Fundamentals of

Biochemistry
Fourth Edition

Donald Voet • Judith G. Voet •

Charlotte W. Pratt

Chapter 7
Protein Function: Myoglobin and Hemoglobin,
Muscle Contraction, and Antibodies

Copyright © 2013 by John Wiley & Sons, Inc. All rights reserved.
 Chapter 7
Oxygen Binding to Hemoglobin & Myoglobin

Key Concepts 7.1

• Myoglobin, with its single heme prosthetic group, exhibits a
hyperbolic O2-binding curve.
• Hemoglobin can adopt the deoxy (T) or oxy (R)
conformation, which differ in O2-binding affinity.
• Oxygen binding triggers conformational changes in
hemoglobin so that oxygen binds to the protein
cooperatively, yielding a sigmoidal binding curve.
• The Bohr effect and BPG alter hemoglobin’s O2-binding
affinity.
• Mutations can change hemoglobin’s O2-binding properties
and cause disease.
 Structure of Myoglobin

 153-residue monomeric protein consists of eight

alpha helices and contains a single heme group.
Myoglobin Contains a Heme Prosthetic Group and Binds to O2

The heme group

 Heme is an O2 binding chemical compound with an Fe(II) ion in the center.

 The Fe(II) atom at the center of heme is coordinated by the four porphyrin N atoms and one N from
the His F8 side chain.
 A molecule of O2 can act as a sixth ligand to the Fe(II) atom.
 His E7 hydrogen bonds to the O2.
 Two hydrophobic side chains Val E11 and Phe CD1 help hold the heme in place.
 Protein portion of myoglobin prevents Fe(II) oxidation to Fe(III).
 Other small molecules such as CO, NO, and H 2S can bind to heme groups in proteins
 Myoglobin function

 The major physiological role of myoglobin is to facilitate oxygen diffusion

in muscle.
 Myoglobin increases the effective solubility of O2 in muscle cells, acting

as a kind of molecular bucket brigade to boost the O2 diffusion rate.

 The oxygen-storage function of myoglobin is probably significant only in
aquatic mammals such as seals and whales, whose muscle myoglobin
concentrations are around 10-fold greater than those in terrestrial.
 Experiments with “knocked out” mice suggests that myoglobin is not
required by muscles under normal metabolic conditions.
 Myoglobin binds to O2 in a hyperbolic trend.
Fractional Saturation (Y):
the proportion of myoglobin
molecules that have bound O2
Bound Mb
Y=
Total Mb
[MbO2]
Y=
[Mb] + [MbO2]
[Mb] [O2]
K =
[MbO2]
pO2
Y=
K + pO2

The dissociation constant K can be operationally defined as the value of pO 2 at which Y = 0.5,
that is, the oxygen pressure at which myoglobin is 50% saturated.
K = p50
Mb and Hb are only ~18% identical in primary
sequence.

Invariant Identical in all Identical in Hb

 Mb and Hb are similar in their
secondary and tertiary structures.
Heme

Even though
myoglobin and hemoglobin have
only ~18% identical residues,
their secondary and tertiary
structures overlap almost
perfectly when superimposed!

Hb has quaternary
structure, but Mb
does not.
Myoglobin
α-Subunit of Hemoglobin
β-Subunit of Hemoglobin
 The similarities in structure and
sequence between myoglobin and
hemoglobin indicate a common
evolutionary origin.
 Hemoglobin is a Tetramer with Two Conformations

Deoxyhemoglobin (T state) Oxyhemoglobin (R state)

 Hemoglobin is the intracellular protein which transport oxygen in blood and gives red blood cells their color.
 Mammalian hemoglobin is an α2β2 tetramer. The α and β subunits are structurally and evolutionarily related
to each other and to myoglobin.
 The interactions between subunits are predominantly by hydrophobic effect, although numerous H-bonds
and several ion pairs are also involved.
 Oxygen binding alters the structure of the entire hemoglobin tetramer. The structures of deoxyhemoglobin
and oxyhemoglobin are noticeably different.
 The structural rearrangement is a crucial element of hemoglobin's O 2-binding behavior.
 Oxygen Binds Cooperatively to Hemoglobin
Hemoglobin has a p50 of 26 torr which is nearly 10 times greater than the p50 of myoglobin.
Oxygen binding to hemoglobin is described by a sigmoidal curve. This permits the
hemoglobin to deliver much more O2 to the tissues.

hyperbolic
binding

In any binding system, a sigmoidal

curve is diagnostic of a cooperative
interaction between binding sites.
Binding of O 2 to one subunit
increases the O 2 affinity of the
Tissue Lung remaining subunits. The fourth O2
binds to hemoglobin with 100-fold
greater affinity than the first.

