Chapter 7: Hemoglobin and Myoglobin pt.1 Flashcards by Jolin Labberton

Myoglobin relating to 3D structure

1st protein whose 3D structure was determined (by x-ray crystallography)

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John Kendrew

used sperm whale myoglobin to determines myoglobin structure
-Mb has O2 storage/transport protein

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Myoglobin facts

-monomer
-8 alpha-helices labeled A through H
-globular

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Hemoglobin facts

-tetramer of 4 Mb like homologous subunits
*2 Alpha-globin and 2 Beta-globin subunits

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Max Perutz (and Kendrew)

solved Hb’s crystal structure

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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

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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

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How to determine oxy and deoxy Hb

-they have different wavelengths so diff colors
-color changes due to presence or absence of oxygen

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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

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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

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O2 binding pocket in heme: Val E11 and Phe CD1

holds heme in place through hydrophobic interactions

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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

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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)

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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

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diaphorase

enzyme that reduces Fe^3+ to Fe^2+
*allows oxygen to bind back to heme

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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 1. 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 2. 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) 3. in T-state the Fe2+ is 0.6A out of heme plane 4. Through the increase of [O2], the 1st oxygen is able to bind and pulls Fe2+ back into the heme plane 5. His F8 is attached to Fe2+ so it also gets pulled towards the heme plane 6. 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 7. 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) 8. 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 9. 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