Hemoglobin Function

Evolution has honed the Hb tetramer into a molecule ideally suited for its tasks. Because human Hb
must behave differently than that of altitude dwelling species or species inhabiting hypoxic locales,
many different variants of the same basic molecular design have evolved. Because of the exigencies
of molecular evolution, we find in the genome of all animals, including humans, attempts by nature
to propagate a variety of different globin genes. The crystallographic studies of Perutz et al defined
the oxygenated and deoxygenated structures of Hb at Ångström-unit resolution and provided an
exquisitely detailed picture of how the globin chains and individual amino acid residues respond to
the loading and unloading of oxygen. All of these, however, share the properties of highly reversible
oxygen binding and high solubility in cytoplasm. We know more about the function of Hb than about
virtually any other protein, and the knowledge of this mechanism provides a beautiful and
intellectually satisfying culmination to decades of study by many investigators.

The oxygen dissociation curve of Hb, shown in Fig. 33.3 , describes the percent saturation of Hb with
oxygen at different oxygen tensions. The sigmoidal shape of this curve is a result of interaction
among the subunits of Hb. Communication within the tetramer is called heme–heme interaction or
cooperativity. This implies that the four heme groups do not undergo simultaneous oxygenation or
deoxygenation but rather that the state of each heme unit with regard to the presence or absence of
bound oxygen influences the binding of oxygen to other heme groups. Myoglobin, a heme-
containing protein with virtually the same tertiary structure as globin, exists in muscle as a
monomer. The oxygen equilibrium curve of myoglobin is a rectangular hyperbola; in physiologic
terms, it rapidly becomes fully saturated at low oxygen tensions and remains saturated as the
oxygen tension plateaus. The difference in the oxygen equilibrium curves of myoglobin and Hb lies in
the tetrameric nature of the Hb molecule and the cooperativity permitted by the association of
similar but unlike subunits. Compared with Hb, myoglobin has a very low P 50 (i.e., oxygen partial
pressure at which the molecule is one-half saturated). It therefore has an extremely high oxygen
affinity and would not be useful for delivering oxygen to tissues. The oxygen in myoglobin is passed
on to the mitochondria, where oxidative metabolism occurs. The sigmoidal shape of the oxygen
dissociation curve of Hb indicates that the totally deoxygenated Hb tetramer is slow to become
oxygenated, but as oxygenation proceeds, the reaction of heme with oxygen accelerates. Perutz has
drawn an analogy in which the “appetite” of heme for oxygen grows with the “eating,” and
conversely, loss of oxygen by heme lowers the oxygen affinity of the remaining heme groups. The
Hill coefficient, n, which can be calculated from plots of oxygen equilibrium curves, is a description of
heme–heme interaction or cooperativity that explains in part the oxygen-binding properties of Hb
and myoglobin. The Hill coefficient for myoglobin is 1, indicating no cooperativity; n is approximately
3 for the normal human HbA molecule.

The oxygen affinity of Hb within the erythrocyte does not depend solely on the intrinsic properties of
the tetramer. The position of the Hb oxygen dissociation curve, and therefore the P 50 , can be
influenced by a number of heterotropic modifiers, including temperature, pH, and small organic
phosphate molecules in the cell. The effects of these modifiers on P 50 are shown in Fig. 33.3 .

Hb is the prototype of an allosteric protein; its structure and function are influenced by other
molecules. The major intracellular modulator of Hb–oxygen affinity in human erythrocytes is 2,3-
BPG, an intermediate product of glycolysis that is present within the erythrocyte at concentrations
equimolar to Hb. The synthesis of 2,3-BPG is enzymatically regulated, and its levels can change
depending on the conditions extant. 2,3-BPG is able to bind stereospecifically within the central
cavity of the Hb tetramer. Hb prepared in the absence of 2,3-BPG has a very high oxygen affinity, but
as 2,3-BPG is added to a Hb solution, the oxygen affinity progressively decreases. 2,3-BPG is a
polyanion that binds strongly to the deoxygenated form of Hb but poorly to its oxygenated or other
liganded forms. Specific amino acids are involved in the binding of 2,3-BPG; these β-chain residues
include the N-terminal valines, the H21 histidine (position 143), and the EF6 lysine (position 82). In
oxyhemoglobin, the H helices of the β-chains are insufficiently spread to permit firm binding of 2,3-
BPG; this, along with other conformational changes, favors the binding of this anion to the
deoxygenated rather than the oxygenated form of Hb. The binding of 2,3-BPG stabilizes the tense (T)
structure of the deoxygenated form at the expense of the relaxed (R) structure of the
oxyhemoglobin tetramer.

