Vertebrate Hemoglobin’s: Structure, Function and Action (With Diagram)

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Our understanding of the structure of the hemoglobin tetramer and the individual globin chains has advanced rapidly during the past 15 years, primarily because information about this molecule has been acquired through a variety of independent methods of investigation.

These include:

(1) X-ray crystallographic studies of hemoglobin crystals, providing atomic reso­lution on the order of just a few angstroms;

(2) Bio­chemical studies of amino acid sequences in isolated globin chains;

(3) Determination of the nucleotide se­quences in globin chain genes (from these sequences the primary structures of the globin chains can be in­ferred);

(4) Electron spin resonance (ESR) studies of the heme groups and their manner of interaction with the globin chains;

(5) Radioactive tracer studies of the mechanism of globin chain biosynthesis; and

(6) Physiological studies of the behavior of the molecule. Most notable among those scientists who have contrib­uted to our present understanding of the structure and function of hemoglobin is M. F. Perutz, whose work with this protein began nearly 50 years ago. In 1962, Perutz, together with his colleague J. C. Ken- drew, received the Nobel Prize for his studies of hemo­globin and a closely related protein, myoglobin.

The following discussion of the anatomy of hemoglo­bin molecules will illustrate a number of general features that are characteristic of protein structure and function. There is, however, a more far-reaching lesson to be learned from these studies of hemoglobin.

Hemoglobin, like many other proteins (especially en­zymes), is widespread in nature and the primary structure of the molecule is now known for several dozen different species of organism. Although differ­ences in composition do exist, there are overriding similarities among all of the hemoglobin’s. These simi­larities are not merely chemical curiosities; rather, they provide us with an insight into the nature of evolution as it proceeds at the molecular level.

General Structure of the Hemoglobin’s:

The hemoglobin’s are conjugated globular proteins, that is, they contain some non-protein parts. In verte­brates, the function of hemoglobin is to transport oxy­gen in the blood from the lungs to the tissues, and therefore hemoglobin molecules can exist in two states: oxygenated and un-oxygenated. In all but the lowest vertebrates, hemoglobin is a tetramer. In lamp­reys, however, hemoglobin is monomeric, that is, it contains a single globin chain. In this regard, lamprey hemoglobin is similar to myoglobin.

In hu­mans, the most common type of hemoglobin is hemo­globin a (abbreviated HbA), which consists of 574 amino acids and has a molecular weight of 64,500. Its secondary, tertiary, and quaternary structure is typi­cal of all higher vertebrate hemoglobin’s.

The protein portion of the molecule, called globin, is composed of four polypeptide chains, each of which is also globular in shape. The four globin chains consist of two identi­cal pairs: two alpha chains (141 amino acids each) and two beta chains (146 amino acids each). The non-pro­tein portion of hemoglobin consists of four heme groups, one associated with each of the four globin chains.

Cysteine residues are not uncommon among the various hemoglobin’s; for example, there are six in HbA. Yet, despite the fact that disulfide bridges be­tween cysteine residues play an important role in the maintenance of secondary, tertiary, and quaternary structure in many proteins, there are no such linkages in the hemoglobin’s. Instead, the tertiary structure of each chain and the quaternary structure of the whole tetramer are maintained by non-covalent linkages.

Hemoglobin molecules are highly symmetric. The molecule can be divided into two identical halves, each consisting of an αβ dimer (called a “hy­brid” or “asymmetric” dimer). The complete tetra­mer is similar to a mildly flattened sphere (i.e., an ob­late ellipsoid) having a maximum diameter of about 5.5 nm.

The four polypeptides are arranged in such a manner that unlike chains have numerous stabilizing interactions, whereas like chains have few. That is, each alpha chain has many contacts with both betas, and each beta chain has many contacts with both al­phas; however, alpha-alpha and beta-beta contacts are few. The like chains face each other across an axis of symmetry such that rotation of any chain 180° about this axis would make it congruent with its iden­tical partner. A cavity about 2.5 nm long and varying in width from about 5 to 10 Å passes through the mol­ecule along the axis.

The interior of each subunit contains many amino acids with hydrophobic side chains; these form hydro­phobic bonds with one another. Amino acids with ionized side chains predominate at the surface of the subunits and at the surface of the molecule as a whole. Amino acids with nonpolar side chains may be found along a polar segment at the surface, but in such cases the side chains are buried in a crevice formed in the molecule’s surface and thereby have little contact with the surrounding water.

As noted above, contacts be­tween like subunits are rare. In the oxygenated mole­cule, ionic bonds (salt bridges) occur between the polar amino and carboxyl terminals of the respective chains and also between a few other residues. Con­tacts between unlike subunits are much more numer­ous and include 17 to 19 hydrogen bonds between about 35 amino acids of each hybrid dimer. Far fewer bonds link alpha and beta chains of different dimers and no covalent bonds link any of the subunits to­gether.

Association of the Globin Chains with Heme and the Heme with Oxygen:

All of the globin chains have the same fundamental secondary structure (Fig. 4-28). The helical regions are alpha helices, although there may be small excur­sions into pi and 3₁₀ conformations. The heme group that is associated with each globin chain consists of an organic portion and an iron atom (Fig. 4-29).

