HEMOGLOBIN

AND

MYOGLOBIN

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 Hemoglobin
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 Nature of Hb
– a tetramer of two -chains (141 amino acids each) and
two -chains (153 amino acids each); 22

– the function of Hemoglobin is to transport oxygen

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 Hemoglobin- Key Properties
• Ubiquitous O2 transport protein
• A globular soluble protein, 2X2 chains (164 kDa)
•  and  chains are 44% identical
• All Helical Secondary Structure (like Myoglobin)
•  quaternary structure
-subunit 141 residues
 -subunit 153 residues
• Extensive contacts between subunits
Mix of Hydrophobic, H-bond, and Ionic Interactions
 11 (22)- 35 residues, 12 (21)- 19 residues
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 Structure of Hemoglobin
2 Heme

1
2

1
Inter-subunit contacts

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 The Globin Fold

 Eight -Helices (A-H)

connected by turns and
loops.
 Heme binds between
Helices E and F.
 Histidine residues from
Helices E and F are
situated near the Heme
Iron.

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 Hemoglobin Structure
Strongest subunit interactions are highlighted:

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 Heme Structure

methylene

P rotop orp hy rin I X

 The Heme Prosthetic Group
The O2 carrier in Myoglobin and Hemoglobin

• Protoporphyrin with Fe(II)

• Covalent attachment of Fe via His F8 side chain
• Additional stabilization via hydrophobic interaction
• Fe(II) state is active, Fe(III) [oxidized]
• Fe(II) atom in Heme binds O2

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 Heme is the Ligand Binding Site
The heme group. Porphyrin occupies 4 of the 6
coordination sites of the iron:

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 Oxygen Binding of Hb
– each chain has 1 heme group; hemoglobin can bind up
to 4 molecules of O2
– binding is cooperative; when one O2 is bound, it
becomes easier for the next O2 to bind
– the structure of oxygenated Hb is different from that of
unoxygenated Hb
– H+, CO2, Cl-, and 2,3-BisPhosphoGlycerate (BPG) affect
the ability of Hb to bind and transport oxygen

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 Oxygen-Binding Site

- One His residue

coordinates the iron.
-The second one assists
with stabilizing the O2-
bound form and also
destabilizing the CO-
bound form.
- CO binding only 200x
stronger (compare to
20,000x to free heme).

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 Oxygen-binding
site for myoglobin

The porphyrin ring occupies

four of the six coordination
sites of the Fe(II).

Histidine F8 occupies
the fifth coordination
site of the iron
 Binding of O2 to Heme
• Binding of O2 to a free heme group is irreversible
• Enclosure in a protein allows reversible binding
 O2 has only limited solubility (1 X 10-4 M) in water
 Solubility problem overcome by binding to proteins
 Also increases diffusion
• Binding of O2 alters heme electronic structure
 Causes changes in heme electronic spectrum (Vis)
 Bright scarlet color of blood in arteries
 Dark purple color of blood in veins

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 Oxygen Binding of Hb

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Oxygen Binding of Hb
• The effect of pH on the oxygen-binding ability of Hb is
called the Bohr effect
Hb O2 + H+ Hb H+ + O2

as pH decreases (more acidic), oxygen is released

• CO2 promotes release of O2 from HbO2

carbonic
anhy drase
CO 2 + H 2 O H2 CO 3

H2 CO 3 H+ + HCO 3 -
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Hb-oxygen dissociation curve
Hb-oxygen dissociation curve
 The normal position of curve depends on

 Concentration of 2,3-DPG
 H+ ion concentration (pH)
 CO2 in red blood cells
 Structure of Hb
 Deoxygenated Hb

Oxygenated Hb

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Oxygen Binding of Hb
Release of oxygen influenced by carbon dioxide

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 Hemoglobin also carries CO2 and H+

 The amino-termini of the four chains

become carbamylated at low pH (7.2) in the
tissues [Hb-NH-COO-]
 Helping to transport about 20% of the
carbon dioxide formed in the tissues to the
lungs and kidneys
 But what happens to the rest of the CO2?
 …and how else is the “pH” involved?

