Hemoglobin (Molecular Biology)

Hemoglobin (Hb) is one of the heme-containing oxygen-binding proteins, that generally contain a globin polypeptide chain and a protoheme IX prosthetic group that contains a ferrous iron atom. It is found in circulating fluids (blood or hemolymph) of animals, where its fundamental function is transport of molecular oxygen. Its phylogenetic distribution is wide, but somewhat capricious. All of the vertebrates, except icefish, have a Hb within their red blood cells that is a tetramer made up of two pairs of two different subunits, each a globin polypeptide chain that contains one heme group. In contrast, invertebrate Hbs are strikingly variable, in their architecture and size, and also in their function. They can be either intracellular or extracellular. Generally, an intracellular Hb is relatively small, whereas extracellular Hbs have a molecular mass ranging from 250 to 3500 kDa. The latter are polymeric and are dissolved directly in the blood plasma, hemolymph, or coelomic fluid. The invertebrate extracellular Hbs have been known traditionally as erythrocruorins, but this term lost its significance when it became apparent that there is no particular distinction between the subunits of the extracellular and of the vertebrate Hbs in their prosthetic group, amino acid sequence homology, or three-dimensional protein structure, the globin fold.

1. Vertebrate Hemoglobin

1.1. Structure

The adult form of human Hb, HbA, is composed of two a-subunits and two b-subunits, each of which has one bound heme group (Fig. 1). The a- and b-chains consist of 141 and 146 amino acid residues, respectively. All of the vertebrate Hb chains have similar numbers of residues and homologous amino acid sequences. All adopt very similar globin folds comprised of eight alpha-helices, designated A through H, although a-chains lack a-helix D. Early in development, related polypeptide chains replace the a- and b-chains.

Figure 1. The structure of the vertebrate hemoglobins. The molecule is a tetramer composed of two pairs of a- and b-subunits. The a- and b-chains are homologous, have similar globin folds, and each has a bound heme group. How the individual chains are distinguished is described in the text.

Within the a2b2 tetramer, there are relatively few contacts between the pair of a-subunits and the pair of b-subunits, whereas contacts are extensive between the unlike subunits (Fig. 1). If the a-subunits are designated a1 and a2, the b-subunits are numbered so that the distance between the heme iron atoms of the a1/b2 pair is shorter than that in the a1/b1 pair. Because of symmetry, the a1/b1 pair is identical to a2/b2, and a1/b2 is the same as a2/b1 (see Quaternary Structure and Oligomeric Proteins). The tetrameric assembly is stabilized by weak noncovalent bonds, by many van der Waals interactions, a few hydrogen bonds between unlike subunits, and several salt bridges between the two b-subunits.

1.2. Ligand Binding

One molecule of O2 binds to the sixth coordination site of the iron atom which is on the distal side of the heme group (see Globins and Myoglobin). The heme pocket in the globin fold provides a highly hydrophobic environment that maintains the heme iron atom in the ferrous state. Oxidization of the iron atom to ferric to produce methemoglobin destroys its ability to bind oxygen. Upon binding oxygen, the quaternary structure undergoes an extensive change in the relative orientations of the four subunits, especially due to changes in the a1/b2 and a2/b1 interfaces. This switch between two alternative quaternary structures is believed to be involved in the cooperativity of oxygen binding. Hb was the primary inspiration of the concerted model of allostery, and it remains one of the proteins that best fits that model.

Several other classes of ligand are bound at other specific sites on the deoxy quaternary structure of hemoglobin. These ligands include protons (see Bohr Effect), carbon dioxide, chloride ions, and 2,3-diphosphoglycerate (2,3-DPG). As the ligands bind more avidly to the deoxy form of Hb, they compete in effect with oxygen for binding, and the presence of one of these ligands affects the binding of the others.

1.3. Physiological Function

The physiological functions of vertebrate Hb are (1) transport of oxygen from the lungs to peripheral tissues; (2) transport of carbon dioxide and enhancement of its transport by the blood plasma; and (3) pH regulation (acid-base balance) of the blood.

