Hemoglobin: A Paradigm for Cooperativity

and Allosteric Regulation

After this lecture you will have learned:

• The Similiarities and differences of oxygen binding to myoglobin vs hemoglobin

• How Hemoglobin is able to transport oxygen

• What allosteric regulation is, and the specific example of cooperativity.

Cellular Requirement for O2

Catabolism

(Oxidation)

O2

ADP

ATP

NADP+

NADPH

Intermediates

Anabolism

(Biosynthesis)

Proteins

Fats

Carbohydrates

(Nutrients)

Waste

(CO2/Urea/etc.)

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 has limited solubility in Blood and Cytosol

–Use Oxygen Carriers

Myoglobin and Hemoglobin

• Myoglobin (Mb) – Increases O2 solubility in tissues (muscle)

– Facilitates O2 diffusion

– Stores O2 in tissues (in marine mammals)

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

tissues (in erythrocytes)

8 helices (A-H) and loops in between

The Globin Fold

• permanent, non-proteinaceous • Incorporated during folding • • responsible for reversible O2

binding

• Fe2+ has 6 coordination sites

• 4 with N of pyrrole rings, • 2 perpendicular to ring

• 6th coordination site: none deoxyhemoglobin O2 oxyhemoglobin CO carboxyhemoglobin

The Heme Prosthetic Group

5th coordination site is occupied with proximal His

Heme – Binding of CO vs. O2

• free heme binds C0 105 times better than O2

• kinked binding topology in Mb/Hb

favors O2 (100-fold)

TOTAL: CO binding ~ 230 fold stronger than O2

binding (Carbon monoxide poisoning)

Ligand Binding

• Small molecules (such as metals or hormones) that bind to proteins by non-covalent interactions

• usually transient and reversible interaction • 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

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

[L][P][PL] aK]PL[

[L][P]1

a

dK

K

[P][PL]

[PL]

]sites binding total[

sites] binding [occupied

Ligand Binding Curve

[L]

[L]

d

K

If [L] = Kd = 0.5 Kd corresponds to the ligand concentration at which 50% of the binding sites are occupied

Some Examples for Dissociation Constants

Figure 7-1

Myoglobin

• Small Intracellular Protein in Vertebrate Muscle

• Single polypeptide (153 aa) with one bound heme

• Facilitate O2 Diffusion in Muscle

• O2 Storage (aquatic mammals)

Myoglobin – Oxygen Binding Curve

• binds oxygen at high pO2, releases it at really low pO2

Mb + O2 MbO2

[L]

[L]

d

K

250

2

O

O

pp

p

pO2 in tissue ~ 4 kPa

Myoglobin – Diffusion/Oxygen Storage!

pO2 in lung ~ 13 kPa

Saturation of Mb depends on: the binding constant of Mb for O2

the concentration of O2 (pO2)

KD = p50 = 0.4 kPa

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 • arranged as a pair of identical subunits

(2 subunits) • Hb F (fetal): two and two chains

1 2

2 1

Hemoglobin – 3D Structure

Each subunit has 1 heme, which binds 1 O2

Lehninger, Figure 7-5, 7-6

O2

Heme

Function of Hemoglobin: 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

• O2 binding in lungs

• O2 release in tissues

Oxygen binds to Hemoglobin and Myoglobin differently

0

0.2

0.4

0.6

0.8

1

0 20 40 60 80 100

Frac

tio

n S

atu

rate

d

pO2 (torr)

Myoglobin

Hemoglobin

Hb’s p50 for O2 is higher than Mb.

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)

T R

The Concerted Model All or nothing mechanism

Lehninger, Figure 7-14

The Sequential Model

T R

Hb follows a little of both

Lehninger, Figure 7-14

Figure 7-8

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

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

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

T vs R State

(1) Change at interface between 12 and 21

(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

0

0.5

1

Frac

tio

nal

Sat

ura

tio

n (θ)

pO2

Hb

R state

T state

Lungs Tissues

When the partial pressure of O2 in venous blood is 30 torr, the saturation of myoglobin with O2 is ______ while the saturation of

hemoglobin with O2 is ______.

A) 0.55, 0.91

B) 0.91, 0.55

C) 2.8 torr, 26 torr

D) 0.91, 0.97

Cooperativity is measured by the Hill coefficient (HC): HC greater than 1 is for positive cooperativity, less than 1 for negative

cooperativity, and 1 for non-cooperative systems. What is the HC for Hemoglobin?

A. 3

B. 1

C. 0

D. -1

MCAT

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

Bohr effect

pH Dependence of O2 Binding to Hb

Mechanism of Bohr Effect

Protonation of side chains

His-146+ forms salt bridge with nearby Asp-94 stabilizes low affinity T-state O2 is released as pH drops

Figure 7-12

Roles of Hemoglobin and Myoglobin in O2 and CO2 Transport

High pH (7.6) Low [CO2]

Low pH (7.2) High [CO2]

2,3-BPG is a negative regulator 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 Lungs At Sea Level

pO2 In Tissues

No BPG

5mM BPG

Oxygen transport at high altitude

At Sea Level pO2

In Tissues At 10,000 FT

pO2 In Lungs

0

0.2

0.4

0.6

0.8

1

0 20 40 60 80 100

Frac

tio

nal

Sat

ura

tio

n (

)

pO2 (torr)

Oxygen transport at high altitude

0

0.2

0.4

0.6

0.8

1

0 20 40 60 80 100

Frac

tio

nal

Sat

ura

tio

n (

)

pO2 (torr)

At Sea Level pO2

In Tissues At 10,000 FT

pO2 In Lungs

Oxygen transport at high altitude

0

0.2

0.4

0.6

0.8

1

0 20 40 60 80 100

Frac

tio

nal

Sat

ura

tio

n (

)

pO2 (torr)

At Sea Level pO2

In Tissues At 10,000 FT

pO2 In Lungs

5mM BPG

8mM BPG

39% 32%

Oxygen transport at high altitude

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

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

Oxygen triggers Hb to switch from its low affinity (T) state to its high affinity (R) state. What kind of allosteric effector is

oxygen relative to Hb?

A. Heteroallosteric; positive effector

B. Homoallosteric; inhibitor

C. Heteroallosteric; negative effector

D. Homoallosteric; activator

The major focus of oxygen transport in the blood compartment is the hemoglobin

contained in red blood cells. In contrast, the carriage of carbon dioxide by the blood

is predominantly in the form of:

A. Dissolved gas

B. Hemoglobin-bound gas

C. Albumin-attached gas

D. Bicarbonate ion

MCAT

Table 7-1

Hemoglobin Variants

Sickle Cell Anemia

• Glu ——> Val

– (residue 6 of -chain)

• Leads to hydrophobic interactions between hemoglobin molecules

Sickle Cell anemia

• Hemoglobin fibers

• Sickling of erythrocytes

• Increased resistance to malaria

Figure 7-17a

Erythrocytes

Normal Sickled

Figure 7-20

Correspondence between Malaria and Sickle-Cell Gene