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Regulation of O2 binding
and release

Structure is key to function

Structure without functional meaning is useless

Example of function we will discuss here: binding
Myoglobin (Mb): binding and storage of oxygen
Hemoglobin (Hb): binding and transport of of oxygen, as well
as CO2
Each erythrocyte contains about 300 millions Hb molecules
Both Mb and Hb are developed to bind the same molecule,
but with a different purpose
Also called Antibobies (Ab): developed to bind a multitude of
molecules, antigens, to regulate immuno-response

Prof. G. Gilardi - Biological Chemistry

Globins: Mb and Hb

Myoglobin: monomeric
Hemoglobin: tetrameric,
each chain resembles
How can nearly identical
structures perform such
different functions
(storage versus

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The O2 binding site

O2 is bound very efficiently by

transition metals, such as Cu(I) or
For this reason proteins have
learned to incorporate prosthetic
groups, such as heam;
In Mb and Hb the Fe(II) is
chelated by a tetrapyrrole ring
system, the protoporphyrin IX
This is strongly colored, red when
Fe(II), green when Mg(II) as in
Mb and Hb contain haem b-type

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Il pirrolo un composto aromatico eterociclico avente formula bruta C4H5N.
A temperatura ambiente un liquido giallo, infiammabile, dall'odore tenue.
insolubile in acqua, ma solubile in etanolo e nei pi comuni solventi organici.
La molecola del pirrolo ciclica e planare ed ha la forma di un pentagono
grossomodo regolare. L'anello composto da 4 atomi di carbonio ed uno di
azoto; aromatico perch attraverso gli orbitali p dei cinque atomi vengono
condivisi 6 elettroni: uno da ciascun atomo di carbonio e due dall'azoto.
Essendo il doppietto elettronico dell'azoto impegnato nell'anello aromatico,
questi non disponibile per essere ceduto a specie acide, quindi il pirrolo
mostra una basicit molto bassa.
In natura il pirrolo spesso parte di sistemi aromatici pi complessi quali, ad
esempio, le porfirine dell'emoglobina e della clorofilla o la vitamina B12.

Prof. G. Gilardi - Biological Chemistry

Prof. G. Gilardi - Biological Chemistry
Haem in Mb and Hb
Haem b is bound non-covalently in a
highly hydrophobic pocket of the protein;
In both the oxygenated and non-
oxygenated forms of the protein, the Fe(II)
is coordinated by the proximal (in direct
contact) His 93 in Mb = 5th ligand
In deoxy-myoglobin the 6th ligand is H2O
In oxy-myoglobin the 6th ligand is O2 that
is sandwiched between the Fe(II) and the
distal His 64
The equivalent ligands are found in Hb
Normally O2 bound to Fe(II) would oxidise
it to Fe(III): this happens with heam free in
solution the role of the protein through
its hydrophobic pocket is to protect the
haem so that O2 can be delivered as
Fe(II) can also bind similar small
molecules with similar electronic
configuration: CO and CN bind with much
higher affinity than O2 and this is why they
are poisons! Prof. G. Gilardi - Biological Chemistry
O2 binding to Mb
Henrys law: [O2] = PO2 partial O2 pressure;
Binding curve: dependence of O2 binding () on PO2
Association const. K, or affinity const. is also an
equilibrium const.:
Mb + O2 MbO2
K = [MbO2]/[Mb][O2]
Since the fraction () of sites with bound O2 is:
= n. sites occupied / total n. sites
= [MbO2]/([Mb] + [MbO2])
= K[Mb][O2]/([Mb] + K[Mb][O2])
= K[O2]/(1 + K[O2])
=[O2]/([O2]1/2 + [O2])
That is:
=PO2/(P50 + PO2)
This equation describes the hyperbolic function of the
figure on the left
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Dettaglio derivazione matematica

Mb + O2 MbO2
Ma per
K = [MbO2]/[Mb][O2]
quindi [MbO2]= K [Mb][O2]
= 0.5 (50% Mb legata allO2)

= [MbO2]/([Mb] + [MbO2]) eq. 1

0.5 =[O2]/(Kd+ [O2]) quindi
0.5Kd + 0.5 [O2] = [O2]
Sostituiamo nelleq. 1 [MbO2]=K [Mb][O2]
e otteniamo Da cui [O2] = Kd
= K[Mb][O2]/([Mb] + K[Mb][O2])
Possiamo quindi semplificare [Mb] in tutti I termini e si Quindi
= K[O2]/(1 + K[O2]) e dividendo tutto per K =PO2/(P50 + PO2)

=[O2]/(1/K+ [O2]) ossia

=[O2]/(Kd+ [O2])

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Dynamics of
O2 binding to Mb
The curve determined experimentally shows a
P50 = 4 mm Hg
Because the PO2 in the capillaries is about 30
mm Hg, the Mb gets saturated with O2
O2 is stored till the PO2 in the tissues/ cells
goes lower than 10 mm Hg
Because the affinity constant K is also an
equilibrium constant:
K = k1 / k-1
The role of the protein is also to regulate the
k1 (rate const. for binding) and the k-1 (rate
const. for release) by opening a pathway for
the O2 to escape from the heam
Dynamics of internal protein motions does
exactly that!
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All higher animals contain an O2

transport protein: in vertebrates this
is hemoglobin (Hb);
All Hb are multi-subunit: 22

