Protein Function

Globins and Antibodies

3/10/2003
 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
 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.
The Backbone structure of Myoglobin 4
The Heme complex in myoglobin
 Hemoglobin

Spherical 64 x 55 x 50 Å two fold rotation of

symmetry  and  subunits are similar and are placed
on the vertices of a tetrahedron. There is no D helix in
the  chain of hemoglobin. Extensive interactions
between unlike subunits 2-2 or 1-1 interface
has 35 residues while 1-2 and 2-1 have 19
residue contact.
Oxygenation causes a considerable structural
conformational change
Quaternary structure of deoxy- and oxyhemoglobin

T-state R-state
Oxygenation rotates the 11 dimer in relation to 22
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?
 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.
Binding of the oxygen on one heme is more difficult
but its binding causes a shift in the 1-2 contacts
and moves the distal His E7 and Val E11 out of the
oxygen’s path to the Fe on the other subunit. This
process increases the affinity of the heme toward
oxygen.

The 1-2 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
 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 1C-2FG contact one turn
along the 1C helix.
•The inflexibility of the 1-1 and the 2-2 interfaces requires
that this shift simultaneously occur at both the 1-2 and 2-1
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
a. Free energy changes with fractional saturation
b. Sigmoidal binding curve as a composite of the R
state binding and the T state binding.
 Hemoglobin function
2,2 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.
 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
 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
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
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.
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.
 O2 binding to myoglobin
Written backwards we can
Mb  O 2  MbO 2 get the dissociation constant

[Mb][O 2 ]
Kd 
[MbO 2 ]
Fractional Saturation solve for [MbO2] and plug in

[MbO 2 ] [O 2 ]
YO 2  
[Mb]  [MbO 2 ] Kd  [O 2 ]
How do you measure the concentration of oxygen?
Use the partial pressure of O2 or O2 tension. = pO2

pO 2
YO 2  P50 = the partial oxygen
K d  pO 2 pressure when YO2 = 0.50

pO 2
YO 2  What is the shape of the
P50  pO 2 curve if you plot YO2 vs.
pO2

What does the value of P50 tell you about the O2 binding
affinity?
 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?
 The Hill Equation

E = enzyme, S = ligand, n= small number

E  nS  ESn This is for binding of 1 or

more ligands
O2 is considered a ligand

[E][S] n n[ESn]
1. K  2. Ys 
n  [E]  [ESn]
[ESn]
Fractional Saturation = bound/total
As we did before, combine 1. + 2. = 3.

n
[E][S] n
K [S]
Ys  3. Ys 
[E]

1  [S] n
 or K  [S] n

Look similar to Mb + O2 except for the n

 Continuing as before:

 pO 2 
n

K   P50  
n
4. YO 2
 P50    pO 2 
n n

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 < 1, negative cooperativity
 Hill Plot
Rearrange equation 4.

 Ys 
Log   nLog[S]  logK
 1 - Ys 
y = mx + b

n = slope and x intercept of -b/m

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 G of 11.4 kJ•mol -1 in the binding affinity for oxygen
When one molecule binds, the rest bind and when one is
released, the rest are released.
Contrast Mb O2 binding to Hemoglobin

YO2 = 0.95 at 100 torr

but

0.55 at 30 torr
a YO2 of 0.40
Understand Fig 9-3

Hb gives up O2 easier than Mb and the binding is

Cooperative!!
 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.
 A heme dimer is formed
Fe O O Fe which leads to the
formation of Fe(III)

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
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.
 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

Hb O 2  n H x  O 2  Hb O 2  n 1  xH 

Where n = 0, 1, 2, 3 and x  0.6 A shift in the equilibrium

will influence the amount of oxygen binding. Bohr protons
 Origin of the Bohr Effect

The T  R transition causes the changes in the pK’s

of several groups. The N-terminal amino groups are
responsible for 20 -30% of the Bohr effect. His146
accounts for about 40% of the Bohr effect salt
bridged with Asp 94. This interaction is lost in the
R state.
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!!

 -
CO 2  H 2 O  H  HCO3
As oxygen is consumed CO2 is released. Carbonic
Anhydrase catalyzes this reaction in red blood cells.
 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.
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.
 Lecture 3/12/2003
Protein Function II
Globins and Antibodies