Transcript Myoglobin and Hemoglobin Lecture 8, Medical Biochemistry Lecture 8 Outline • Cooperativity of oxygen binding to hemoglobin • Stuctural basis for sickle-cell anemia • Modulators.

Myoglobin and Hemoglobin
Lecture 8, Medical Biochemistry
Lecture 8 Outline
• Cooperativity of oxygen binding to
hemoglobin
• Stuctural basis for sickle-cell anemia
• Modulators of oxygen binding to
hemoglobin and the Bohr Effect
Oxygen
Saturation
Curves for
Myoglobin &
Hemoglobin
Conformational Changes in
Hemoglobin upon binding of O2
• The heme iron atom in deoxyHb is slightly out of
the plane of the porphryin ring and bound to a
histidine imidazole sidechain. This is referred to as a
T (“taut”) state by protein chemists
• When oxygen binds to the heme on the side opposite
the histidine, the iron atom’s electron cloud becomes
slightly smaller, and the iron moves into the plane of
the porphryin ring, also pulling the histidine side
chain in the same direction
Molecular Shift at the Heme
Group after Oxygen Binding
Blue: deoxy
Red: oxy
Conformational Changes (cont)
• The movement of the histidine causes the
movement of the F-helix. This causes a
corresponding rearrangement of the other
helices in the protein subunit. The
conformational change of the subunit causes
it to move away from its partner in the ab
pair. Movement of one subunit in the ab
pair causes a corresponding conformational
adjustment in the paired subunit, enhancing
the ability of the latter to bind oxygen.
Dynamics of H-bond and
ionic interactions (dotted
lines) and van der Waals
forces (dashed lines) at the
dimer interface when
oxygen is bound (blue lines)
or unbound (black lines)
Quaternary conformational
changes in the oxygen
bound (red) and
deoxyhemoglobins (blue)
Hemoglobin: Axis of Symmetry
Channel View
a2
b2
b1
a1
a2
b2
b1
a1
The Oxy form (red) forms a more condensed channel along the
interface as compared to the Deoxy form (blue)
Sickle Cell Anemia
Sickled RBC
Normal RBC
Ruptured, sickled RBC
Sickle Cell Anemia
• The most common form of sickle cell anemia is
caused by a single amino acid substitution of valine
for glutamate at position 6 on the b-subunit of
hemoglobin. This defect is an autosomal (non-sex
chromosome) recessive inherited disease, meaning
both parents must be heterozygous carriers to
produce a homozygous child. Even heterozygous
carriers can experience sickle cell symptoms after
vigorous exercise or unpressurized travel at high
altitudes.
Sickle Cell Anemia (cont)
• The Glu to Val mutation in sickle cell hemoglobin
(termed Hb-S) reduces the solubility of
deoxyhemoglobin and allows formation of fibrous
polymeric filaments of deoxyhemoglobin that
precipitate in the red blood cells. This precipitation
leads to an ultrastuctural deformity of the red blood
cell, the "sickle" shape, which gives these cells a
tendency to get hung up and accumulate in the
narrow capillaries (thus leading to the associated
peripheral pain and many other complications).
Glu to Val Mutation in HbS
Position 6 - b-subunits
Subunit interactions
that promote
polymerization of
deoxy-HbS
Sickle Cell Anemia (cont)
• Upon exposure of oxygen at the lungs, the HbS
filaments immediately dissolve. Thus, situations
that slow down flow of blood through the capillaries
(normally 0.5 to 2 secs) can lead to the periodic
sickle cell anemia “crises”. Conditions like oxygen
deprivation (such as high altitudes or strenuous
exercise) and dehydration can contribute to slower
capillary passage of erythrocytes. The longer times
allow deoxy-HbS polymerization to occur, and thus
further contribute to the blockage of the affected
area.
