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Structures of Myoglobin and
Hemoglobin
• Myoglobin (Mb) - monomeric protein that
facilitates the diffusion of oxygen in
vertebrates
• Hemoglobin (Hb) - tetrameric protein that
carries oxygen in the blood
• Heme consists of a tetrapyrrole ring system
called protoporphyrin IX complexed with iron
• Heme of Mb and Hb binds oxygen for
transport
Heme Fe(II)-protoporphyrin IX
Protein component of Mb and
Hb is globin
• Myoglobin is composed of 8 a helices
• Heme prosthetic group binds oxygen
• His-93 is complexed to the iron atom, and
His-64 forms a hydrogen bond with oxygen
• Interior of Mb almost all hydrophobic amino
acids
• Heme occupies a hydrophobic cleft formed by
three a helices and two loops
Sperm whale oxymyoglobin
Hemoglobin (Hb)
• Hb is an a2b2 tetramer (2 a globin subunits,
2 b globin subunits)
• Each globin subunit is similar in structure to
myoglobin
• Each subunit has a heme group
• The a chain has 7 a helices, b chain has
8 a helices
Hemoglobin tetramer
(a) Human oxyhemoglobin (b) Tetramer schematic
Oxygen Binding to Mb and Hb
• Oxymyoglobin - oxygen bearing
myoglobin
• Deoxymyoglobin - oxygen-free myoglobin
• In oxymyoglobin, six ligands are
coordinated to the ferrous ion in octahedral
symmetry
• Oxygen is coordinated between the iron
and the imidazole sidechain of His-64
Oxygen-binding site of
whale oxymyoglobin
• Octahedral geometry of
coordination complex (six
ligands around iron)
• His-93 (proximal histidine)
liganded to Fe
• His-64 (distal histidine)
Oxygen-binding curves
(a) Comparison of O2binding to Mb and Hb
Oxygen-Binding Curves of
Myoglobin and Hemoglobin
• Curves show reversible binding of O2 to Mb
and Hb
• Fractional saturation (Y) is plotted versus the
partial pressure of oxygen, pO2 (oxygen
concentration)
• The shape of the Hb curve shows a positive
cooperativity in the binding of 4 O2 molecules
(i.e. the O2 affinity of Hb increases as each O2
molecule is bound)
Oxygen-binding curves
(a) Comparison of O2binding to Mb and Hb
O2 binding curves (continued)

Mb-O2 binding curve is hyperbolic, indicating
a single equilibrium constant for binding O2

Hb-O2 binding curve is sigmoidal, and reflects
the binding of 4 molecules of O2, one per each
heme group
Oxygen-binding curves
(a) Comparison of O2binding to Mb and Hb
Oxygen-binding curves
Binding of the R
(high-affinity) and T
(low affinity) forms
of Hb
Conformational changes in a
hemoglobin chain induced by
oxygenation
• Oxygen binding
to Fe pulls the His
toward ring plane
• Helix with His
shifts position,
disrupting some
ion pairs
between subunits
(blue to red position)
Oxygen-binding site of
whale oxymyoglobin
• Octahedral geometry of
coordination complex (six
ligands around iron)
• His-93 (proximal histidine)
liganded to Fe
• His-64 (distal histidine)
Hemoglobin is an Allosteric
Protein
• Oxygen binding and release from Hb are
regulated by allosteric interactions
• Allosteric effectors (modulators) bind to
a protein at a site separate from the
functional binding site (may be activators or
inhibitors)
• The activity of an allosteric protein is
regulated by allosteric effectors
Two conformations of
hemoglobin: T and R
• Active (R state) and inactive (T state)
forms are in rapid equilibrium in allosteric
proteins
• Binding of substrates and allosteric
activators stabilize the R state and shift the
equilibrium in the R direction
• Allosteric inhibitors stabilize the T state and
shift the equilibrium in the T direction
Bisphospho-D-glycerate
(2,3BPG)
• 2,3BPG is an allosteric effector of Hb
• 2,3BPG lowers the affinity of deoxyHb for
oxygen (raises the P50 of Hb from ~12 to
~26 torr)
• Negatively charged 2,3BPG is bound to six
(+) charged groups of deoxyhemoglobin
Bisphospho-D-glycerate
(2,3BPG)
Binding of 2,3BPG to
deoxyhemoglobin
• (-) Charges on
2,3BPG pair
with (+) charges
lining the central cavity,
stabilizing the
DeoxyHb form
• a-Subunits pink,
b-subunits blue,
heme groups red
Oxygen-binding curves
(a) Comparison of O2binding to Mb and Hb
Bohr effect
• Lowering the pH
decreases
the affinity of Hb
for oxygen
Carbamate adduct
• Carbon dioxide is transported
from the tissues to the lungs in
two ways:
(1) Dissolved bicarbonate ions
(2) Carbamate adducts of
hemoglobin (N-terminal globin
residues react with CO2 to form
carbamates)
Review of Relevant Parameters
(1) Low P50 indicates high O2 affinity
(2) Low pH (through CO2 intake) stabilizes 2,3BPG and lowers O2 affinity
(3) Raising P50 causes unloading of O2
Oxy-Hb
Deoxy-Hb
(R)
(T)
Case Studies
Shock victims are given intravenous HCO3Why?
HCO3- generates CO2 to the tissues
and lowers the O2 affinity of Hb,
thus releases O2 from HbO2 to the tissues
Case Studies
Fetal Hb (HB-F) contains ser in place of the cationic his
at position 143 of the b chains of adult Hb (HB-A).
Residue 143 faces the central cavity between the b chains
Outcomes:
his in Hb-A is protonated and thus binds more tightly to
negatively charged 2,3-BPG;
ser in Hb-F is not protonated and does not bind to 2,3-BPG as
strongly; thus Hb-F has a greater fraction of HbO2
Case Studies
Fetal Hb (HB-F) contains ser in place of the cationic his
at position 143 of the b chains of adult Hb (HB-A).
Residue 143 faces the central cavity between the b chains
Outcomes:
Hb-F has a greater fraction of HbO2 which means greater O2
affinity and lower P50 (18 torr)
Since average P50 for Hb-A is 26 torr, oxygen can efficiently
be transferred from maternal blood to fetus
Antibodies Bind Specific
Antigens
• Vertebrate immune systems synthesize
protein antibodies (immunoglobulins) to
eliminate bacteria, viruses, other foreign
substances
• Antibodies specifically recognize and bind
antigens
• Antibodies are synthesized by lymphocytes
(white blood cells)
Human antibody structure
• Heavy chains
(blue)
and light chains
(red)
• Disulfide bonds
(yellow)
• Variable domains
colored darker
Stereo view of the
immunoglobin fold
• Two antiparallel b sheets linked by nonrepetitive segments
Binding of three different
antibodies to an antigen