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

R-NH2 + CO2 R-NH-COO- + H+

Carbamate Formation (N-termini)

DeoxyHb binds more CO2 as carbamatethan does oxyHb

Comparison of the O2-dissociation curves of “stripped” Hb and whole blood in 0.01M NaCl at pH 7.0.

The effect of2,3-BPG onHb oxygen

affinity

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).

Structure of sperm whale myoglobin (Mb)

Contains 8helices: A-H

Contains some 310 helices

Subunits of Hbare similar to Mb

The Amino Acid Sequences of the and Chains of Human Hemoglobin and of Human Myoglobin

The Amino Acid Sequences of the and Chains of Human Hemoglobin and of Human Myoglobin

Stereo drawings of the heme complex in oxyMb.

Heme located ina hydrophobic pocket

formed mainly by helices E and F

Fe(II) is 0.22 Å outof the heme plane in

oxyMb on the proximalHis side; O2 in bent

geometry

Fe(II) is 0.55 Å outof plane in deoxyMb

Structures of oxyMb anddeoxyMb are superimposable

The X-ray structure of deoxyHb as viewed down its exact 2-fold axes.

Contains two protomers

Tertiary structuresof and subunitsare similar to each

other and to Mb

There is extensiveinteractions between

unlike subunits (1-1 and 2-2);

hydrophobicin character

Contacts between likesubunits few and polar

The X-ray structure of oxyHb as viewed down its exact 2-fold axes.

Extensive quaternarystructural changes occurto Hb upon oxgenation

Changes occur at the1-2 and 2-1 interfaces

The major structural differences between the quaternary conformations of (a) deoxyHb and (b) oxyHb

Oxygenation rotatesthe 1-1 dimer by 15o withrespect to the 2-2 dimer;

two-fold symmetryis maintained

4o forms:deoxyHb = T state (tense)oxyHb = R state (relaxed)

The heme group and its environment in the unliganded chain of human Hb.

Explainingcooperativity:

Perutz mechanism(based on X-ray analyses)

Note out-of-planeFe(II) in deoxyHb;

ion moves in-plane inoxyHb, and pulls on the proximal

His; F helix is moved

Triggering mechanism for the T R transition in Hb (T = blue; R = pink)

The 1C–2FG interface of Hb in (a) the T state and (b) the R state.

No stable intermediatestates are allowed:

a binary switch

The hemoglobin12 interface as viewed perpendicularly to Fig. 10-13.

black: deoxyHbblue: oxyHb

Networks of salt bridges and hydrogen bonds in deoxyHb. (a) Last two residues of the chains.

Salt bridgesmust break in T to

R transition

Val-1 on 2:Bohr effect

Networks of salt bridges and hydrogen bonds in deoxyHb. (b) Last two residues of the chains.

His-146 on 2:Bohr effect

Free energy and saturation curves for O2 binding to hemoglobin

Relative free energies of theT and R states vary with

fractional saturation

Overall binding curve for Hbis a composite of the hyperbolicbinding curves for pure T and R

Reaction of cyanate with the unprotonated (nucleophilic) forms of primary amino groups.

Hb with carbamoylated subunits (N-terminal aminogroups) lacks 20-30% of the Bohr effect.

Binding of BPG to deoxyHb: selective stabilization of the T form

BPG binding pocket is linedwith positive charge (Lys, His, N-termini):

complementary to BPG’snegative charge

BPG preferentiallybinds to deoxyHb: centralcavity is smaller in oxyHb

Mutations stabilizing the Fe(III) oxidation state of heme. (a) Alterations in the heme pocket of the subunit on changing from deoxyHbA to Hb Boston.

Abnormal Hemoglobins:Hemoglobinopathies -

860 variant Hbs in humans

Mutations stabilizing the Fe(III) oxidation state of heme. (b) The structure of the heme pocket of the subunit in Hb Milwaukee.

Electron micrograph of deoxyHbS fibers spilling out of a ruptured erythrocyte.

Sickle-CellAnemia: HbS

Single-sitemutation:

Valine replacesGlu A3(6)

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.

Structure of the deoxyHbS fiber: a schematic diagram indicating the intermolecular contacts in the crystal structure of deoxyHbS.

Intermolecular associationVal 6 involving 2;Val 6 of 1 - pocket

Structure of the deoxyHbS fiber: the mutant Val 62 fits neatly into a hydrophobic pocket formed mainly by Phe 85 and Leu 88 of an adjacent 1 subunit.

MolecularBasis for

Fibril FormationIn HbS

Time course of deoxyHbS gelation: the extent of gelation as monitored calorimetrically (yellow) and optically (purple).

Note delay, td

1/td = k(ct/cs)n: concentration dependence of the delay time

Time course of deoxyHbS gelation: a log–log plot showing the concentration dependence of 1/td for the gelation of deoxyHbS at 30°C.

Implies a 30th powerconcentration dependence

Double nucleation mechanism for deoxyHbS gelation

The species and reactions permitted under the symmetry model of allosterism

Allosteric regulation: two general models

Monod, Wyman, Changeux:symmetry model

conformational change altersaffinity for ligand: molecular

symmetry conserved

Models of ligand binding

The sequential model of allosterism

Koshland, Nemethy, Filmer

Binding to T-state induces conformational changes inunliganded subunits (intermediate affinity between T and R)

Sequential binding of ligand in the sequential model of allosterism

Ligand affinity varies with number of bound ligands; intermediate conformations

The sequential and the symmetry models of allosterism can provide equally good fits to the measured O2-dissociation curve of Hb.

Free energy penalties for binding O2 to various ligation states of Hb tetramers relative to O2-binding to noncooperative Hb dimers.

More complexmodel of Hballosterism

END

Adair Constants for Hemoglobin A at pH 7.40.