Hemoglobin

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Hemoglobin Structure FunctionChapter
15Pages 467-4788/15/11Thomas Ryan, Ph.D.
Biochemistry and Molecular Geneticstryan_at_uab.edu
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Introduction

• Hemoglobin Structure and Function
• Hemoglobinopathies
• Thalassemia
• Sickle Cell Disease
• UAB Animal Models

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• Red Blood Cells Contain Hemoglobin

Human Body 1 x 1014 Total Cells 2.5 x
1013 Total RBCs 25 of Total Cells
95 of cytosolic protein in RBC is hemoglobin 85
of total body heme is in RBC 70 of total body
iron is in RBC
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Erythroid Development
Erythropoiesis
HSC
BFU-E CFU-E
Pro-
Basophilic
Polychromatic
Orthochromatic
Reticulocyte
Mature Red Cell
Erythroblasts
gt95 protein is hemoglobin
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• Red Blood Cells Contain Hemoglobin

RBC
Sickle Cell Anemia is caused by single mutation
in b globin Cooleys Anemia is caused by the
absence of b globin chains
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The structure of myoglobin is similar to that of
the hemoglobin monomer
Figure 15.21 The myoglobin and hemoglobin
structures. Myoglobin is monomeric Hemoglobin
is tetrameric
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Hemoglobin Myoglobin

• Hemoglobin and myoglobin are oxygen transport and
  storage proteins
• Compare the oxygen binding curves for hemoglobin
  and myoglobin
• Hemoglobin is a classic example of allosteric
  regulation
• Myoglobin is monomeric hemoglobin is tetrameric
• Mb 153 aa, 17,200 MW
• Hb two a chains of 141 residues, 2 ß chains of
  146 residues

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Globin Subunit Conformational Structures
153 aa 141 aa
146 aa
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Myoglobin Structure

• Mb is a monomeric heme protein
• Mb polypeptide "cradles" the heme group
• Fe in Mb is Fe2 - ferrous iron - the form that
  binds oxygen
• Oxidation of Fe yields 3 charge - ferric iron
• Mb with Fe3 is called metmyoglobin and does not
  bind oxygen

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Figure 15.22 Mb and Hb use porphryins to bind Fe2
Heme is formed when protoporphyrin IX binds Fe2
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Intracellular Free Heme Level Control
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Fe2 is coordinated by His F8

• Iron interacts with six ligands in Hb and Mb
• Four of these are the N atoms of the porphyrin
• A fifth ligand is donated by the imidazole side
  chain of amino acid residue His F8
• (This residue is on the sixth or F helix, and
  it is the 8th residue in the helix, thus the
  name.)
• When Mb or Hb bind oxygen, the O2 molecule adds
  to the heme iron as the sixth ligand
• The O2 molecule is tilted relative to a
  perpendicular to the heme plane

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Globin Heme Iron Liganding Six Positions
Histidine at this position is invariant
throughout globin gene evolution!
Imidazole Ring of Histidine F8
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O2 Binding Alters Mb Conformation

• In deoxymyoglobin, the ferrous ion actually lies
  0.055 nm above the plane of the heme
• When oxygen binds to Fe in heme of Mb, the heme
  Fe is drawn toward the plane of the porphyrin
  ring
• With oxygen bound, the Fe2 atom is only 0.026 nM
  above the plane
• For Mb, this small change has little consequence
• But a similar change in Hb initiates a series of
  conformational changes that are transmitted to
  adjacent subunits

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Oxygen Binding Causes Conformational Change
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The Conformation Change

• The secret of Mb and Hb
• Oxygen binding changes the Mb conformation
• Without oxygen bound, Fe2 is out of heme plane
• Oxygen binding pulls the Fe2 into the heme plane
  
• Fe2 pulls its His F8 ligand along with it
• The F helix moves when oxygen binds
• Total movement of Fe2 is 0.029 nm i.e., 0.29 Å
  
• This change means little to Mb, but lots to Hb!

