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CHAPTER 28
Hemoglobin
A shift in the dissociation curve can be very important.
In chronic conditions in which the supply of oxygen to the
lungs is normal and arterial blood is saturated with oxygen
but the ability of the blood to deliver oxygen to the tissues
is impaired because of a low hemoglobin concentration
(anemia) or a low cardiac output (cardiac insufficiency),
the intraerythrocytic 2,3-DPG concentration increases. A
rightward shift of the dissociation curve will not affect the
percent saturation during loading in the lungs (since the
upper part of the curve is quite flat), but it will decrease
the percent saturation in the tissues, thereby providing a
useful increase in moles of oxygen per unit time available
for metabolism.
Respiration in a normal individual provides another ex-
ample. Since Pco, is higher (and pH lower) in the tissues
than in the lungs, oxygen unloading is facilitated at the tis-
sues, and loading occurs more readily in the lungs. The de-
crease in oxygen saturation at constant Po
2
(i.e., decrease
in oxygen affinity) with increasing PCo
2
or decreasing pH
above pH 6.3 is known as the
alkaline Bohr effect.
An
acid Bohr effect occurs below pH 6.3 and consists of an
increase in oxygen affinity with decrease in pH; it is due to
a chloride-induced proton uptake that is greater for oxy-
hemoglobin than for deoxyhemoglobin. It is not physio-
logically significant because a serum pH below ~
6.8
is
not compatible with human life.
Mechanism of Oxygenation
Hemoglobin has two quaternary structures that corre-
spond to the deoxygenated (deoxy; five-coordinate iron;
T-state) and the oxygenated (oxy; six-coordinate iron;
R-state) forms. X-ray crystallographic studies have de-
monstrated that the coordination number of the iron is the
crucial difference between these forms. All six-coordinate
hemoglobins
(oxyhemoglobin,
carbon
monoxyhemo-
globin, and methemoglobin) are in the R-state, whereas
deoxyhemoglobin is in the T-state. Binding of one mole-
cule of oxygen to deoxyhemoglobin causes a change in
the tertiary structure of the binding subunits, which results
in a change in quaternary structure (T-to-R transition)
that enhances the binding of oxygen to the remaining
subunits. Thus, up to four molecules of O
2
per molecule
of Hb can be bound.
The “machinery” for this transition is composed of the
globin peptide chain, amino acid side chains, and heme.
In deoxyhemoglobin, the bond between the nitrogen of
histidine F
8
and the iron atom of heme is tilted at a slight
angle from the perpendicular to the plane of the porphyrin
ring. The porphyrin ring is domed “upward” toward the
histidine, with the iron at the apex of the dome. Thus, the
iron is in an unfavorable position for binding of a sixth
ligand, such as oxygen. This is a primary cause of the
weak binding of O
2
to T-state hemoglobin (Figure 28-5).
Deoxyhemoglobin is in an unstrained conformation, as
expected for a stable molecular form, despite its desig-
nation as the T (“tense”) state. It is stabilized by hydro-
gen bonds, salt bridges, and van der Waals contacts be-
tween amino acid side chains on the same subunit and on
different side chains. Breaking of many of these bonds
occurs during oxygen binding, destabilizing the deoxy
structure and causing the release of the Bohr protons (see
below). Oxyhemoglobin is also stabilized by several non-
covalent bonds, formation of which aids in the T-to-R
transition.
Upon binding of the first molecule of oxygen to com-
pletely deoxygenated hemoglobin, strain is introduced
into the molecule as a result of competition between main-
tenance of the stable deoxy form and formation of a stable
iron-C
>2
bond. “Strain” refers to “long” bonds that have
higher energy than do normal-length, minimal-energy
bonds. When possible, the “stretched” bonds shorten to
lower their energy and thereby produce movement within
the molecule. In an effort to reduce the strain and return
to an energetically more favorable state, the F helix slides
across the face of the heme, causing the bond between his-
tidine F
8
and the iron atom to straighten toward the per-
pendicular (Figure 28-5b), and the heme rotates and sinks
further into the heme pocket. These movements occur in
both
a
and /3 subunits but are greater in the
fi
subunits.
The change in radius of the iron upon O
2
binding, origi-
nally thought to “trigger” the conformational changes, is
now considered to be of minor importance in the T-to-R
transition.
The movement of heme and surrounding amino acid
residues accomplishes two things. First, it relieves the
strain on the six-coordinated heme iron, increasing the
strength of the Fe-C
>2
bond (i.e., increasing oxygen affin-
ity). Second, it strains other noncovalent bonds elsewhere
in the subunit, causing some of them to break. This is
the beginning of the quaternary structure transition from
T-state to R-state. The energy needed for the conformation
changes is provided by the energy of binding of oxygen
to the heme iron and by the formation of new, noncova-
lent bonds typical of oxyhemoglobin. Under normal con-
ditions of PCo
2
and Po, in the lungs, more energy is re-
leased by binding four oxygen molecules than is needed
to break the noncovalent bonds in deoxyhemoglobin. The
opposite situation prevails in extrapulmonary capillary
beds.
The binding of an oxygen molecule in one subunit
induces strain in another subunit and causes some non-
covalent bonds to break. Thus, Hb(C>
2
), № (
02
)
2
, and
Hb(
0 2)3
represent different transient structures. Which