The or­ganic part is called protoporphyrin IX and is formed from four pyrrole rings linked together by methene bridges. All of the atoms of this tetrapyrrole lie in a single plane; however, when associated with globin, the plane of the tetrapyrrole becomes slightly dome- shaped. Four methyl, two vinyl, and two propionic acid groups are attached to the tetrapyrrole as side chains.

The iron atom is linked to each pyrrole ring, and depending on whether or not the hemoglobin is oxygenated, the iron lies either in the same plane as the tetrapyrrole or just above it. Each globin chain en­velopes its heme group in a deep cleft, exposing only the propionic acid side chains to the surrounding water.

Within each cleft, the heme groups interact with the R-groups of 16 amino acids from seven segments of the globin chain. Except for the iron atom, the por­tion of the heme group that is enclosed in the cleft is strongly hydrophobic and most of the bonds that are formed with the heme involve hydrophobic side chains of amino acids such as leucine, valine, and phenyl­alanine. The tertiary structure of an alpha globin chain together with its heme group is depicted stereoscopically in Figure 4-30.

Although each iron atom can form up to six bonds, only five occur in the un-oxygenated form of hemoglo­bin. Four of these bonds are formed with the pyrrole nitrogen atoms and the fifth with the side chain of a histidine residue at position F8 (called the “proximal” histidine). In the un-oxygenated state, the bond be­tween iron and the proximal histidine “lifts” the iron atom out of the plane of the tetrapyrrole. Histidine F8 is said to be “asymmetrically tilted” in that its bond with iron is not perpendicular to the heme plane. A second histidine residue at position E7 (the “distal” histidine) interacts with the iron but does not form a bond (Fig. 4-31).

During oxygenation of hemoglobin, molecular oxygen (i.e., O₂) enters the heme cleft on the distal side, where one of the two oxygen atoms then forms the sixth bond with iron. Oxygen binding causes the iron atom to be pulled back into the heme plane. This movement also draws the proximal histi­dine closer to the heme group, straightens the histidine-iron atom bond, and precipitates a train of additional small changes in the conformation of the polypeptide (Fig. 4-31). The changes that occur within a subunit as a result of oxygen binding lead to changes in subunit-subunit interaction and are funda­mental to the “cooperativity” exhibited by hemoglobin.

Families of Globin Chains and Globin Chain Homologies:

Myoglobin is a protein that is closely related functionally and structurally to hemoglobin. In myoglobin, however, there is a single polypeptide chain and a single heme group. Thus, three “families” of globin chains exist in nature. These are (1) the al­pha and (2) beta families of globin chains that com­prise all of the hemoglobins, and (3) the myoglobin family of globin chains.

At the present time, about 60 different vertebrate alpha family globin chains, 66 beta family chains, and 60 myoglobin family chains have been fully sequenced. In addition to human he­moglobin and myoglobin, the known sequences include those of the chimpanzee, gorilla, monkey, rabbit, mouse, horse, pig, cow, dog, sheep, camel, goat, llama, kangaroo, echidna, platypus, whale, chicken, turtle, frog, carp, shark, and lamprey.

All of these globin chains share a fundamentally similar secondary and tertiary structure. Because the primary structures of these globin chains are known, it is possible to analyze the relationship between their common secondary and tertiary structures and their respective primary structures.

If two (or more) chains of the same family are found to have the same amino acid occupying a given posi­tion in the secondary structure (e.g., position B6, E7, FG2, and so on), then the chains are said to be homolo­gous at that position. Such comparisons reveal an in­teresting relationship between the extent of homology of the globin chains of the various vertebrate species and the evolutionary relatedness of the organisms.

Moreover, certain positions of the secondary structure are invariably occupied either by a specific amino acid or by a limited number of different amino acids, im­plying that a high degree of conservation has occurred. Such conservation is clearly related to the constraints that must be satisfied for the assumption of a specific and common tertiary structure among the various globin chains.

Highly Conserved Amino Acid Positions:

Among the 60 alpha family chains that have been sequenced, iden­tical amino acids are found at 23 of the 141 positions. The 66 beta family chains that have been sequenced have identical amino acids at 20 of their 146 positions. For the myoglobins, 27 of the 153 amino acid positions are occupied by the same amino acid.

If one compares all 186 globin chains, five positions are found to be to­tally invariant (Table 4-7). An additional 10 positions are invariably occupied by one of two alternative amino acids whose side chains have the same proper­ties (e.g., they are hydrophobic, positively charged, negatively charged, etc.) Of the two alternative occu­pants of a given position, one is overwhelmingly fa­vored.

Clearly, these 15 residues play an important part in maintaining the three-dimensional shape pecu­liar to the globins. For some of these positions, the role is readily explained; for example, amino acid F8 (totally invariant) is the proximal histidine and E7 (in­variant in all but a few globins) is the distal histidine.

There are 33 positions in the interior of the globin chains that do not contact the surrounding environ­ment; among these, 30 positions are invariably occu­pied by amino acids with nonpolar side chains. There are also 10 positions in crevices at the surface of the molecule invariably occupied by nonpolar amino acids.