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O2 binding to Hb:
cooperative binding and allostery

The challenge faced by

To bind O2 in the lungs
at PO2 100 mmHg;
To release O2 in the
tissues at PO2 30 mmHg;
The hyperbolic function
of Mb is not adequate;
A sigmoidal curve is
( = fraction of binding)
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Cooperative interaction

The switch from weak

binding to strong binding
cannot be obtained from
monomeric molecules
such as Mb;
For this reason we need
cooperative interaction
that can be obtained from
a multimeric protein such
as Hb that can bind 4 O2
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The Hill plot
Cooperativity is described by the Hill
log /(1- ) = nlog PO2 - nlog P50
The plot will be a straight line with
slope n = 1 in the case of non-
cooperative binding at the strong or
weak binding extremes;
The transition between states has a
maximum slope of n = 3.5;
For Hb the maximum theoretical
slope should be n = 4 because there
are 4 binding sites, but these are
never in reality fully saturated;

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Allostery: models
Allostery is an important mechanism for regulation of protein
It is the ability of the uptake of one ligand to influence the action of
the protein on another ligand. This is also true for enzymes.

Theories that describe

Filmer model (KNF
model):Subunits change
conformation one at the
time upon O2 binding

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Monod-Wyman-Changeux (MWC model): the entire
Hb exists in one of 2 forms: T, deoxy, weak binding,
and R, oxy, strong binding

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Changes in Hb structure

22 in urea gives 2
suggesting strong
interactions between
rather than and
X-ray diffraction has
showed that in fact the DEOXY OXY
dimer rotates and slides
with respect to one
another narrowing the
central cavity;
This is consistent with the
R and T states
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Molecular details:

Deoxy form:
The C-ter. of 2 (res. 146) lies on top of the C-
ter. helix of 1

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The basis for the
conformational change

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Confirmation by
site-directed mutagenesis
DNA Protein, Codon Amino-
Oligonucleotide with mismatch used
to start polymerisation of a mutant
Here His Gly, then add imidazole
In this way the Fe was still coordinated
as normal, but no connection with the FG
loop was present
No cooperative effects were found in this
case. This confirmed the model.

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The Bohr effect

Low pH = need of O2:

Anaerobic metabolism = production of lactic
acid = low pH;
Accumulation of CO2 (carbonic anhydrase
CO2 + H2O HCO3- + H+
Bohr effect:
Hb4O2 + nH+ HbnH+ + 4O2
where n > 2
Sites of protonation / deprotonation = His 146
at the C-ter of each -chain and the NH2-ter of
the chains;
Note from the graph that a change of pH of
0.8 units changes the PO2 from 20 to 40

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CO2 transport

This is done in 2 ways:

Formation of bicarbonate and transport dissolved in
blood serum;
Linkage to Hb N-ter of the protein chains to form

Note that here the release of H+ furher helps in the Bohr effect;
It follows that CO2 transport stimulates O2 release: for
this reason hyperventilation, breathing too rapidly with
excess release of CO2 leads to dizziness and
unconsciousness; this can be easily corrected by
breathing into a paper bag to bring up the level of CO2
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Biphosphoglycerate - BPG

This is another effector, like H+ and CO2;

It binds to Hb to lower its affinity to O2:
The higher the BPG in the blood, the more this will bind to the deoxy-form,
stabilising it and decreasing the affinity for O2;
Higher levels of BPG are found in people at high altitude, where the lower loading
of O2 in the lungs is compensated with more efficient unloading in the tissues;

BPG binds in the

cavity between
the -chains
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Fetal Hb

Problem: fetal blood must have higher affinity for O2 to be able

to obtain it from the mothers blood through the placenta;
For this reason the fetus Hb has a different Hb, of the type 22 .
The difference if that the -chain has a Ser instead of the His
143 present in the -chains of the adult;
This creates a site for BPG with much less affinity, there will be
less BPG bound, hence the Hb will have a much higher affinity.

Prof. G. Gilardi - Biological Chemistry

Possibili domande
1. Descrivi nel dettaglio le proprieta funzionali della mioglobina e dellemoglobina
facendo chiaro riferimento al ruolo svolto dalla struttura terziaria e quaternaria nella
funzione di queste proteine.
2. Rispondi alle seguenti domande:
a) Scrivi la formula del gruppo prostetico dellemoglobina;
b) Descrivi con luso di formule il meccanismo di legame dellossigeno al gruppo
prostetico dellemoglobina;
c) Descrivi in termini molecolari la sua curva di legame dellossigeno.
3. Evoluzione molecolare dellemoglobina ed effetti patologici di alcune forme varianti
4. Rispondi alle seguenti domande:
a. Scrivi la formula del gruppo prostetico eme legato nella mioglobina
b. Discuti in termini di struttura e funzione la mioglobina e lemoglobina
c. Descrivi almeno un meccanismo di regolazione dellemoglobina da parte di
piccole molecole
d. Discuti in termini strutturali e funzionali le differenze tra emoglobina adulta ed
emoglobina fetale
Prof. G. Gilardi - Biological Chemistry