Roles of haemoglobin and myoglobin in O2 delivery

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Roles of haemoglobin and myoglobin in O2 delivery

• Mitchell Guss
• School of Molecular Microbial Biosciences
• M.Guss_at_mmb.usyd.edu.au

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Some questions?

• Why do we use proteins to transport and store O2?
• Why do we have 2 proteins haemoglobin (Hb) and
  myoglobin (Mb)?
• What is the structural basis of Hb function,
  including the Bohr effect, BPG?
• How do foetuses get their O2?
• What are Hb-opathies?

Reference appropriate chapters in any general
Biochemistry text. Eg those by Voet Voet,
Garrett Grisham, Lenninger, Stryer.
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The role of proteins

• Use of O2 increases extraction of energy from
  glucose 18x
• aerobic respiration is more efficient
• Blood carries up to 0.01 M O2 at 37C, plasma
  dissolves only 0.0001 M O2, 100x less
• Hb transports O2 in red blood cells and
  facilitates the transport of CO2
• Mb in muscle gets O2 to mitochondria

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Oxygen Binding

• Mb binds O2 tightly over wide pO2 range
  releases O2 at very low pO2
• Hb high affinity for O2 at high pO2 (lungs),
  low affinity for O2 at low pO2 (tissues)

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Oxygen Binding

• Hb functions to transport O2 from a region of
  high pO2 (lungs) to one of relatively low pO2
  (resting muscle)
• Mb binds O2 tightly at a point when Hb is binding
  less tightly but will eventually release the O2
  if the pO2 falls to a low enough level (working
  muscle)

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Mb and Hb proteins are related

• Mb is a monomer (1 polypeptide, 1 haem)
• Hb is like 4xMb (4 polypeptides , 4 haems) there
  are 2 copies of 2 different polypeptides, ? ?.
  The ? ? chains of Hb each resembles the single
  chain of Mb.

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O2 binding to Mb

• O2 binds to the Fe2 in a pocket
• protected from solvent
• prevents oxidation to Fe3
• reduces affinity for carbon monoxide (CO) binds
  25000x less strongly to Mb than to free heme!!
• Haem is covalently attached via the Fe

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What happens when O2 binds to Mb?

• O2 binding causes the Fe to change its spin state
  (electronic organisation)
• The Fe gets smaller and moves into the plane of
  the haem which becomes flat
• The part of the protein connected to the Fe moves
  as well
• This has no further consequence for Mb

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O2 Binding to Hb
High affinity form

• Sigmoidal curve indicates cooperativity
• 2 conformations of the protein
• one has low O2 affinity, other has high affinity
• As one O2 binds it makes other subunits convert
  to high affinity

Low affinity form
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O2 Binding induces conformation change
Low affinity
High affinity

• Fe moves on binding O2 in one subunit
• This motion is magnified and transmitted to the
  neighbouring subunits
• sufficiently large change to cause Hb crystals to
  crack on exposure to air

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Quaternary change in Hb
15 degree rotation of dimers Oxy form has smaller
hole in the centre
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Quaternary change in Hb
15 degree rotation of dimers Oxy form has smaller
hole in the centre
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Why does Hb behave this way?

• Remember Hb has 4 protein chains and 4 haems.
• The arrangement of the 4 molecules (quaternary
  structure) has two forms
• R (relaxed) which binds O2
• T (tense) which does not bind O2
• The equilibrium between the forms is affected by
  various ligands O2, H, CO2, BPG
• This cooperative binding is termed allostery

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Structural effects O2 binding to Hb

• Initial cause is change in Fe size and movement
  of Fe
• This movement is amplified propogates through
  the molecule
• The ultimate effect is a change in the interfaces
  between the protein chains and a smaller cavity
  in the centre

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Bohr Haldane Effects

• A pH drop in the blood in the capillaries lowers
  the oxygen affinity of haemoglobin, allowing even
  more efficient release of the last traces of
  oxygen. The response of hemoglobin to changes in
  pH is called the Bohr effect.
• At any given pCO2 venous blood (deoxygenated) can
  carry more CO2 than arterial (oxygenated) blood.
  This observation is termed the Haldane effect.

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pH dependence of O2 binding to Hb

• Oxygen binding to Hb is critically dependent on
  the pH (i.e. the H ion concentration. O2
  binding to Hb releases protons.
• HbO2 H lt-gt Hb O2
• Hb O2 lt-gt HbO2 H

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CO2 transport

• Increased CO2 leads to decreased pH
• CO2 H2O lt-gt HCO3- H
• and release of O2
• HbO2 H lt-gt Hb O2
• Transport of CO2 and release of O2 are linked
• In addition 15 of HCO3- combines with N-terminal
  ?-amino group to form carbamate group.
• --N3H HCO3- ?? --NHCOO-

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Bisphosphoglycerate -BPG

• Binding of BPG to Hb causes low O2 affinity
• BPG binds in the cavity between ?-Hb subunits
• Stabilizes T-conformation

• BPG involved acclimation to high altitude
• Foetal Hb (?2?2) has low affinity for BPG, allows
  foetus to compete for O2 with mothers Hb (? 2?2)
  in placenta.

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Foetal Hb

• Foetal maternal circulations are separate - O2
  diffuses across
• Adult (HbA - ?2?2) and foetal (HbF - ?2?2) Hbs
  are different
• HbF binds O2 more tightly because it binds BPG
  less tightly - ve charges are missing in cavity

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Hemoglobinopathies

• Diseases of Hb
• Caused by single site mutations e.g. sickle cell
  anaemia OR
• Gene deletions e.g. thalassaemia

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Sickle cell anaemia

• Hb polymerizes to form long filaments
• Cause sickling of cells
• Sickle cell trait offers advantage against
  malaria
• fragile sickle cells have shorter life time which
  may not support parasite life cycle
• heterzygous in 25 of African black people and
  10 of American black people
• homozygotes have severe anaemia

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Sickle cell anaemia

• A single mutation of Glu6 to Val6 in one ? chain
• Causes Val6 to bind to hydrophobic pocket in
  deoxy-Hb (not present in oxy-Hb)
• Polymerizes to form long filaments

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Summary

• We have two proteins to transport and store O2
• The structure of each defines its properties
• O2 binding to Hb is controlled by exterior
  factors BPG, H etc
• Delivery of O2 and removal of CO2 are synergistic
  processes
• Transfer of O2 from HbA to HbF can be explained
  by relative BPG affinities
• Genetic diseases of Hb can be explained by the
  molecular structures