Hemoglobin:A Paradigm for Cooperativity and

Allosteric Regulation

Why do we breathe?

http://www.uni.edu/schneidj/webquests/spring04/tvbroadcast/circulatorysystem.html

Cellular Requirement for O2

Catabolism

(Oxidation)

O2

ADP

ATP

NADP+

NADPH

Intermediates

Anabolism

(Biosynthesis)

ProteinsFats

Carbohydrates(Nutrients)

Waste

(CO2/ Urea/ etc.)

Oxygen Carriers

Diffusion

Limited solubility of O2 in Blood and Cell Water

Myoglobin and Hemoglobin

• Myoglobin (Mb)– Increases O2 solubility in tissues

(muscle)

– Facilitates O2 diffusion

– Stores O2 in tissues

• Hemoglobin (Hb)– Transports O2 from lungs to peripheral

tissues (erythrocytes)

Oxygen Transport

O2

O2 O2

deoxyHb

deoxyMbMbO2

Hb(O2)n Hb(O2)n

deoxyHb

deoxyHb

LUNGS MUSCLE CELL

pO2 = ~20- 30 torr

RED BLOOD CELLS

O2 + 4e– + 4H+ 2H2O

pO2 = 100 torr

Myoglobin

Small Intracellular Protein in Vertebrate Muscle

Function(s) of Myoglobin

Facilitate O2 Diffusion in Muscle

O2 Storage (aquatic mammals)

Figure 7-1

Structure of Sperm Whale Myoglobin

Figure 7-2

The Heme Prosthetic Group

Properties of Heme Prosthetic Group in Myoglobin

• Tightly bound

• Synthesized separately from myoglobin

• Fe2+ Coordination– Nitrogens of heme (4)

– His (F8): proximal histidine

• His (E7): distal histidine

• Ligands: O2, CO, and NO

Ligands

Small molecules that bind to proteins by non-covalent

interactions(e.g. O2 to myoglobin)

Ligand Binding

•usually transient and reversible interaction with others molecule (= ligands) such as metals, hormones

•often involves “molecular breathing” of the protein, i.e. ability to undergo small conformational changes

•often induces molecular rearrangements in the protein

• ligand binding sites are- highly conserved- complementary in size, shape, and charge

• prosthetic (permanent, non-proteinaceous)

•group of Mb and Hb

• incorporated into Hb and Mb during folding

• responsible for reversible O2 binding

• responsible for red color of blood and muscles

Heme

central Fe2+

Heme – Structure

2 vinyl groups (buriedin protein)

4 methyl groups

2 propionate groups(exposed)

•Fe2+ has 6 coordination sites

•4 with N of pyrrole rings,

•2 perpendicular to ring

•Mb/Hb: 5th coordination site is occupied with proximal His

•6th coordination site:O2 oxyhemoglobinnone deoxyhemoglobinCO carboxyhemoglobin

Heme – Iron Coordination

Heme – Binding of CO vs. O2

• free heme binds C0 105 times better than O2

•kinked binding topology in Mb/Hbfavors O2 (100-fold)

TOTAL: CO binding ~ 230 fold stronger than O2 binding (Carbon monoxide poisoning)

Function(s) of Myoglobin

Facilitate O2 Diffusion in Muscle

O2 Storage (aquatic mammals)

Myoglobin (Mb)

•primarily found in muscle (highly abundant in marine mammals such as whales)

•single polypeptide (153 aa) with one bound heme

•very simple oxygen binder: binds oxygen at high pO2, releases it at low pO2

Mb + O2 MbO2

• typical globin fold

8 helices (A-H) and loops in between

20MCDB310 – Chapter 5: Protein Function

The Globin Fold

Myoglobin – Oxygen Binding Curve

Binding/Association Constant Ka

Quantitatively describes the affinity of a protein P for its ligand L

P + L PL

the higher the binding affinity, the higher Ka

[L][P]

]PL[

aK

Dissociation Constant Kd

P + L PL

the higher the binding affinity, the smaller Kd

]PL[

[L][P]1

ad KK

Example: Ka = 106 M-1 Kd = 10-6 M

Degree of Saturation,

0 1

[P][PL]

[PL]

]sites binding total[

sites] binding [occupied

Fraction of binding sites that are occupied by ligand at any given ligand concentration

Degree of Saturation,

Using

[L]

[L]

[L]1

[L]

da

a

KK

K

If [L] = Kd = 0.5

Kd is the ligand concentration at which 50% of the binding sites are occupied

[L][P]

]PL[

aK [L][P][PL] aK

Ligand Binding Curve

[L]

[L]

d K

Some Examples

with KD = 1 µM

Question: What fraction of the protein has ligand bound when the [L] is 1 µM or 10 µM?

[L] = 1 µM:

[L]

[L]

d K

5.0μM1μM1

μM1

[L]

[L]

d

K

[L] = 10 µM: 91.0μM10μM1

μM10

[L]

[L]

d

K

Some Examples for Dissociation Constants

Myoglobin – Oxygen Binding Curve Revisited

[L]

[L]

d K

When ligand is a gas, partial pressures = concentrations

250

2

O

O

pp

p

Saturation of Mb depends on

•the binding constant of Mb for O2 (KD = p50 = 2.8 torr)

•the concentration of O2 (pO2)

Question: What is the fractional saturation of Mb?

pO2 = 1 torr:

pO2 = 10 torr:

Myoglobin – Oxygen Binding Curve Revisited

26.0torr8.2torr1

torr1

78.0torr8.2torr10

torr10

[L]

[L]

d K

pO2 in tissue ~ 4 kPa

Myoglobin – An Oxygen Storage!

pO2 in lung ~ 13 kPa

10 kPa = 76 torr

Hemoglobin(22)

Hemoglobin (Hb)

