Protein Function

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Protein Function

• Globins and Antibodies
• 3/10/2003

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Hemoglobin and Myoglobin

• Because of its red color, the red blood pigment
  has been of interest since antiquity.
• First protein to be crystallized - 1849.
• First protein to have its mass accurately
  measured.
• First protein to be studied by ultracentrifugation
  .
• First protein to associated with a physiological
  condition.
• First protein to show that a point mutation can
  cause problems.
• First proteins to have X-ray structures
  determined.
• Theories of cooperativity and control explain
  hemoglobin function

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The structure of myoglobin and hemoglobin
Andrew Kendrew and Max Perutz solved the
structure of these molecules in 1959 to 1968.
The questions asked are basic. What chemistry
is responsible for oxygen binding, cooperativity,
BPG effects and what alterations in activity does
single mutations have on structure and
function. Myoglobin 44 x 44 x 25 Å single
subunit 153 amino acid residues 121 residues are
in an a helix. Helices are named A, B, C, F.
The heme pocket is surrounded by E and F but not
B, C, G, also H is near the heme. Amino acids
are identified by the helix and position in the
helix or by the absolute numbering of the residue.
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The Backbone structure of Myoglobin
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The Heme complex in myoglobin
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Hemoglobin
Spherical 64 x 55 x 50 Å two fold rotation of
symmetry a and b subunits are similar and are
placed on the vertices of a tetrahedron. There is
no D helix in the a chain of hemoglobin.
Extensive interactions between unlike subunits
a2-b2 or a1-b1 interface has 35 residues while
a1-b2 and a2-b1 have 19 residue contact.
Oxygenation causes a considerable structural
conformational change
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Quaternary structure of deoxy- and oxyhemoglobin
R-state
T-state
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Oxygenation rotates the a1b1 dimer in relation to
a2b2 dimer about 15
The conformation of the deoxy state is called the
T state The conformation of the oxy state is
called the R state individual subunits have a t
or r if in the deoxy or oxy state. What causes
the differences in the conformation states? It is
somehow associated with the binding of oxygen,
but how?
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The positive cooperativity of O2 binding to Hb
arises from the effect of the ligand-binding
state of one heme on the ligand-binding affinity
of another.
The Fe iron is about 0.6 Å out of the heme plane
in the deoxy state. When oxygen binds it pulls
the iron back into the heme plane. Since the
proximal His F8 is attached to the Fe this pulls
the complete F helix like a lever on a fulcrum.
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Binding of the oxygen on one heme is more
difficult but its binding causes a shift in the
a1-b2 contacts and moves the distal His E7 and
Val E11 out of the oxygens path to the Fe on the
other subunit. This process increases the
affinity of the heme toward oxygen. The a1-b2
contacts have two stable positions. These
contacts, which are joined by different but
equivalent sets of hydrogen bonds and act as a
binary switch between the T and the R states
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• The energy in the formation of the Fe-O2 bond
  formation drives the T? R transition.
• Hemoglobins O2 -binding Cooperativity derives
  from the T ? R Conformational shift.
• The Fe of any subunit cannot move into its heme
  plane without the reorientation of its proximal
  His so as to prevent this residue from bumping
  into the porphyrin ring.
• The proximal His is so tightly packed by its
  surrounding groups that it can not reorient
  unless this movement is accompanied by the
  previously described translation of the F helix
  across the heme plane.
• The F helix translation is only possible in
  concert with the quaternary shift that steps the
  a1C-b2FG contact one turn along the a1C helix.

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• The inflexibility of the a1-b1 and the a2-b2
  interfaces requires that this shift
  simultaneously occur at both the a1-b2 and a2-b1
  interfaces.
• No one subunit or dimer can change its
  conformation.
• The t state with reduced oxygen affinity will be
  changed to the r state without binding oxygen
  because the other subunits switch upon oxygen
  binding. Unbound r state has a much higher
  affinity for oxygen, and this is the rational for
  cooperativity

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a. Free energy changes with fractional
saturation b. Sigmoidal binding curve as a
composite of the R state binding and the T state
binding.
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Hemoglobin function

• a2,b2 dimer which are structurally similar to
  myoglobin
• Transports oxygen from lungs to tissues.
• O2 diffusion alone is too poor for transport in
  larger animals.
• Solubility of O2 is low in plasma i.e. 10-4 M.
• But bound to hemoglobin, O2 0.01 M or that of
  air
• Two alternative O2 transporters are
• Hemocyanin, a Cu containing protein.
• Hemoerythrin , a non-heme containing protein.

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Myoglobin facilitates rapidly respiring muscle
tissue The rate of O2 diffusion from capillaries
to tissue is slow because of the solubility of
oxygen. Myoglobin increases the solubility of
oxygen. Myoglobin facilitates oxygen
diffusion. Oxygen storage is also a function
because Myoglobin concentrations are 10-fold
greater in whales and seals than in land mammals
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The Heme group

• Each subunit of hemoglobin or myoglobin contains
  a heme.
• Binds one molecule of oxygen
• Heterocyclic porphyrin derivative
• Specifically protoporphyrin IX