Other Types of Hemoglobin
Mutations (Examples)
• Higher O2 affinity, b143 His to Gln; 2,3DPG
binding decreased
• Lower O2 affinity, b102 Asn to Thr; T-form
stabilized
• Methemoglobinemias, b63 His to Tyr; increased
stability for Fe3+ caused by Tyr residue
 a-helix disruptors, b63 His to Pro; Helix E
disrupted, heme pocket opened, MetHb forms
• Decreased stability of Hb, b43 Phe to Val; heme
pocket destabilized
Sickle Cell Anemia Structure/Function Concepts
• Hb-S polymerization illustrates how one amino acid
change from a charged to non-polar residue can lead
to powerful, cumulative hydrophobic interactions.
These are the same forces that hold the Hb monomer
cores together, while the salt bridges and hydrogen
bonds stabilize the surface interactions between
subunits. Loss of the charged surface Glu allows
more stable hydrophobic interactions to form in the
deoxy-Hb conformation.
2,3 Diphosphoglycerate (DPG)
2,3 Diphosphoglycerate (DPG) (or called 1,3 bisphosphoglycerate
(BPG)) binds tightly to the channel interface of deoxyHb, a binding
stabilized by interactions with positively charged sidechains. DPG
has a poor binding affinity for oxyHb due to the compacted channel
Physiological Function of DPG
• The presence of DPG in the erythrocyte creates a dynamic
competition for oxygen binding to deoxy-Hb. DPG bound
to deoxy-Hb reversibly stabilizes the deoxy conformation.
As [DPG] increases, the greater the percentage of oxygen
that will be released by Hb at the peripheral tissues. This
process occurs naturally due to the differences in oxygen
pressures between the lung and periphery, plus the presence
of DPG further promotes this oxygen release. This is one
mechanism to increase oxygen delivery to tissues during
exercise and/or at high altitudes
Model of Hemoglobin Forms
“Taut” T-forms, lowest O2 affinity
T
R
“Relaxed” R-forms, highest O2 affinity
= DPG
Effects of DPG, pH or CO2
Bohr Effect
• Hemoglobin releases H+ when it binds
O2
• Hemoglobin binds H+ when it releases
O2
• HbO2 + H+
HbH+ + O2
Hemoglobin and the Bohr Effect
• The Bohr Effect is the direct result of the conformational
changes that occur in Hb during oxygen binding. About
50% of the effect is caused by a change in pKa of His 146
(on b subunit). In the T-state, the His is H-bonded to Asp
94. When Hb shifts from the T to R conformation, the Hbond to Asp 94 is disrupted, allowing a proton on the His to
readily dissociate. The remainder of the effect results from
similar pKa changes of other ionizable groups as a
consequence of the T to R transition. This is the O2 bind/H+
release portion of the effect
Hb and the Bohr Effect (cont)
• An equivalent amount of H+ released (about 0.31
H+ per oxygen bound) is picked up by Hb as acid
(H+) produced from tissue metabolism.
• At the lungs, when oxygen rebinds, the H+
released is converted to carbon dioxide and
exhaled. About 40% of acid produced by the
tissues is buffered in this way by Hb
Physiological Bohr Effect
Hb and Carbon Dioxide
Transport
• Some of the dissolved CO2 in the blood
reacts with amino groups on Hb (and other
proteins). This reaction occurs with Nterminal amino groups in the NH2 form; at
blood pH, the side chain groups are in the
NH3+ form and will not react. About 15% of
tissue-produced CO2 is bound by protein in
this way (termed carbamino-CO2)
Interactions between DPG,
Carbamino-CO2 and Bohr Effect
• The carbamino-CO2 can be bound to some of the
same groups involved in DPG binding, thus
diminishing DPG binding; the reverse can occur,
DPG can block CO2
• CO2 also binds to some of the groups involved in
the Bohr effect; thus bound CO2 can diminish the
H+ buffering capacity of hemoglobin (again the
reverse effect can occur, bound H+ blocking CO2
Bohr Effect Summary