Hemoglobin
Tetrameric protein
Found in red blood cells.
Main function: O2 transport
Hemoglobin monomers contain:
•heme prosthetic group that
contains iron
•globin polypeptide chain.
The presence of the heme group
is what gives blood cells their
characteristic color.
It is the heme group that binds
oxygen.
Globin polypeptide: optimal
environment for heme function.
•Globin - polypeptide chain
Globin fold
– 75% 〈-helix (8 helices, A- H)
– Adults have two major subtypes,
〈-globin, and -globin
– Adult hemoglobin contains:
• two 〈-globin poly-peptide chains
• two -globin poly-peptide chains
– Other globin subtypes are expressed
during development. The expression
of globin subtypes is developmentally
regulated.
〈-globin
-globin
4
Heme
•Planar prosthetic group
•Purpose: oxygen binding
Heme is a:
Fe-containing porphyrin
Two main components:
1) organic:
protoporphyrin IX
2) inorganic:
central iron ion:
Ferric Fe3+
Ferrous Fe2+
7
4 coordination sites
One at each pyrrole N
Polar propionates face out of the oxygen binding cleft
Non-polar vinyls face in toward the hydrophobic binding cleft
Only two polar residues in the binding cleft: Both Histidines
Distal
Proximal
Dioxygen and Superoxide Ion
Ferrous Iron
Ferric Iron
Upon binding dioxygen, an unpaired electron of
oxygen oxidizes the ferrous iron to ferric iron and
oxygen is reduced to superoxide.
This results in no net unpaired electrons and a
thus a lower energy state.
Resonance Structure of Dioxygen and Superoxide Ion
Ferrous Iron
Ferric Iron
Dioxygen
O2 released as dioxygen, not superoxide.
Superoxide release:
Release of highly reactive oxygen species
Production of Fe3+ which cannot bind O2.
Dioxygen and Superoxide Ion: Leaving as O2
Ferrous Iron
Ferric Iron
Dioxygen
The environment stabilizes oxygen.
The distal histidine
reduces chance of
superoxide release, and
acts as a gate keeper for
the iron binding site
lowering the possibility of
CO binding
Heme in deoxyhemoglobin is non-planar. When oxygen binds
to Fe2+, and an electron is partially transferred from ferrous
iron to oxygen, this results in Fe3+ and .O2- (superoxide). This
changes the organization of electrons in iron, causing the iron
atom to become smaller. Ferric iron can now enter the center
of the porphyrin ring. With oxygen bound and iron in the
ferric oxidation state, the heme group is planar.
Proximal
Histidine
The Oxidation State of Iron
Oxyhemoglobin = Hemoglobin with O2 bound: Fe3+
De-oxyhemoglobin = Hemoglobin with no O2 bound: Fe2+
Methemoglobin = Hemoglobin with no O2 bound: Fe3+
Because of the oxidation state of iron, methemoglobin cannot
bind O2, as Fe2+ is required for oxygen binding.
At any time there is a very small (less than 2%) of
hemoglobin is methemoglobin, an increase in this
proportion is not good.
Myoglobin Oxygen
Saturation Curve
Y = the fraction of
possible oxygen
binding sites filled, on
a scale of 0-1, where
1 = 100% of sites
filled.
P50 = The partial pressure of oxygen at which 50% of
available binding sites are filled
The oxygen binding curve of myoglobin indicates simple
equilibrium.
The oxygen binding curve for hemoglobin is sigmoidal, not
reflecting simple equilibrium but allostery,cooperativity
23
Tense State
Relaxed State
Oxygen bound
Tense State
Relaxed State
Upon oxygen binding, 〈11 rotates 15 degrees relative to
〈22. This changes intermolecular interactions between 〈11
and 〈22 , increasing affinity for oxygen.
Relaxed State
Hyperbolic curves reflect a simple chemical equilibrium
Sigmoidal curves are indicative of more complicated
kinetics, in which allostery has an effect.
Allostery, - in this case, refers to the fact that oxygen binding
to one subunit, causes the binding of oxygen to another
subunit of the same tetramer to be more energetically
favorable.
Oxygen binding to one hemoglobin subunit increases the
oxygen affinity of other subunits in the same tetramer.
This is cooperative binding.
Hemoglobin
without 2,3-BPG
is greedy. It binds
oxygen very well,
but does not
release it to the
tissues well at all,
and appears like a
myoglobin
saturation curve.
The small molecule, 2,3,-BPG, stabilizes interactions
between the 4 subunits favoring the tense state, and
oxygen release to the tissues.
-globin has a serine in place of histidine 143 in the. This
chain binds 2,3,-BPG less tightly and so fetal Hb (HbF) has a
higher O2 affinity than HbA.
His143 of -globin is
replaced by a Serine in
-globin. Fetal Hb has
a lower affinity for
2,3-BPG. Fetal Hb
(2〈21) has a higher
affinity for O2 than adult
Hb (2〈2 ).
Protonation of histidine residues in the
globin carboxyl termini occurs at
decreased blood pH as seen during
high metabolic conditions. This
protonation introduces increased
interactions between C-terminal
histidines and a specific aspartate that
favor the tense state, oxygen release.
Salt bridge (ionic interaction)
Increase in [CO2], decrease in pH
Both shift the O2 saturation curve to the right.
Amino terminal alpha amino group + CO2
R-NHCO2-
Bohr effect
Blood in tissue capillaries: High CO2, Low pH
CO2 transported into RBC
H2O + CO2 <=>H2CO3 (carbonic anhydrase catalyzed rxn)
H2CO3 dissociation => HCO3- + H+
HCO3- out of RBC, Cl- in, by way of anti-porter
HCO3- transported in serum to capillaries in lungs
Majority CO2 transport: HCO3-, Minor: CO2(d), carbamate
Bohr effect
In alveoli: low CO2
1. HCO3- transported into RBC, Cl- out, by way of anti-porter
2. HCO3- + H+ <=> H2CO3
3. H2CO3 <=> H2O + CO2 (carbonic anhydrase catalyzed)
4. CO2 transported from RBC
• CO2 exhaled.
Small changes in primary sequence
can have a large effect on protein
function: HbS
Glutamate to Valine substitution
Micrograph showing
RBC rupture, and
leaking of rod-shaped
HbS multimers.
Sickeling can occur
in response to low
oxygen saturation
or high Hb
concentration as
both will increase
the pool of
deoxyHb
Plasmodia infected red cells, showing
rupture of RBC membrane