Transition from the deoxy (T) to the oxy (R) form of Hb is accompanied by rotation of the αβ dimers
along the α 1 –β 2 contact region ( Fig. 33.5 ). The T structure is stabilized by salt bridges, which are
broken as the molecule switches into the R structure. Some abnormal Hbs with an intrinsically high
oxygen affinity, or low P 50 , occur as a result of an amino acid substitution that leads to loss of bonds
that stabilize the tetramer in the T conformation. Hydrogen ions, chloride ions, and carbon dioxide
all decrease the affinity of Hb for oxygen by strengthening the salt bridges that lock the molecule
into its T conformation. The corollary of the lowering of Hb oxygen affinity by protons is the
combination of Hb with protons on deoxygenation. This is known as the Bohr effect and is
responsible for carbon dioxide transport in blood, another critical function of the Hb molecule.
Deoxyhemoglobin binds the hydrogen ion liberated by the reaction of carbon dioxide with water,
increasing the concentration of bicarbonate. Within the lungs, hydrogen ions are lost as Hb binds
oxygen; therefore, carbon dioxide leaves solution and is excreted from the body through the lungs.
Deoxyhemoglobin can also directly bind carbon dioxide; however, this process involves the minority
of carbon dioxide exchanged by the RBCs.

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Fig. 33.5

SUBUNIT MOTION IN THE HEMOGLOBIN TETRAMER.

The relative motion of hemoglobin subunits on oxygenation and deoxygenation is shown. The α 1 β 1
dimer (black) is moving relative to the α 2 β 2 dimer (shaded) . The oxyhemoglobin tetramer (R state)
is more compact than the deoxyhemoglobin configuration (T state).

(Reproduced with permission from Dickerson RE, Geis I: Hemoglobin: Structure, function, and
evolution pathology . Menlo Park, CA, 1983, Benjamin-Cummings.)

RBCs containing high levels of Hb F have high oxygen affinity because it binds 2,3-BPG poorly.
Physiologically, this predicts that the Hb of fetuses should be oxygenated at the expense of the
maternal HbA. The high oxygen affinity of HbF is accounted for by a single change in its primary
structure, the presence of a serine residue at helical position H21 in place of the histidine found in
the β-globin chain. This weakens the binding of 2,3-BPG and leads to stabilization of the molecule in
its R state.

Interactions of Hb with NO have been a recent focus of investigation. NO, generated from l -arginine
by NO synthases, activates soluble guanylate cyclase to produce the second messenger cyclic
guanosine monophosphate. As a potent vasodilator, NO is an important regulator of vascular tone.
The reaction of free NO with erythrocytes is diffusion limited. Normally, the primary NO–Hb adduct
is nitrosyl (heme) Hb (HbFe[II]NO). Within the erythrocyte, β93 cysteine is reduced and seems
incapable of NO storage and delivery by S -nitrosohemoglobin as originally proposed. NO was
thought to form S -nitrosylhemoglobin in the lungs, where Hb is in its R or oxygenated state, and
liberate NO in the microcirculation, where the transition of the R to T conformation induced by
deoxygenation released NO from Hb. However, studies suggest that NO binding to heme groups is
physiologically a rapidly reversible process. This view supports a model of Hb delivery of NO distinct
from its dissociation from the β93 cysteine residues. Small nitrosothiol molecules could also be
involved in NO transfer. The thiol groups of Hb can exchange NO with small nitrosothiols derived
from free cysteine and glutathione. Accordingly, the thiol groups of Hb could bind and transfer NO or
exchange NO with small shuttle molecules, increasing perfusion of hypoxic tissues. It has been
suggested that cytoskeletal and other erythrocyte proteins slow NO influx into the cell and, coupled
with NO heme binding, preserve NO bioactivity. NO–Hb interactions, whether through S -
nitrosohemoglobin formation at the β93 cysteine or the formation of nitroso intermediates, are
likely to be physiologically important. Hb liberated from the intravascularly hemolyzed RBCs rapidly
inactivates NO. As the RBC lyses, arginase is also released and destroys the substrate for NO
synthases, l -arginine. Together, this leads to a reduction in biologically active NO. With hemolysis as
in sickle cell disease or thalassemia, reduced NO bioavailability is associated with disease
complications such as pulmonary hypertension, leg ulcers, priapism, and perhaps increased risk of
stroke. Lactic dehydrogenase also released from the RBC in hemolytic anemia is an excellent marker
of these complications.

