His E7: forces CO and any small molecules to bind Fe^2+ at a bent angle
*reduces their affinity for heme
(CO still poisonous but it would be worse without His E7)
Methemoglobin (metHb) and Metmyoglobin (metMb)
-what it is called (final) when Fe^2+ in presence of O2 gets oxidized to Fe^3+
-makes it so heme can not bind oxygen
diaphorase
enzyme that reduces Fe^3+ to Fe^2+
*allows oxygen to bind back to heme
Heme dimerization
-what the process ic called: Fe^2+ oxidized to Fe^3+ and harmful oxygen free radicals
-2 hemes can auto-oxidize through an intermediate where O2 bridges btwn two Fe (II) centers (heme dimers)
*Fe-O=O-Fe
*does not allow oxygen to bind
How is heme dimerization prevented?
bulky groups (Val E11 and Phe CD1) in hydrophobic cleft prevent oxidation of Fe (II)
*allows for reversible oxygen binding
O2 binding equation
YO2= pO2/ p50+pO2
*pO2 is the partial pressure
*p50=kd (amt of saturation req to get half oxygenation
-as p50 decreases, the affinity for oxygen increases
-creates a hyperbolic binding curve
p50 of myoglobin
2.8 torr
O2 binding equation: at half saturation
half saturation pO2= Kd= YO2
Function of myoglobin
-it is a O2 storage protein
*gives up little O2 over normal physiological range
-under extreme conditions when O2 transport is not fast enough to meet the cells need - PO2 falls really low- allows Mb to release O2
Hemoglobin function
transports oxygen from lungs to tissues
-dimer of dimers: can function like 2 subunits (AlphaBeta)2 dimer
what does urea do to hemoglobin?
it break Hb into dimers (alpha1-beta1) and (alpha2-beta2)
What did Perutz find in hemoglobin’s response to oxygen binding
-found change btwn the two states in Hb quaternary structure upon oxygen binding
*T-state and R-state
*switch occurs btwn surface of alpha1-beta1 and alpha2-beta2
*has a 15 degree twist between alpha-beta dimer pairs
T-state
-“tense state”
-low affinity for oxygen
*no oxygen binded
-only state that has H-bonds and ion pairs
R-state
-“relaxed state”
-high affinity for oxygen
*oxygen is binded
Mechanism for Hb’s positive binding O2 binding cooperativity (perutz)
proposed that Hb’s cooperativity is from the mechanical movement of the protein scaffold
-like a domino affect
- T-state is locked in a tense conformation through H-bonds and ion pairs (not in R-state_
*get energy from binding Fe2+ and oxygen to break the bonds - Difficult for the T-state to bind the 1st oxygen bc His E7 and Val E11 block the 1st oxygen’s access to heme (sterically hinder)
- in T-state the Fe2+ is 0.6A out of heme plane
- Through the increase of [O2], the 1st oxygen is able to bind and pulls Fe2+ back into the heme plane
- His F8 is attached to Fe2+ so it also gets pulled towards the heme plane
- Bc His F8 is part of the F helix, it pulls the F helix like a lever on a fulcrum
*F helix moves 1 A - When F helix moves, the alpha-beta pairs rotate 15 degrees
*change in quaternary structure
*at the alpha1-beta2 and alpha2-beta1 contacts, different but equivalent sets of H-bonds and ion pairs act like knobs on a molecular switch (look at pic S.18) - T–>R shift in quaternary struc causes His E7 and Val E11 to move out of the way
*oxygen has clear access to the heme on the other 4 subunits - The energy in the formation of the Fe-O2 bond formation drives the T–>R transition
explanation of the sigmoidal shaped O2 binding curve
-T-state has a low affinity for O2
*low hyperbole
-R-state has a high affinity for O2
*high hyperbole
- Hb is first in the T-state but then switches to the R-state- gives the oxygen saturation/binding curve
positive cooperativity
O2 binding causes a shift from T to R-state in Hb
*need more than 1 subunit?
myoglobin and cooperativity
does not exhibit any positive cooperativity bc it does not have a quaternary structure
-has hyperbolic saturation curve
-p50: 2.8 torr
*low p50=the faster the protein gets saturated=will bind “tighter”
what is hemoglobins p50?
26 torr
Perutz proof of hemoglobin binding oxygen mechanism
-he replaced His F8 with Glycine
-added Imidazole to heme
Found:
-normal Hb: cooperativity
-muated: no cooperativity (t-state)
Hb function in O2 transport
IN LUNGS (pO2=100 torr):
-Hb is in R-state
-loads up on O2
IN CAPILLARIES (pO2=30 torr)
-Hb switches to T-state
-releases O2
