Protein Function
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
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.
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.
Quaternary structure of deoxy- and oxyhemoglobin
Oxygenation rotates the 11 dimer in relation to 22 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?
The positive cooperativity of O
2binding 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.
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
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
•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
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.
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
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
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
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
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!!
O
2binding to myoglobin
2
2
MbO
O
Mb
]
[MbO
]
[Mb][O
Kd
2 2
]
[O
Kd
]
[O
]
[MbO
[Mb]
]
[MbO
Y
2 2 2 2 O2
Written backwards we can get the dissociation constant
How do you measure the concentration of oxygen?
Use the partial pressure of O2 or O2 tension. = pO2
2 d 2 O
pO
K
pO
Y
2
P50 = the partial oxygenpressure when YO2 = 0.50
2 50 2 O
pO
P
pO
Y
2
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?
The Hill Equation
E = enzyme, S = ligand, n= small number
ESn
nS
E
This is for binding of 1 or more ligandsO
2is considered a ligand
ESn]
[
[E][S]
K
n
1.
n
[E]
[ESn]
n[ESn]
Ys
2.
As we did before, combine
1. + 2. = 3.
K
S]
[
1
]
E
[
K
[E][S]
Ys
n n
or n n[S]
K
[S]
Ys
Look similar to Mb + O2 except for the n
Continuing as before:
n50
P
K
n2 n 50 n 2 O
pO
P
pO
Y
2
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
Hill Plot
Rearrange equation 4.
logK
nLog[S]
Ys
-1
Ys
Log
y = mx + b
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
Contrast Mb O
2binding to Hemoglobin
YO2 = 0.95 at 100 torr
but
0.55 at 30 torr
a YO2 of 0.40
Understand Fig 9-3
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
Fe O O Fe
A heme dimer is formed 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
The globin surrounds the heme like a hamburger is surrounded by a bun. Only the propionic acid side chains are exposed to the solvent.
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
O
Hb
O
xH
H
O
Hb
2 n x 2 2 n 1Where n = 0, 1, 2, 3 and x 0.6 A shift in the equilibrium
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
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.
-3 2
2
H
O
H
HCO
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-NH
2+ CO
2
R-NH-COO
-+ H
+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
