Transcript Figure 10-1 The heme group.

The heme group.
The visible absorption spectra of oxygenated
and deoxygenated hemoglobins.
Oxygen dissociation curves of Mb and of Hb in whole blood.
Hill plots for Mb and purified (“stripped”) Hb.
A picket-fence Fe(II)–porphyrin complex with bound O2
(prevents auto-oxidation via dimerization)
Effect of pH on the O2-dissociation curve of Hb: the Bohr effect.
Hb(O2)nHx + O2  Hb(O2)n+1 + xH+
x ≈ 0.6
CO2 + H2O  H+ + HCO3-
catalyzed by carbonic anhydrase in erythrocytes
Carbamate Formation (N-termini)
R-NH2 + CO2  R-NH-COO- + H+
DeoxyHb binds more CO2 as carbamate
than does oxyHb
The effect of
2,3-BPG on
Hb oxygen
affinity
Comparison of the O2-dissociation curves of “stripped” Hb and
whole blood in 0.01M NaCl at pH 7.0.
The effects of 2,3-BPG and CO2, both separately and
combined, on hemoglobin’s O2-dissociation curve compared
with that of whole blood (red curve).
The effect of high-altitude exposure on the p50 and the BPG
concentration of blood in sea level–adapted individuals.
The O2-dissociation curves of blood adapted to sea level (black
curve) and to high altitude (red curve).
Contains 8
helices: A-H
Contains some
310 helices
Subunits of Hb
are similar to Mb
Structure of sperm whale myoglobin (Mb)
The Amino Acid Sequences of the a and b Chains of Human
Hemoglobin and of Human Myoglobin
The Amino Acid Sequences of the a and b Chains of Human
Hemoglobin and of Human Myoglobin
Heme located in
a hydrophobic pocket
formed mainly by
helices E and F
Fe(II) is 0.22 Å out
of the heme plane in
oxyMb on the proximal
His side; O2 in bent
geometry
Fe(II) is 0.55 Å out
of plane in deoxyMb
Structures of oxyMb and
deoxyMb are superimposable
Stereo drawings of the heme complex in oxyMb.
Contains two
ab protomers
Tertiary structures
of a and b subunits
are similar to each
other and to Mb
There is extensive
interactions between
unlike subunits
(a1-b1 and a2-b2);
hydrophobic
in character
Contacts between like
subunits few and polar
The X-ray structure of deoxyHb as viewed down its
exact 2-fold axes.
Extensive quaternary
structural changes occur
to Hb upon oxgenation
Changes occur at the
a1-b2 and a2-b1 interfaces
The X-ray structure of oxyHb as viewed down its exact 2-fold axes.
Oxygenation rotates
the a1-b1 dimer by 15o with
respect to the a2-b2 dimer;
two-fold symmetry
is maintained
4o forms:
deoxyHb = T state (tense)
oxyHb = R state (relaxed)
The major structural differences between the quaternary
conformations of (a) deoxyHb and (b) oxyHb
Explaining
cooperativity:
Perutz mechanism
(based on X-ray analyses)
Note out-of-plane
Fe(II) in deoxyHb;
ion moves in-plane in
oxyHb, and pulls
on the proximal
His; F helix is moved
The heme group and its environment in the unliganded
a chain of human Hb.
Triggering mechanism for the T  R transition in Hb
(T = blue; R = pink)
No stable intermediate
states are allowed:
a binary switch
The a1C–b2FG interface of Hb in (a) the T state and (b) the R state.
black: deoxyHb
blue: oxyHb
The hemoglobin a1b2 interface as viewed
perpendicularly to Fig. 10-13.
Salt bridges
must break in T to
R transition
Val-1 on a2:
Bohr effect
Networks of salt bridges and hydrogen bonds in deoxyHb.
(a) Last two residues of the a chains.
His-146 on b2:
Bohr effect
Networks of salt bridges and hydrogen bonds in deoxyHb.
(b) Last two residues of the b chains.
Relative free energies of the
T and R states vary with
fractional saturation
Overall binding curve for Hb
is a composite of the hyperbolic
binding curves for pure T and R
Free energy and saturation curves for O2 binding to hemoglobin
Reaction of cyanate with the unprotonated (nucleophilic) forms of
primary amino groups.
Hb with carbamoylated a subunits (N-terminal amino
groups) lacks 20-30% of the Bohr effect.
BPG binding pocket is lined
with positive charge
(Lys, His, N-termini):
complementary to BPG’s
negative charge
BPG preferentially
binds to deoxyHb: central
cavity is smaller in oxyHb
Binding of BPG to deoxyHb: selective stabilization of the T form
Abnormal Hemoglobins:
Hemoglobinopathies 860 variant Hbs in humans
Mutations stabilizing the Fe(III) oxidation state of heme. (a)
Alterations in the heme pocket of the a subunit on changing from
deoxyHbA to Hb Boston.
Mutations stabilizing the Fe(III) oxidation state of heme. (b) The
structure of the heme pocket of the b subunit in Hb Milwaukee.
Sickle-Cell
Anemia: HbS
Single-site
mutation:
Valine replaces
Glu A3(6)b
Electron micrograph of deoxyHbS fibers spilling out of a
ruptured erythrocyte.
220-Å in diameter fibers of deoxyHbS: an electron
micrograph of a negatively stained fiber
220-Å in diameter fibers of deoxyHbS: a
model, viewed in cross section, of the HbS
fiber.
Structure of the deoxyHbS fiber: arrangement of the
deoxyHbS molecules in the fiber.
Intermolecular association
Val 6 involving b2;
Val 6 of b1 - pocket
Structure of the deoxyHbS fiber: a schematic diagram indicating
the intermolecular contacts in the crystal structure of deoxyHbS.
Molecular
Basis for
Fibril Formation
In HbS
Structure of the deoxyHbS fiber: the mutant Val 6b2 fits neatly into
a hydrophobic pocket formed mainly by Phe 85 and Leu 88 of an
adjacent b1 subunit.
Note delay, td
1/td = k(ct/cs)n: concentration
dependence of the delay time
Time course of deoxyHbS gelation: the extent of gelation as
monitored calorimetrically (yellow) and optically (purple).
Implies a 30th power
concentration dependence
Time course of deoxyHbS gelation: a log–log plot showing the
concentration dependence of 1/td for the gelation of deoxyHbS at
30°C.
Double nucleation mechanism for deoxyHbS gelation
Allosteric regulation:
two general models
Monod, Wyman, Changeux:
symmetry model
conformational change alters
affinity for ligand: molecular
symmetry conserved
The species and reactions permitted under the
symmetry model of allosterism
Models of ligand binding
The sequential model of allosterism
Koshland, Nemethy, Filmer
Binding to T-state induces conformational changes in
unliganded subunits (intermediate affinity between T and R)
Ligand affinity varies with number of bound ligands;
intermediate conformations
Sequential binding of ligand in the sequential model of allosterism
The sequential and the symmetry models of allosterism can provide
equally good fits to the measured O2-dissociation curve of Hb.
More complex
model of Hb
allosterism
Free energy penalties for binding O2 to various ligation states of Hb
tetramers relative to O2-binding to noncooperative Hb ab dimers.
END
Adair Constants for Hemoglobin A at pH 7.40.