The iron must be in the Fe(II) form or reduced
form. (ferrous oxidation) state. Loss of
electrons oxidation LEO Gain of electrons
reduction GER Leo the lion says GER
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The visible absorption spectra for hemoglobin The
red color arises from the differences between the
energy levels of the d orbitals around the
Ferrous atom. There is an energy difference
between them, which determines the size of the
wavelength of the maximal absorbance band.
Fe(II) d6 electron configuration Low spin
state
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Binding of oxygen rearranges the electronic
distribution and alters the d orbital
energy. This causes a difference in the
absorption spectra. Bluish for deoxy Hb Redish
for Oxy Hb Measuring the absorption at 578 nM
allows an easy method to determine the percent of
Oxygen bound to hemoglobin.
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When Fe(II) goes to Fe(III), oxidized, it
produces methemoglobin which is brown and
coordinated with water in the sixth position.
Dried blood and old meat have this brown color.
Butchers use ascorbic acid to reduce
methemoglobin to make the meat look fresh!! There
is an enzyme methemoglobin reductase that
converts methemoglobin to regular hemoglobin.
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O2 binding to myoglobin
Written backwards we can get the dissociation
constant
Fractional Saturation solve for MbO2 and plug
in
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How do you measure the concentration of
oxygen? Use the partial pressure of O2 or O2
tension. pO2
P50 the partial oxygen pressure when YO2 0.50
What is the shape of the curve if you plot YO2
vs. pO2
What does the value of P50 tell you about the O2
binding affinity?
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P50 value for myoglobin is 2.8 torr or 1 torr 1
mm Hg 0.133 kPa 760 torr 1 atm of pressure Mb
gives up little O2 over normal physiological
range of oxygen concentrations in the tissue i.e.
100 torr in arterial blood 30 torr in
venous blood YO2 0.97 to YO2 0.91 What is the
P50 value for Hb? Should it be different than
myoglobin?
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The Hill Equation
E enzyme, S ligand, n small number
This is for binding of 1 or more ligands
O2 is considered a ligand
2.
1.
Fractional Saturation bound/total
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As we did before, combine 1. 2. 3.
3.
or
Look similar to Mb O2 except for the n
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Continuing as before
4.
n Hill Constant, a non integral parameter
relating Degree of Cooperativity among
interacting ligand-binding sites or subunits
The bigger n the more cooperativity (positive
value) If n 1, non-cooperative n negative cooperativity
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Hill Plot
Rearrange equation 4.
y
mx b n slope and x
intercept of -b/m
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Things to remember Hb subunits independently
compete for O2 for the first oxygen molecule to
bind When the YO2 is close to 1 i.e. 3 subunits
are occupied by O2 , O2 binding to the last site
is independent of the other sites However by
extrapolating slopes the 4th O2 binds to
hemoglobin 100 fold greater than the first O2 A
DDG of 11.4 kJmol -1 in the binding affinity for
oxygen When one molecule binds, the rest bind and
when one is released, the rest are released.
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Contrast Mb O2 binding to Hemoglobin
YO2 0.95 at 100 torr but 0.55 at 30 torr a
DYO2 of 0.40 Understand Fig 9-3 Hb gives up O2
easier than Mb and the binding is Cooperative!!
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Function of the globin
Protoporphyrin binds oxygen to the sixth ligand
of Fe(II) out of the plane of the heme. The
fifth ligand is a Histidine, F8 on the side
across the heme plane. His F8 binds to the
proximal side and the oxygen binds to the distal
side. The heme alone interacts with oxygen such
that the Fe(II) becomes oxidized to Fe(III) and
no longer binds oxygen.
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A heme dimer is formed which leads to the
formation of Fe(III)
Fe O O Fe
By introducing steric hindrance on one side of
the heme plane interaction can be prevented and
oxygen binding can occur.

• The globin acts to
• a. Modulate oxygen binding affinity
• b. Make reversible oxygen binding possible

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The globin surrounds the heme like a hamburger is
surrounded by a bun. Only the propionic acid
side chains are exposed to the solvent. Amino
acid mutations in the heme pocket can cause
autooxidation of hemoglobin to form
methemoglobin.
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The Bohr Effect
Higher pH i.e. lower H promotes tighter
binding of oxygen to hemoglobin and Lower pH i.e.
higher H permits the easier release of oxygen
from hemoglobin
Where n 0, 1, 2, 3 and x ? 0.6 A shift in the
equilibrium will influence the amount of oxygen
binding. Bohr protons
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Origin of the Bohr Effect
The T ? R transition causes the changes in the
pKs of several groups. The N-terminal amino
groups are responsible for 20 -30 of the Bohr
effect. His146b accounts for about 40 of the
Bohr effect salt bridged with Asp 94b. This
interaction is lost in the R state.
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To help you understand look at the relation
between pH and the P50 values for oxygen binding.
As the pH increases the P50 value decreases,
indicating the oxygen binding increases. The
opposite effect occurs when the pH decreases. At
20 torr 10 more oxygen is released when the pH
drops from 7.4 to 7.2!!
As oxygen is consumed CO2 is released. Carbonic
Anhydrase catalyzes this reaction in red blood
cells.
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About 0.8 mol of CO2 is made for each O2
consumed. Without Carbonic Anhydrase bubbles of
CO2 would form. The H generated from this
reaction is taken up by the hemoglobin and causes
it to release more oxygen. This proton uptake
facilitates the transport of CO2 by stimulating
bicarbonate formation.
R-NH2 CO2 ? R-NH-COO- H
Carbomates are formed with the interaction of CO2
with the N-terminal amino groups of proteins.
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About 5 of the CO2 binds to hemoglobin but this
accounts for the 50 of the exchanged CO2 from
the blood. As oxygen is bond in the lungs the
CO2 comes off.
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Lecture 3/12/2003 Protein Function II Globins
and Antibodies