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Figure 15.20 O2-binding curves for hemoglobin and
myoglobin
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Cooperative Binding of Oxygen Influences
Hemoglobin Function

• Mb, an oxygen storage protein, has a greater
  affinity for oxygen at all oxygen pressures
• Hb is different it must bind oxygen in lungs
  and release it in capillaries
• Hb becomes saturated with O2 in the lungs, where
  the partial pressure of O2 is about 100 torr
• In capillaries, pO2 is about 40 torr, and oxygen
  is released from Hb
• The binding of O2 to Hb is cooperative binding
  of oxygen to the first subunit makes binding to
  the other subunits more favorable

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Oxygen Binding by Hb Induces a Quaternary
Structure Change

• When deoxy-Hb crystals are exposed to oxygen,
  they shatter. Evidence of a large-scale
  structural change
• One alpha-beta pair moves relative to the other
  by 15 degrees upon oxygen binding
• This massive change is induced by movement of Fe
  by 0.039 nm when oxygen binds

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Hemoglobin is a Tetramer Composed of 2 ab Dimers
Figure 15.24 An aß dimer of Hb, with packing
contacts indicated in blue. The sliding contacts
made with the other dimer are shown in yellow.
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Fe2 Movement by Less Than 0.04 nm Induces the
Conformation Change in Hb
Figure 15.26 Changes in the position of the heme
iron atom upon oxygenation lead to conformational
changes in the hemoglobin molecule.
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Fe2 Movement by Less Than 0.04 nm Induces the
Conformation Change in Hb

• In deoxy-Hb, the iron atom lies out of the heme
  plane by about 0.06 nm
• Upon O2 binding, the Fe2 atom moves about 0.039
  nm closer to the plane of the heme
• As if the O2 is drawing the heme iron into the
  plane
• This may seem like a trivial change, but its
  biological consequences are far-reaching
• As Fe2 moves, it drags His F8 and the F helix
  with it
• This change is transmitted to the subunit
  interfaces, where conformation changes lead to
  the rupture of salt bridges

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R vs. T Conformational State of Hb
T (Tense) DeoxyHb
R (Relaxed) OxyHb
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Oxygen Binding Curves for Mb and Hb
P50 of HbA is 26 mmHg
P50 is the partial pressure of oxygen at which Hb
is 50 saturated. The P50 is inversely
proportional to the oxygen affinity.
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The Physiological Significance of the HbO2
Interaction

• Hb must be able to bind oxygen in the lungs
• Hb must be able to release oxygen in capillaries
• If Hb behaved like Mb, very little oxygen would
  be released in capillaries - see Figure 15.20!
• The sigmoid, cooperative oxygen binding curve of
  Hb makes its physiological actions possible!

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H Promotes Dissociation of Oxygen from Hb

• Binding of O2 to Hb is affected by several
  agents, including H, CO2, and chloride ions
• The effect of H is particularly important
• Deoxy-Hb has a higher affinity for H than oxy-Hb
• Thus, as pH decreases, dissociation of O2 from
  hemoglobin is enhanced
• Ignoring the stoichiometry of O2 and H, we can
  write

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Antagonism of O2 Binding by H is Termed the Bohr
Effect

• The effect of H on O2 binding was discovered by
  Christian Bohr (the father of Neils Bohr, the
  atomic physicist)
• Binding of protons diminishes oxygen binding
• Binding of oxygen diminishes proton binding
• Important physiological significance

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Bohr Effect Increased H Shifts O2 Binding
Curve to the Right
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CO2 Also Promotes the Dissociation of O2 from
Hemoglobin

• Carbon dioxide diminishes oxygen binding
• Hydration of CO2 in tissues and extremities leads
  to proton production
• These protons are taken up by Hb as oxygen
  dissociates
• The reverse occurs in the lungs

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Summary of the Physiological Effects of H and
CO2 on O2 Binding by Hemoglobin