The transport of oxygen by Hb is well described by its oxygen dissociation curve (or oxygen equilibrium curve), which expresses the dependence of oxygen saturation of Hb (Y) upon the partial pressure of oxygen (P^) at chemical equilibrium (Fig. 2). The general features of this curve are (1) its sigmoid shape and (2) its shift to the right to higher oxygen partial pressures at higher temperatures or in the presence of the nonheme ligands that bind specifically to deoxy-Hb, protons, CO2, Cl-, and 2,3-DPG. The sigmoid shape of the binding curve is ascribed to stepwise enhancement of the oxygen-binding affinity:

Here, P =and Kj (7 = 1,2,3,4) are the intrinsic oxygen association constants for the 7th molecule to bind. If the four heme groups of a2b2 Hb were equivalent and independent of each other, which would produce a hyperbolic oxygen dissociation curve, like that of myoglobin (Mb) (Fig. 2). Mb is a monomeric globin that contains a single heme group. In contrast, HbA gives a sigmoidal curve in whichand the ratio of K 4 to Kl is 500 (2) (Fig. 3).

This cooperative binding of oxygen produces the sigmoid curve, in which apparent interactions between the four heme groups of the molecule are mediated by conformational changes in the protein (3). The sigmoidal shape of the oxygen-binding curve contributes to increasing the difference in oxygen saturation between arterial and venous blood and enhances the amount of oxygen released at the tissues (Fig. 2).

Figure 2. Oxygen dissociation curves for hemoglobin (Hb) and myoglobin (Mb). The oxygen concentration is specified by its partial pressure in mm of Hg. The dissociation curve for the monomeric Mb has the expected hyperbolic shape, whereas that for Hb is sigmoid, indicating positive cooperativity in binding the four oxygen molecules by each Hb tetramer. The dissociation curve for Hb is shifted to the left or right upon changing the solution conditions or temperature, as indicated, whereas that for Mb is insensitive to changes in conditions, except for the temperature. The middle of the three curves for Hb is for standard conditions: 37°C, pH 7.4, 40 mm Hg CO2, and 5 mM 2,3-DPG. The partial pressure of oxygen for arterial blood, 100 mm Hg, and that for mixed venous blood, 40 mm Hg, produce a difference in oxygen saturation of 23%, whereas there would be a much smaller release of oxygen for a hyperbolic curve like that of Mb.

Figure 3. Changes in oxygen affinity of human hemoglobin with oxygen binding at the four heme groups and those upon addition of the cofactors 0.1 M Cland 2 mM 2,3-DPG. The abscissa indicates the ith oxygen molecule to bind, and the ordinate gives the logarithm of the intrinsic association constant for the ith step. Open circles correspond to the absence of cofactors, closed circles to their presence. The four-step oxygen binding is illustrated schematically below the graph. Squares and circles represent deoxy and oxy subunits, respectively.

Under physiological conditions, the nonheme ligands lower the oxygen affinity and increase the cooperativity of oxygen binding by HbA. The effect of these ligands on the intrinsic binding constants, Kp is not uniform with respect to i (Fig. 3). The shift of the oxygen binding curve to higher partial oxygen pressures, that is, the lowering of the overall oxygen affinity upon acidification or upon increasing the CO2 concentration or temperature has physiological consequences because this enhances the release of oxygen at the peripheral tissues, where such changes result from active metabolism. The effect of pH on the oxygen affinity is known as the Bohr effect. In some cases of chronic hypoxia found in anemia, chronic heart and lung diseases, etc., there is an increase in the intracellular concentration of 2,3-DPG, a decrease in Hb’s oxygen affinity, and consequently an enhancement of oxygen release at tissues (4).

Carbon dioxide produced in tissue cells diffuses into the red blood cells and reacts with water, catalyzed by carbonic anhydrase:

The HCO-3bicarbonate ions thus produced move to the blood plasma in exchange for chloride ions and are transported to the lungs. Reaction 3 is greatly shifted to the right when Hb absorbs the protons produced. Moreover, dissolution of the carbon dioxide is greater in the tissue capillaries than in the alveolar because of the uptake of protons upon oxygen release by Hb:

This oxygen-linked proton binding known as the Haldane effect is the reciprocal expression of the Bohr effect. The release of oxygen in the tissue capillaries enhances the uptake of protons by Hb, thereby causing a further shift of Eq. 3 to the right. Part of the CO2 is transported linked directly to Hb as a carbamino moiety:

where -N^is the a-amino groups of the a- and b-chains. This CO2 binding is also oxygen-linked. Dissociation of oxygen in tissue capillaries enhances the CO2 binding by Hb, increasing the CO2 content of the venous blood. The remainder of the CO2 is dissolved directly in the blood plasma. The major portion (85%) of the total CO2 is transported as bicarbonate, 10% as the carbamino form, and the remaining 5% as free CO2. Consequently, as a result of its allosteric properties, Hb plays a key role in CO2 transport and oxygen transport.

The properties of HbA described here also apply to other vertebrate Hb, except for some slight differences. In some species, 2,3 DPG is replaced by other cellular metabolites, such as ATP, GTP, or inositol pentaphosphate, and CO2 is replaced by bicarbonate ion.

