Myoglobin Key Properties

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Myoglobin- Key Properties

• An O2 transport protein in muscle
• A globular soluble protein, 151 residues (16
  kDa)
• 8 a-helices (A,B,C,..H)- first protein crystal
  structure!
• Contains a heme prosthetic group
• Fig. 7-1

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The Heme Prosthetic GroupThe 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 Figs.
7-2 7-3
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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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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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Myoglobin O2 Binding Properties
Mb O2 ? MbO2 Dissociation constant, Kd
Mb O2 / MbO2
Quick Review from enzymes If Kd
(dissociation) is higher, binding is weaker If
binding curve is less steep, binding is weaker
Half-saturation is the point where O2 Kd Kd
Mb O2 / MbO2 Mb Kd / MbO2 Kd / Kd
Mb / MbO2 1 Mb MbO2
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Myoglobin O2 Binding Properties
Mb O2 ? MbO2, Kd Mb O2 / MbO2 To
think about O2 transport, express binding in
terms of the fraction of occupied binding sites,
i.e. Fractional Saturation (YO2) YO2 MbO2 /
(Mb MbO2) Substituting with Kd, allows
understanding of transport properties in terms of
O2 availability YO2 O2 / (Kd O2)
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Myoglobin O2 Binding Properties

• YO2 O2 / (Kd O2)
• O2 is a gas express O2 as partial pressure
  (pO2)
• Common to call pO2 oxygen tension
• YO2 pO2 / (Kd pO2)
• As pO2 increases, binding increases
• Binding curve is hyperbolic, not linear (just
  like enzymes!!!) Draw
• Low pO2 ? little binding to protein
• High pO2 ? saturation of protein
• At half-saturation pO2 Kd, field uses term P50
• P50(myoglobin) 2.8 torr / pO2 10-100 torr
  in-vivo!!

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Hemoglobin- Key Properties

• Ubiquitous O2 transport protein
• A globular soluble protein, 2X2 chains (164 kDa)
• a and b chains 44 identical
• All helical secondary structure (like myoglobin)
• abab quaternary structure
• a-subunit 141 residues
• b-subunit 146 residues
• Extensive contacts between subunits
• Mix of hydrophobic, H-bond, and ionic
  interactions
• a1b1 (a2b2)- 35 residues, a1b2 (a2b1)- 19
  residues

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Structure of Hemoglobin
Heme
a2
b1
b2
a1
Inter-subunit contacts
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Hemoglobin O2 Binding Properties
Hb nO2 ? Hb(O2)1 Hb(O2)2 Hb(O2)3
Hb(O2)4 Kd Hb O2n / Hb(O2)1 Hb(O2)2
Hb(O2)3 Hb(O2)4 Hows that for
complicated!?!?! To think about O2 transport,
express binding in terms of the fraction of
occupied binding sites, i.e. Fractional
Saturation (YO2) YO2 Hb(O2)1 . / (Hb
Hb(O2)1 .)
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Hemoglobin O2 Binding Curve

• 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
The sigmoidal shape is a consequence of the 4
subunits of hemoglobin "cooperating" 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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The Hill Equation

• Simplification is required to solve binding
  equation
• assume O2 binding cooperativity is infinite
• Binding curve can be expressed in terms of
  O2
• YO2 O2n / (Kd O2n)
• O2 is a gas substitute pO2 for O2 and use Kd
  P50
• YO2 (pO2)n / (P50 (pO2))n
• This expression for the degree of saturation of
  Hb is known as the Hill Equation
• The quantity n is called the Hill Constant

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Interpretation of the Hill Equation

• YO2 (pO2)n / (P50 (pO2))n
• n the degree of cooperativity in ligand
  binding
• n 1 non-cooperative
• n gt 1 positive cooperativity implies binding of
  a ligand increases affinity for next ligand.
• n lt 1 negative cooperativity implies binding of
  a ligand decreases affinity for next ligand.
• Rearranging the Hill equation allows Hill Plots
  to be made to facilitate analysis of binding
  curves.

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Binding of O2 to the Heme Changes the Whole
Structure of Hemoglobin
R state
T state
b chains further apart
Shifts at the ab 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.
• H. Lower pH favors the T state which causes Hb
  to release bound O2. This is known as the Bohr
  Effect.
• CO2. Release of CO2 lowers pH via conversion to
  HCO3- CO2 H2O ? HCO3- H. Reinforces Bohr
  Effect
• 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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Towards a More Complete Picture
Model for disucssion
HEMOGLOBIN at the pH (7.6) found in the
lungs. HEMOGLOBIN at the pH (7.2) found in
peripheral tissues. MYOGLOBIN in muscle (a
peripheral tissue).
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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.
• B. pO2 decreases because O2 USED by the tissues.
• C. 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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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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Defects from Hemoglobin Mutations

• Weakened heme binding.
• Disruption of secondary structure.
• Disruption of quaternary structure.
• Defective oxygen transfer.
• Altered affinity for oxygen.
• Oxidation of Fe(II) to Fe(III).
• 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.