Oxygen-binding curve of myoglobin and hemoglobin

 Hill Equation

n = Hill Constant, a non integral parameter relating

Degree of Cooperativity among interacting ligand-
binding sites or subunits
The bigger n the more cooperativity (positive value)
If n = 1, non-cooperative
n < 1, negative cooperativity
Hemoglobin’s Two Conformations Exhibit Different Affinities for Oxygen
Oxygen Binding to Hemoglobin Triggers a Conformational Change from T to R.
T & R states are conformations of deoxyhemoglobin & oxyhemoglobin respectively.
The changes in tertiary structure are coupled to a shift in the arrangement of
hemoglobin's four subunits.

The conformational shift in the T → R

transition tears away the ion pairs formed by C-
terminal residues of each subunit in a
process that is driven by the energy of
formation of the Fe—O2 bonds.

Movements of the heme and the F helix

during the T → R transition in
hemoglobin
 The Bohr Effect
Higher pH i.e. lower [H+] promotes tighter binding of oxygen to hemoglobin
and
Lower pH i.e. higher [H+] permits the easier release of oxygen from hemoglobin

 The Bohr effect is a physiological phenomenon first described in 1904 by the Danish
physiologist Christian Bohr, stating that hemoglobin's oxygen binding affinity is
inversely related both to acidity and to the concentration of carbon dioxide.
 The conformational changes in hemoglobin by oxygen binding decreases the pK’s of
several groups.
 The Bohr Effect Enhances Oxygen Transport
.

 The Bohr effect has important physiological functions in transporting O2 from the lungs to

respiring tissue and in transporting the CO2 produced by respiration back to the lungs.
 Increasing the pH stimulates hemoglobin to bind more O2 at lower O2 pressures
 CO2 also modulates O2 binding to hemoglobin by combining reversibly with the N-terminal

amino groups of blood proteins to form carbamates. The T (deoxy) form of hemoglobin binds
more CO2 and transport it to lung.
 Bisphosphoglycerate (BPG) Binds to Deoxyhemoglobin

The presence of BPG in mammalian erythrocytes decreases

hemoglobin's oxygen affinity by keeping it in the deoxy
conformation.

The BPG concentration in erythrocytes can be adjusted more

rapidly than hemoglobin can be synthesized for example
increases as a result of high-altitude adaptation.

Fetal hemoglobin has low BPG affinity to facilitate the transfer

of O2 from maternal hemoglobin to Fetal hemoglobin.

The effects of BPG and CO2 on hemoglobin's

O2 dissociation curve
 Hemoglobin O2 Binding Affinity

Lung Tissue

Vein

pO2 low
Capillary CO2 high
pH low
BPG high

Artery

High O2 affinity Low O2 affinity

 Symmetry Model of Allosterism
Allosteric effect, in which the binding of a ligand at one site affects the binding of
another ligand at another site, generally require interactions among subunits of
oligomeric proteins. Oxygen binds cooperatively to hemoglobin is a classic model.

[Link] allosteric protein is an oligomer of

symmetrically related subunits.

[Link] oligomer can exist in two conformational

states, designated R and T; these states are in
equilibrium.

[Link] ligand can bind to a subunit in either

conformation. If a ligand binds more tightly to the R
state than to the T state, it will promote T to R shift,
thereby increasing the affinity of the unliganded
subunits for the ligand.

4.T to R shift occurred simultaneously in all subunits

regardless of the number of ligands bound.
 Sequential Model of Allosterism

According to sequential model, ligand binding induces a conformational

change in the subunit to which it binds, and cooperative interactions arise
through the influence of those conformational changes on neighboring
subunits.

Oxygen binding to hemoglobin exhibits features of both

models.
Mutations May Alter Hemoglobin’s Structure and Function

Normal Human Erythrocytes Sickled Human Erythrocytes

 A single amino acid change Glu to Val at the sixth position of each beta chain
causes sickle-cell anemia.
 Normal erythrocytes are flexible, biconcave disks that can tolerate slight
distortions as they pass through the capillaries.
 Sickled erythrocytes are elongated and rigid and cannot easily pass through the
capillaries.