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Recall aqueous CO2 forms carbonic acid

 Carbon dioxide released in

tissues is insoluble until
hydrated and converted to
bicarbonate by the enzyme
carbonic anhydrase
 But this releases H+
(causing the pH in the
tissues and blood to fall)
 This process is reversed in
the lungs …

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 Role of Hb in carrying H+

 Histidine HC3 of T-state

hemoglobin has a higher pKa
than normal (via interaction
with Asp FG1), and thus gets
readily protonated
 Back at the lungs, return to
the R-state shifts HC3 back
to its normal pKa, releasing
the H+
 Release of H+ promotes
conversion to the R state,
increasing O2 affinity…

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 The Bohr Effect
Effect of pH and pCO2 on Hb oxygen affinity

deoxyHb
(T-state)

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 The Bohr Effect

 This “effect of pH and CO2 concentration on the

binding and release of oxygen by hemoglobin” is thus
dependent on
 The allosteric changes in the tetramer that
interrelate the physiological parameters in tissues,
blood, and lungs
 With the ability to bind oxygen,
 And carry both carbon dioxide and protons away from
tissue and back to the lungs.

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Oxygen Binding of Hb
Summary of the Bohr Effect

L ungs Actively Metaboliz ing Muscle

+
Higher pH than actively L ower pH due to production of H
metaboliz ing tissue

Hemoglobin binds O2 Hemoglobin releases O2

+ +
Hemoglobin releases H Hemoglobin binds H

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 Oxygen saturation curves for myoglobin and Hb
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 Hemoglobin O2 Binding Curv e

 Binding curve is
sigmoidal
 Artery: high pO2, loading
of protein
 Vein: lower pO2,
unloading from protein
 P50(hemoglobin) = 26 torr,
adjusts as needed!!

*Drastic change in pO2 over physiological range*

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 Cooperativity in Binding O2
T he sigmoidal shap e is a conseq uence of the 4 subunits
of hemoglobin "coop erating" in the binding of O2.
• As pO2 increases and [O2] increases, increasing
probability that at least 1 subunit has bound O2.
Binding of O2 to a subunit INCREASES the probability
that empty subunits will be able to bind an O2!!
• As pO2 increases even further, the probability that
remaining binding sites will have O2 bound increases.
• Eventually, a plateau is reached: when most
hemoglobins are filled there are few sites left to bind to,
so not much increase, even if the pO2 is very high.

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Binding of O2 to the Heme Changes
the Whole Structure of Hemoglobin
R state T state

 chains
Shifts at the further apart
 interfaces
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 The T to R State Transition
• Binding of O2 causes a series of shifts in all subunits
• Change in heme structure upon binding O2
• Since His F8 is covalently attached, all of F helix shifts
• Reorganization of helix alters tertiary structure, which
in turn alters the quaternary structure- 4 chains behave
as a single cooperative structural unit
 Changes in packing of hydrophobic side chain
 Changes in pairing of charged side chains
The change in conformation of Hemoglobin from the T to
the R state increases O2 affinity at ALL sites

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 Allosteric Effectors
• The R or T state can be stabilized by the binding of
ligands other than O2.
1. H+. Lower pH favors the T state which causes Hb to
release bound O2. This is known as the Bohr Effect.
2. CO2. Release of CO2 lowers pH via conversion to
HCO3-: CO2 + H2O  HCO3- + H+. Reinforces Bohr Effect
3. Bisphosphoglycerate (BPG). Regulation of activity via
binding more strongly to T state, helps to release O2.

