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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.

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a picket fence fe ii porphyrin complex with bound o 2 prevents auto oxidation via dimerization
A picket-fence Fe(II)–porphyrin complex with bound O2 (prevents auto-oxidation via dimerization)
effect of ph on the o 2 dissociation curve of hb the bohr effect
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

slide7

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

comparison of the o 2 dissociation curves of stripped hb and whole blood in 0 01 m nacl at ph 7 0

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.
slide9

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).

slide10
The effect of high-altitude exposure on the p50 and the BPG concentration of blood in sea level–adapted individuals.
slide11
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 8

helices: A-H

Contains some

310 helices

Subunits of Hb

are similar to Mb

Structure of sperm whale myoglobin (Mb)
stereo drawings of the heme complex in oxymb

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.
the x ray structure of deoxyhb as viewed down its exact 2 fold axes

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.
the x ray structure of oxyhb 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.
the major structural differences between the quaternary conformations of a deoxyhb and b oxyhb

Oxygenation rotates

thea1-b1 dimer by 15o with

respect to the a2-b2dimer;

two-fold symmetry

is maintained

4o forms:

deoxyHb = Tstate (tense)

oxyHb = Rstate (relaxed)

The major structural differences between the quaternary conformations of (a) deoxyHb and (b) oxyHb
the heme group and its environment in the unliganded a chain of human hb

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.
networks of salt bridges and hydrogen bonds in deoxyhb a last two residues of the a chains

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.
free energy and saturation curves for o 2 binding to hemoglobin

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.

Reaction of cyanate with the unprotonated (nucleophilic) forms of primary amino groups.
binding of bpg to deoxyhb selective stabilization of the t form

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
slide28

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.

slide29
Mutations stabilizing the Fe(III) oxidation state of heme. (b) The structure of the heme pocket of the b subunit in Hb Milwaukee.
electron micrograph of deoxyhbs fibers spilling out of a ruptured erythrocyte

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 in diameter fibers of deoxyhbs a n electron micrograph of a negatively stained fiber
220-Å in diameter fibers of deoxyHbS: an electron micrograph of a negatively stained fiber
slide34

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.
slide35

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.

slide36

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).
slide37

Implies a 30th power

concentration dependence

Time course of deoxyHbS gelation: a log–log plot showing the concentration dependence of 1/tdfor the gelation of deoxyHbS at 30°C.
the species and reactions permitted under the symmetry model of allosterism

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
the sequential model of allosterism

Koshland, Nemethy, Filmer

Binding to T-state induces conformational changes in

unliganded subunits (intermediate affinity between T and R)

The sequential model of allosterism
slide43
The sequential and the symmetry models of allosterism can provide equally good fits to the measured O2-dissociation curve of Hb.
slide44

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.