In summary, the primary amino acid structure of α-globin and non–α-globin chains dictates the
inevitable quaternary structure in which resides the ability of Hb to serve as a respiratory protein.
Cooperativity ensures rapid binding of oxygen in the lungs and unloading in tissues. Similarly, carbon
dioxide is transported from tissues to lungs. The function of Hb may be influenced by mutation and
by heterotropic effectors such as protons and 2,3-BPG. The molecule itself changes shape as it
provides oxygen for metabolism; it is a lung in miniature, breathing as it allows the body to respire.
Structure and Function of Hemoglobin

Dalam setiap molekul Hb, satu kelompok heme dimasukkan ke dalam kantung hydrophobic

In each hemoglobin (Hb) molecule, one heme group is inserted into a hydrophobic pocket of one
folded polypeptide chain ( Bunn & Forget, 1986 ). Normal adult HbA consists of four heme groups
and four polypeptide chains (two α-chains and two β-chains), which form a roughly globular
hemoglobin molecule ( Fig. 31-11 ). The ferrous iron atoms have six coordination bonds: four to the
pyrrole nitrogens of heme, one to the imidazole nitrogen of histidine of the globin chain (87-α or 92-
β), and one that is reversibly bound to oxygen. As the oxygen partial pressure increases, each of the
four heme groups sequentially binds one molecule of oxygen. In the process, a change in the overall
configuration of the hemoglobin molecule occurs, which favors the additional binding of oxygen.

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Figure 31-11

The hemoglobin molecule (tetramer, molecular weight 64,500 Da).

The heme group for each monomeric polypeptide chain is depicted as a black disk, connected to an
imidazole group of histidine and located near the surface of the molecule in a “pocket” formed by
the polypeptide chain. The letters A and H designate α-helix segments of each polypeptide chain: A
is the amino-terminal segment, and H is the carboxy-terminal segment. The four monomers are
separated in this drawing but actually make contact along a relatively large area (α 1 β 1 ), which is
thought to be the relatively fixed or stabilizing contact area, and a smaller (α 1 β 2 ) area thought to
be the functional contact area, where movement occurs during oxygenation and deoxygenation,
changing the molecular configuration.

(Redrawn from White JM, Dacie JV: The unstable hemoglobins—molecular and clinical features, Prog
Hematol 7:69, 1971, with permission.)

The sigmoid-shaped oxygen dissociation curve of hemoglobin reflects this increasing affinity for
oxygen with increasing partial pressure of oxygen in the lungs (see Fig. 31-8 ). In the tissues,
conversion of oxygenated Hb to Hb, decreasing pH, and increasing temperature produced by
metabolic processes, as well as the binding of more 2,3-DPG to Hb, result in a shift of the Hb-oxygen
dissociation curve to the right, favoring the release of oxygen from hemoglobin.

Carbon dioxide (CO 2 ) is transported in erythrocytes as well as in plasma. A small part of red blood
cell CO 2 is dissolved and is bound to amino groups of hemoglobin as carbamino-CO 2 , but most is in
the bicarbonate form. The enzyme carbonic anhydrase catalyzes the transformation of carbon
dioxide to bicarbonate in the red blood cell while in the tissue capillary bed and catalyzes the reverse
reaction (the release of carbon dioxide from bicarbonate) in the erythrocyte when it is in the
capillary bed of the lungs.

The porphyrias, abnormalities of heme synthesis, are described in Chapter 32 ( Fig. 31-12 ).

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Figure 31-12

Biosynthetic pathway of heme and pathogenesis of the porphyrias. ADP, Aminolevuoinic acid
dehydratase; AIP, acute intermittent porphyria; ALA, Aminolevulinic acid; CEP, congenital
erythropoietic porphyria; CP III, coproporphyrinogen III; EPP, erythropoietic protoporphyria; HCP,
hereditary coproporhyria; HMB, hydroxymethylbilane; PCT, porphyria cutanea tarda; UP III,
uroporphyrinogen III; VP, variegate porphyria; XLP, X-linked protoporphyria.