Lecture 17:Myoglobin & Hemoglobin

Margaret A. DaughertyFall 2003

Myoglobin

• Mb– Muscle protein;– Single polypeptide,

153 residues (18 kdprotein);

– All α-helical;– Binds heme;– Functional role to

bind and store O2 inmuscle;

Hemoglobin• Multisubunit, plasma protein

– α2β2 (adult)

– other globin subunits found inthe fetus and newborns (zeta,epsilon, gamma, etc).

– α chain = 141AAs;– β chain = 146 AAs;– Binds 4 hemes;– All α-helical protein;

• Body contains 750g of Hb &it is replaced every 120 days;

• Hb also transports H+ andCO2 in addition to O2;

Quaternary structure!

Comparison of structures

Myoglobin ~ Hb alpha ~ Hb beta

The “globin” fold

Myoglobin: hyperbolic curve

tissue lung

Question: What if Mb was a transport protein, not a storage protein?Hemoglobin & Oxygen

hemoglobin

Hemoglobin

What Sigmoidal (S-shaped) binding curves tells us!!!

Hemoglobin Structure: Low resolution

α

β

α

β+

2

22

α1

β1 α2

β2

“dimer of dimers”

The “α1β2” interfaceDenote intersubunit

interactionsAlpha1-Alpha2Alpha1-Beta2Alpha2-Beta1

Protein-protein interactions:Play a major role in hemoglobin functions

Hetero: αβ dimer Homo: 2 dimers form tetramer

Hb as an example of both

α

β

α1

β1

β2

Denote intersubunitinteractionsAlpha-alpha

Alpha1-beta2Alpha2-beta1

α2

Stretching the truth: α = β; so 4 interacting monomers

Homo-interactions Hetero-interactionsVs.

Hb function: cooperative binding &release of oxygen

α

β

α

β

2

2

2

noncooperative

Mb = Myoglobin

+

Mb

α1

β1 α2

β2

cooperative

Frac

tion

al s

atur

atio

n

pO2, mm Hg

Mb, α, β or αβ

Hb

Ligand Binding Curves: Shape

Ligand Binding Curves: Shape & Energetics

pO2 lungspO2 tissue

Frac

tion

al s

atur

atio

n

pO2, mm Hg

∆Gbinding

Weak binding

-5.4 kcal/mol

X = O2

x

-5.7 kcal/molxx

Weak to strong bindingtransition

-6.7 kcal/molxxx

-9.3 kcal/mol

Strong binding

xxx

x

Oxygen binding site Heme site: Protoporphoryn IX + Fe++

Heme is responsible for red color of blood!

Resonance delocalization: All bonds are equivalent in the heme andto the Fe

Heme binds Fe(II); Fe(II) has octahedral coordination ==> 6 ligands

Hemoglobin structure: Oxygen binding site

Helix F

FG corner

CHANGES IN THE HEME UPON OXYGEN BINDING

In deoxy state: heme is domed; Fe interacts with O2 weakly; His F8 is tiltedfrom the perpendicular by 8o

In oxy state: O2 pulls Fe into plane of heme; His F8 becomesperpendicular. Steric strain builds up on Val FG5 which causes structuralrearrangement in Hb.

OXYGEN-INDUCED HEME-SITE CHANGES ARETRANSMITTED TO THE α1β2 INTERFACE

Deoxy form (blue): iron is 0.6 Aabove center of domed porphyrin ring.

Oxy form (red): iron moves into planeof porphyrin ring & binds more tightlyto O2; pulls His 8 along with it----> thiscauses an adjustment of Helix F

Result of adjustmentof Helix F:Rearrangement ofsalt bridges at α1β2

interface

Subunit Interactions Change when Oxygen Binds

• The deoxy structure is stabilized by a network of saltbridges (hydrogen bond pairs)

• When O2 binds:– the α1β1 and α2β2 contacts (30 AAs) change little;

– the α1β2 and α2β1 contacts (19 AAs) undergo a large change(several ion pairs broken);

α1

β1 α2

β2

Hb quaternary structural changes

Low Affinity (T) structure High Affinity (R) structure

Oxygen binding disrupts salt bridges in the α1β2 interfacethus ------ structural rearrangement

Hb quaternary structural changes

Low Affinity (T) structure High Affinity (R) structure

Other Heme Ligands

• Other small molecules can also bind to the heme inMb and Hb

• These include: CO & NO;• CO has a 250 fold greater affinity for Hb than O2

does

Allosteric regulation of oxygen affinity

O2

BPG

Cl-

H+

CO2

Regulation bysmall ligands

binding at sitesother (allo-)

than theoxygen bindingsite (-steric)

The Bohr Effect: allosteric regulation by H+

Chu, Turner & Ackers, Bioch. 23, 604-614 (1984)

BIPHOSPHOGLYCERATE: BPGNegatively charged BPG binds in positively charged central cavityNet result = stabilization of deoxy hemoglobin

Increased concentration in blood at high altitudes Lowers affinity of hemoglobin for oxygen - offsets decrease in O2 uptake

Carbon Dioxide Transport

CO2 + H20 <--> H2CO3<--> HCO3- + H+

Bicarbonate reaction:

-NH3+ + HCO3

- <--> -N-COO- + H20 + H+

HBicarbonate in blood serum reacts with N-terminal groups of Hb

“carbamate”

Three effects:Transport CO2 to lungs!Release of protons contributes to Bohr Effect.Stabilizes the deoxy (T) quaternary structure.

Additive Effects of BPG and CO2

Hb Defects: 1 in 2000 people have a mutation in Hb

2 classes:

Thalassemias: decreasedrate of synthesis of one ormore peptide chains(different chains expressedat different times duringdevelopment).

Hemoglobinopathies:alterations in function orstability of the moleculearising from amino acidchanges in the chains

α1β2 interface mutations

Ackers & Smith, Ann. Rev. Biochem. 54, 597-629 (1985)

Perturbation tocooperativityKcal/mol)

Normal cooperativity: +6.3 kcal/mol

T structure most affected bymost mutations ---> high affinity

Changes in cooperativity results

Hemoglobin changes during development

Insert figure 28.11 & table

Embryonic:Gower I: ζ2ε2Gower II: α2ε2Portland: = ζ2γ2

Fetal:Hb F: α2γ2

Gγ = gly 136Aγ = ala 136

Adult:Hb A: α2β2 (A1; 95%)Hb A2: α2δ2 (< 3.5%)

Expression of hemoglobins during development

Fetal hemoglobin has higher affinity for O2 than Hb A

Hb FβH143S

Loss of the2 positive charges

that stabilize2,3- BPG binding.

SUMMARYMyoglobin has evolved to function as a storage molecule. Hemoglobin hasevolved to function as a transport molecule. Their oxygen binding curvesreflect the significance of their functions.

The sigmoidicity of the hemoglobins’ oxygen binding curve reflects thecooperativity in oxygen binding. The benefit of cooperativity comes in“dumping” off the oxygen to the body, not picking up the oxygen.

Changes in the heme site upon oxygenation are transmitted to thedimer-dimer interface, producing the T --> R quaternary transition.

The deoxy structure is most sensitive to mutations in the dimer-dimerinterface. Additionally, it is the most stabilized by the allostericeffectors.

Keywords: “dimer-dimer”interface,cooperativity,hyperbolic binding curve,sigmoidal binding curve, allosteric regulation, allosteric effectors, T and Rquaternary structures.