Increase in levels of BPG helps adaptation to high

altitude- faster than making more hemoglobin. Also
important in hypoxia diseases (e.g. anemia)
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 Hemoglobin Dynamics
Oxygen binding triggers local conformational changes:

The shift in the iron tugs on the His, which moves helix F
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 Hemoglobin Dynamics

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 Hemoglobin dynamics
And the motion of helix F is translated to global
conformational changes:

1HG A 1B B B

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 Ionic interactions between subunits
These interactions stabilize the T state. They are
disrupted upon oxygen binding, promoting transition to
the R state.

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 Path of O2 Flow
1. O2 diffuses from the alveoli of the lungs into the
capillaries of the bloodstream then into the red blood
cells
2. In the red blood cells, O2 binds to hemoglobin.
3. In parallel, CO2 diffuses from blood into the alveoli.
4. The lower concentration of dissolved CO2 in the
blood causes lower pH (~7.6) in lungs than in the
peripheral tissues (~pH 7.2) where CO2 is being actively
released.

A. High pO2 / high pH

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Why O2 Transport Works
5. Red blood cells (containing O2-hemoglobin) carried
to the peripheral tissues.
- pO2 decreases because O2 USED by the tissues.
- Blood plasma becomes more acidic (lower pH)
because CO2 is released.
The combination of lower pO2 and pH in the peripheral
tissues causes a large decrease in O2 saturation.
O2 is released by hemoglobin!!!!

Note: changes in pO2

and pH are small!

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 Hemoglobin (Hb)
 Hemoglobin in blood is bound to BPG
 interaction is electrostatic, between negative charges
on BPG and positive side chains (e.g., Lys, Arg) of
hemoglobin
 BPG promotes O2 dissociation
 Hb stripped of BPG remains saturated with O2
O O-
C
H C OPO 3 2 -
CH2 OPO 3 2 -
2, 3 - Bisp hosp hogly cerate
( BP G )
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2,3-bisphosphoglycerate binding

Binding of 2,3-BPG - binds to pocket in center of

tetramer; but only in the T state.

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 2,3-bisphosphoglycerate binding

2,3-BPG concentration in blood is 5 mM at sea level,

but 8 mM at high altitudes …
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 BPG

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 Binding of BPG
to deoxyhemoglobin

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 Fetal Hemoglobin, Hb F
 has a higher affinity for O2 than maternal Hb A
 structure is 22
 binds less strongly to
BPG that does Hb A

Oxygen binding capacity of Hb F

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Defects from Hemoglobin Mutations
1. Weakened heme binding.
2. Disruption of secondary structure.
3. Disruption of quaternary structure.
4. Defective oxygen transfer.
5. Altered affinity for oxygen.
6. Oxidation of Fe(II) to Fe(III).
7. Aggregation in the T state (Hemoglobin S). Sickle cell
anemia results from aggregation of Hb into insoluble
fibers causing mishapen blood cells that cannot pass
through capillaries and block blood flow to tissues.
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 Sickle-cell anemia

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 Sickle-cell anemia
A genetic disease resulting from a
mutation that converts Glu6 (acidic)
in the -chains to Val (nonpolar).
This substitution creates
hydrophobic “sticky” patches on the
normally charged surface of the -
chains.

 The oxygenated molecules are soluble, but upon de-

oxygenation, the conformation of HbS differs
considerably from HbA, and it aggregates into
insoluble fibers (as diagrammed on the next slide).
 These fibers deform the RBCs into spiny or sickle-
shaped cells.
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 Sickle-cell anemia

Fig. 7-18b

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 Transversion type of mutation

The gene defect is a known mutation of a single nucleotide (A to T) of

the β-globin gene, which results in glutamate being substituted by valine at
position 6. Hemoglobin S with this mutation are referred to as HbS, as
opposed to the normal adult HbA.
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 Carbon Monoxide Poisoning
• Heme Fe(II) binds many other small molecules with
structures similar to O2 including: CO, NO, H2S
• O2 is actually a fairly weak binder relative to these
other molecules, particularly CO. [Essential for
Biology]
• When exposed to CO, even at low concentrations, O2
transport proteins will be filled with CO  limiting their
vital O2 capacity.