•present in erythrocytes (makes blood look red, 34% of weight is Hb)

Different Hb subtypes:•Hb A (adult): two (141 aa) and two (146 aa)

subunits that are arranged as a pair of identical subunits (2 subunits)

•Hb F (fetal): two and two chains

12

2 1

Hemoglobin – 3D Structure

Each subunit has 1 heme, which binds 1 O2

Lehninger, Figure 7-5, 7-6

O2

Heme

Hemoglobin

Erythrocytes:

•1 ml blood: 5 x 109 erythrocytes•1 erythrocyte: 3 x 108 Hb molecules •Hb is a good marker for number of red blood cells

Homology:

• 50% of AA are identical between and subunits

• 20% of AA are identical between / and Mb

Function of Hemoglobin

O2 binding in lungs

O2 release in tissues

Oxygen Transport

O2

O2 O2

deoxyHb

deoxyMbMbO2

Hb(O2)n Hb(O2)n

deoxyHb

deoxyHb

LUNGS MUSCLE CELL

pO2 = ~20- 30 torr

RED BLOOD CELLS

O2 + 4e– + 4H+ 2H2O

pO2 = 100 torr

Oxygen binds to hemoglobin and myoglobin differently

Myoglobin

Hemoglobin

Oxygen binding to hemoglobin

Θ = fraction of binding sites that are occupiedpO2 = partial pressure of oxygen

p50 is the pO2 where half the binding sites are occupied

p50

Hb has evolved to transport O2

pO2 In Lungs

pO2 In Tissues

p50

38%

Hb gains cooperativity by switching between 2 states

Lehninger Figure 7-10

T state (Low Affinity) R state (high affinity)

The Concerted ModelAll or nothing mechanism

T RLehninger, Figure 7-14

The Concerted ModelAll or nothing mechanism

T RLehninger, Figure 7-14

The Sequential Model

Hb follows a little of both

T RLehninger, Figure 7-14

Figure 7-8

Movements of the Heme and the F Helix During the T —> R Transition

Local structural changes around the Heme are communicated to the rest of Hb

By Janet Iwasa,https://iwasa.hms.harvard.edu/project_pages/hemoglobin/hemoglobin.html

Figure 7-9

Changes in the 1–2 Interface during the T —> R Transition in

Hemoglobin

Figure 7-9 part 1

Changes in the 1–2 Interface during the T —> R Transition in

Hemoglobin

Figure 7-9 part 2

Changes in the 1–2 Interface during the T —> R Transition in

Hemoglobin

Figure 7-10

Networks of Ion Pairs and Hydrogen Bonds in Deoxyhemoglobin

T vs R State

(1) Change at interface between and

(2) R state is more compact, and relaxed(3) T state has additional salt bridges, which makes it more tense

(4) In R state individual O2 sites have higher affinity for O2.

- better Fe-O2 bond length - fewer steric repulsions associated

with oxygen binding.

Without cooperativity Hb could not efficiently transport oxygen

T state

LungsTissues

homotropic, positive (= cooperative binding)

Allosteric regulation of protein function

homotropic, positive (= cooperative binding)

Allosteric regulation of protein function

heterotropic, negative

The Bohr Effect

• H+ and CO2 are negative, heterotropic modulators of Hb

• metabolizing tissue: H+ and CO2 accumulate bind to Hb and lower the affinity of Hb for O2

Hb releases O2

• lungs: CO2 and H+ dissociate from Hb increases the affinity of Hb for O2

Hb binds O2

• increase the efficiency of Hb as O2 transporter

Hb also binds and transports H+ and CO2 from tissue to lungs and kidneys for secretion

The Bohr Effect

Lungs: pO2 = 100 torr, high pH (7.6), low [CO2] Hb has high affinity for O2

Tissue: pO2 = 20 torr, low pH (7.2), high [CO2] Hb has low affinity for O2

CO2 + H2O HCO3- + H+

CO2 + H2O HCO3- + H+

Bohr effect

pH Dependence of O2 Binding to Hb

Mechanism of Bohr Effect1. Protonation of His-146

His-146+ forms salt bridge with nearby Asp-94 stabilizes low affinity T-state

O2 is released as pH drops

61MCDB310 – Chapter 5: Protein Function

Figure 7-12

Roles of Hemoglobin and Myoglobin in O2 and CO2 Transport

Heterotropic Negative Modulator

Or: 2,3-Diphosphoglycerate (DPG)

BPG is negatively charged

BPG binds to the central cavity of Hb

BPG binds to the positively charged central cavity of Hb

By Janet Iwasa,https://iwasa.hms.harvard.edu/project_pages/hemoglobin/hemoglobin.html

BPG allows for release of O2

pO2 In LungsAt Sea Level

pO2 In Tissues

No BPG

5mM BPG

In Class Activity:

Ligand Binding can affect Protein Function

• Cooperativity– 1 ligand bound = higher affinity for more ligands– Concerted vs Sequential

• Allosteric regulation– 1 regulator binding affects binding of ligand – Homotropic vs heterotropic– Positive vs Negative

From Protein Structure to Function

1. Hemoglobin and myoglobin: Principles of reversible ligand binding

2. (Antibodies: Principles of specific, high affinity ligand binding)

3. Myosin and actin: Protein activity modulated by ATP

4. Enzymes

Table 7-1

Hemoglobin Variants

Sickle Cell anemia

• Glu ——> Val (residue 6 of -chain)

• Leads to hydrophobic interactions between hemoglobin molecules

• Hemoglobin fibers

• Sickling of erythrocytes

• Increased resistance to malaria

Figure 7-17a

Normal Erythrocytes

Figure 7-17b

Sickled Erythrocytes

Figure 7-20

Correspondence between Malaria and Sickle-Cell Gene