2. Annelid Hemoglobins

Annelida possess giant extracellular HB of molecular mass 3500 kDa. The molecular architecture is a hexagonal bilayer whose diameter is about 30 nm and whose thickness is about 20 nm. The molecule is composed of 12 identical spherical protein units, known as "submultiples," six of which make one hexagonal layer. These submultiples are assembled to make up an entire molecule using many linker subunits. The submultiple is composed of 16-kDa globin subunits that have the same globin fold as the vertebrate Hb subunits, but the linker subunits are unrelated, heme-free polypeptide chains. The extracellular Hb from the earthworm Lumbricus terrestris has been most extensively studied. Its submultiple is a dodecamer composed of three copies of each of four different kinds of globin chains, and 12 such dodecamers are assembled with 36 linker chains and approximately 57 tightly bound calcium ions. Consequently, there are 12 x 12( = 144)heme groups in each molecule. Some of the globin and linker chains have attached carboyhydrates, which are thought to contribute to stabilization of the quaternary structure through their noncovalent, lectin-like binding (5, 6).

Annelid Hbs demonstrate a striking diversity of oxygen-binding properties. Most demonstrate a Bohr effect and cooperativity of oxygen binding like those of vertebrate Hb, and sometimes of even greater magnitude, but others have none. The annelid Hb do not respond to cofactors, such as 2,3-DPG or other organic phosphate compounds. Instead, their oxygen affinity is increased by divalent calcium and magnesium ions, which are abundant in the annelid blood. There is some evidence that the functional allosteric unit of annelid Hb is the submultiple.

3. Hemoglobins of Other Species

A great variety of Hbs are found in lower organisms. They can be (1) one-domain monomers, that have a single globin chain of about 16 kDa and one heme group; (2) polymers of such single-domain monomers; (3) two-domain polymers; and (4) multiple-domain polymers. Most of the single-domain monomer Hbs are present in the cytoplasm. Some are in noncirculating cells, whereas others are in coelomic cells of annelida, such as Glycera. Although those in noncirculating cells do not apparently participate in oxygen transport, they are still classified as hemoglobins. Leghemoglobin is a monomeric Hb contained in the cytoplasm of root nodules of legumes. It has an extremely high oxygen affinity, which is important for the completely removing of traces of oxygen from the symbiotic bacteria, because nitrogen fixation is a strictly anaerobic process. Consequently, the function of leghemoglobin is to eliminate oxygen, rather than to supply it to tissues.

Finding the single-domain Hb from the gram-negative bacterium Vitreoscila was the first indication that the evolutionary origin of globins may date back to the common ancestor with prokaryotes. Hbs isolated from yeasts and Escherichia coli have two domains, one of which is a globin chain and the other a flavoprotein. The NADH reductase activity of the second domain prevents the heme iron of the first domain from auto-oxidizing to maintain its capability of reversibly binding oxygen. Chironomus larvae possess Hbs that are polymorphic in various species, tissues, and developmental stages. Among the 12 Hbs of C. thummi thummi, seven exist as homodimers, four as monomers, and one as a monomer or dimer (see Chironomus). The nematode Ascaris lumbrodoides Hbs consists of two-domain globin chains, one of which has lost the ability to bind heme. Hbs from nematodes, including Ascaris, have extremely high oxygen affinities. The brine shrimp Artemia salina Hbs have a molecular mass 250 kDa and are composed of two polypeptide chains, each containing eight heme-binding domains. The blood clam Scapharca inaequivalvis has intracellular homodimeric and heterotetrameric Hbs, which bind oxygen cooperatively. The most conspicuous feature of these Hbs is that their globin chains have an additional a-helix at their N-terminus and that the E and F helices are external, exposed to solvent, whereas the G and H helices are internal, involved in subunit interactions. Thus, the clam Hb tetramer is "back to front" relative to the vertebrate Hb tetramer.

The various species of Hb demonstrate oxygen affinities that vary 26,000-fold. The lowest affinity, measured by the value of the partial pressure of oxygen at half saturation, is 26 mm of Hg, that of human Hb at 37°C, whereas that for the highest affinity is only 0.001 mm Hg, for Ascaris Hb at 20°C. The species-dependence of the oxygen affinity of Hb is a manifestation of its adaptation to the environments that the organisms inhabit. All of the Hbs have the same protoheme IX heme group, so this tremendous variation in oxygen affinity arises from species differences in the amino acid sequences of the globin moiety. How this is accomplished is not known.