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 Abnormal Human Hb
– Hb S: substitution of Val for Glu at 26
– Hb E: Glu B8(26) -> Lys; change is on the surface and has
little effect on Hb stability or function
– Hb Savannah: Gly B6(24) -> Val; not enough room for Val
between B-helix and E-helix which disrupts entire structure
– Hb Bibba: Leu H19(136) -> Pro; proline disrupts the H-helix
– Hb M Iwate: His F8(87) -> Tyr; blood contains
methemoglobin and blood is chocolate brown
– Hb Milwaukee: Val E11(67) -> Glu; glutamate side chain
forms an ion pair with heme iron which stabilizes Fe(III) and
prevents O2 binding

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Myoglobin
Myoglobin
 a single polypeptide chain of 153 amino acids
 a single heme group in a hydrophobic pocket
 8 regions of -helix; no regions of -sheet
 most polar side chains are on the surface
 nonpolar side chains are folded to the interior
 two His side chains are in the interior, involved with
interaction with the heme group
 Fe(II) of heme has 6 coordinates sites; 4 interact with N
atoms of heme, 1 with N of a His side chain, and 1 with
either an O2 molecule or an N of the second His side
chain
 Myoglobin- Key Properties
• An O2 transport protein in muscle
• A globular soluble protein, 151 residues (16 kDa)
• 8 -helices (A,B,C,…..H)- first protein crystal structure!
• Contains a heme prosthetic group

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Why Myoglobin in Muscle?
• Under resting conditions, O2 saturation is at point X
on the green curve
• Small changes in pO2 and pH have very little effect on
saturation
• During extremely vigorous exercise, heart pumps
blood fast and breathing is rapid to increase the intake
of O2 . Also, pH is lowered.
• Eventually, transport not fast enough to meet needs,
i.e. pO2 lowered because O2 is used faster than it can
be replenished. [Hemoglobin now no help!]
• Under extreme conditions, shift from point X to Y:
saturation of the myoglobin is lowered = release of O2.

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Why Myoglobin in Muscle?
• Under resting conditions, O2 saturation is at point X
on the green curve
• Small changes in pO2 and pH have very little effect on
saturation
• During extremely vigorous exercise, heart pumps
blood fast and breathing is rapid to increase the intake
of O2 . Also, pH is lowered.
• Eventually, transport not fast enough to meet needs,
i.e. pO2 lowered because O2 is used faster than it can
be replenished. [Hemoglobin now no help!]
• Under extreme conditions, shift from point X to Y:
saturation of the myoglobin is lowered = release of O2.

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Why Myoglobin in Muscle?
• Under resting conditions, O2 saturation is at point X
on the green curve
• Small changes in pO2 and pH have very little effect on
saturation
• During extremely vigorous exercise, heart pumps
blood fast and breathing is rapid to increase the intake
of O2 . Also, pH is lowered.
• Eventually, transport not fast enough to meet needs,
i.e. pO2 lowered because O2 is used faster than it can
be replenished. [Hemoglobin now no help!]
• Under extreme conditions, shift from point X to Y:
saturation of the myoglobin is lowered = release of O2.

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 Oxygen-binding
site for myoglobin

The porphyrin ring occupies

four of the six coordination
sites of the Fe(II).

Histidine F8 occupies
the fifth coordination
site of the iron
Oxygen and carbon monoxide binding to the heme group of myoglobin
 SUMMARY
 Myoglobin is monomeric; hemoglobin is a tetramer of
two subunit types. Despite having different primary
structures, myoglobin and the subunits of hemoglobin
have nearly identical secondary and tertiary structures.
 The O2-binding curve for hemoglobin is hyperbolic,
but for hemoglobin is sigmoidal, a consequence of
cooperative interactions in the tetramer.