• At the tissue-capillary interface, CO2 hydration
  and glycolysis produce extra H, promoting
  additional dissociation of O2 where it is needed
  most
• At the lung-artery interface, bicarbonate
  dehydration (required for CO2 exhalation)
  consumes extra H, promoting O2 binding

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2,3-Bisphosphoglycerate

• An Allosteric Effector of Hemoglobin
• In the absence of 2,3-BPG, oxygen binding to Hb
  follows a rectangular hyperbola!
• The sigmoid binding curve is only observed in the
  presence of 2,3-BPG
• Since 2,3-BPG binds at a site distant from the Fe
  where oxygen binds, it is called an allosteric
  effector

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BPG Binding to Hb Has Important Physiological
Significance
Figure 15.30 The structure, in ionic form of BPG
or 2,3-bisphosphoglycerate, an important
allosteric effector of Hb
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BPG Binding to Hb Has Important Physiological
Significance

• Where does 2,3-BPG bind?
• "Inside" in the central cavity
• What is special about 2,3-BPG?
• Negative charges interact with 2 Lys, 4 His, 2
  N-termini
• Fetal Hb - lower affinity for 2,3-BPG, higher
  affinity for oxygen, so it can get oxygen from
  mother

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2,3-BPG Binding to Hb

• 2,3-BPG is very electronegative
• Binds in central cavity of Hb
• Interacts with amino acid side chains of both
  ?-globin chains Lys ?82, His ?2, His ?143, and
  the amino terminus.

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Oxygen Binding Curves

• Stroma free Hb, P50 lt 8 mmHg
• Deoxy Hb binds CO2 in tissues, transports it to
  the lungs, and releases it.
• R-NH2 CO2 ? R-NH-COO- H
• (Lungs) (Tissues)
• 2,3-Bisphosphoglycerate (2,3-BPG) stabilizes
  deoxy Hb inside red blood cells.
• Electronegative 2,3-BPG binds to positive charged
  functional groups of the beta globin chains in
  the central cleft of the Hb tetramer.
• Cl- ion also stabilizes deoxy Hb.

venous pO2
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Fetal Hemoglobin Has a Higher Affinity for O2
Because it has a Lower Affinity for BPG

• The fetus depends on its mother for O2, but its
  circulatory system is entirely independent
• Gas exchange takes place across the placenta
• Fetal Hb differs from adult Hb with ?-chains in
  place of ß-chains and thus a a2?2 structure
• As a result, fetal Hb has a higher affinity for
  O2
• Why does fetal Hb bind O2 more tightly?
• Fetal ?-chains have Ser instead of His at
  position 143 and thus lack two of the positive
  charges in the BPG binding cavity
• BPG binds less tightly and Hb F thus looks more
  like Mb in its O2 binding behavior (O2 binding
  curve shifted to left)

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Fetal Hemoglobin Has a Higher Affinity for O2
Because it has a Lower Affinity for BPG
Figure 15.32 Comparison of the oxygen saturation
curves of Hb A and Hb F under
similar conditions of pH and BPG.
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Hemoglobin and Nitric Oxide

• Nitric oxide (NO) is a simple gaseous molecule
  that acts as a neurotransmitter and as a second
  messenger in signal transduction (see Chapter 32)
• NO is a high-affinity ligand for Hb, binding to
  the heme iron 10,000 times more tightly than O2
• So why is NO not bound instantaneously to Hb,
  preventing its physiological effects?
• NO reacts with the SH of Cys93ß, forming an
  S-nitroso derivative

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Hemoglobin and Nitric Oxide

• The S-nitroso group is in equilibrium with other
  S-nitroso compounds formed by reaction of nitric
  oxide with small-molecule thiols such as free Cys
  or glutathione
• These small-molecule thiols transfer NO from
  erythrocytes to endothelial receptors, where it
  exerts its physiological effects

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BREAK
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Hemoglobinopathies Thalassemia8/15/11Thomas
Ryan, Ph.D. Biochemistry and Molecular
Geneticstryan_at_uab.edu