Thus, more than one-fourth of the amino acid posi­tions in globin are invariably nonpolar. These posi­tions may be occupied by any one of several amino acids. It is generally agreed that the invariably nonpo­lar positions, together with the nonpolar portions of the heme group, play a major role in determining the shape of the globin chain. Internally, the hydrophobic interactions between side chains form the skeletal framework on which the shape is founded.

The substi­tution through genetic mutation of a single polar amino acid in a position that is invariably nonpolar usually is sufficient to disrupt the normal organization of the polypeptide and render it biologically nonfunc­tional. Ionic and hydrogen bonds between residues of different chains (hemoglobin) and between residues and the surrounding water and ions (hemoglobin and myoglobin) also contribute to the stability and univer­sal shape of the molecule.

Function and Action of Hemoglobin: Cooperativity in Proteins:

The function of hemoglobin is to reversibly bind mo­lecular oxygen, that is,

The manner in which oxygen is bound by and released from hemoglobin reveals yet another characteristic of protein (especially enzyme) action, namely that of co-operativity. Hemoglobin is contained within the red blood cells (erythrocytes) and is oxygenated as the blood circulates through the capillary networks of the lungs.

On leaving the lungs, virtually every hemoglo­bin molecule has combined with four molecules of oxy­gen. In this state, the hemoglobin is said to be 100% saturated. Later, when the blood is circulated to the other body tissues, hemoglobin releases its bound oxygen.

The amount of oxygen that is released is determined in part by the concentration of dissolved oxygen gas (i.e., the partial pressure) in the surround­ing plasma and body fluid. In muscle, for example, it would not be unusual for the percentage saturation of hemoglobin to fall to 40% or lower as the released oxy­gen diffuses from the erythrocytes into the plasma and then into the muscle tissue.

The closely related and structurally similar oxygen- binding protein myoglobin, which acts to temporarily store oxygen in certain muscle tissues, consists of a single polypeptide chain and one heme group. Conse­quently, myoglobin can reversibly bind only one mole­cule of oxygen. Figure 4-32 compares the oxygen association/dissociation curves of hemoglobin and myoglobin.

Referring to Figure 4-32, as the oxygen partial pressure rises between 0 and 20 mm Hg, myoglobin rapidly combines with oxygen and quickly approaches complete saturation. The association/dissociation curve takes the form of a rectangular hyperbola. The behavior of hemoglobin is considerably different and much more complex.

At low oxygen partial pressure, the affinity of hemoglobin for oxygen is considerably less than that of myoglobin. For example, at 20 mm Hg partial pressure, hemoglobin is only about 21% saturated. However, between 20 and 60 mm Hg oxy­gen partial pressure, the affinity of hemoglobin for oxygen is greatly increased and the hemoglobin ap­proaches saturation.

Above 60 mm Hg, hemoglobin binds only small quantities of additional oxygen. Un­like the myoglobin curve, the oxygen association/ dissociation curve of hemoglobin is sigmoid. From a physiological standpoint, the unique oxygen-binding characteristics of hemoglobin are crucial. The partial pressure of oxygen in the blood leaving the lung capil­laries is generally in excess of 100 mm Hg, but by the time the blood reaches the capillaries of the various body tissues, the partial pressure has fallen to 80 mm Hg. An examination of Figure 4-32 shows that in this interval hemoglobin will have released only a small percentage of its oxygen.

However, in the tissue capil­laries, where the oxygen partial pressure often falls below 40 mm Hg, hemoglobin releases much of its bound oxygen. In actively exercising muscle, where the partial pressure may drop to 20 mm Hg, still greater quantities of oxygen would be released. Thus, within the range of 60 to 20 mm Hg (a range within which most of the tissues of the body operate), a rela­tively small decrease in oxygen partial pressure is ac­companied by a quantitative release of hemoglobin- bound oxygen.

In contrast, myoglobin retains nearly all of its bound oxygen even at a partial pressure of 20 mm Hg. Indeed, the greater affinity for oxygen displayed by myoglobin at all partial pressures provides for the ef­ficient transfer of oxygen from the blood to myoglobin-containing musculature. If the hemoglobin oxygen association/dissociation curve was hyperbolic (like myoglobin’s) instead of sigmoid, then inadequate amounts of oxygen would be released from the blood to the tissues, leading to asphyxiatioft even during moderate exercise.

The difference between the behaviors of these closely related proteins results from the subunit interactions that are possible in the tetrameric hemoglobin molecule but that cannot occur in the monomeric myoglobin. The subunit interactions exhibited by hemoglobin belong to a general class of interactions that are possible in multi-subunit pro­teins; the phenomenon is called co-operativity.

Cooperativity in Hemoglobin:

The complex behavior of hemoglobin, which is precisely what is required of an efficient oxygen-transporting system, may be at­tributed to its quaternary structure, for when hemo­globin is dissociated into its subunits, the separate subunits exhibit the oxygen-binding behavior of myoglobin (i.e., a hyperbolic curve). Li the intact tetramer, the various subunits exhibit cooperativity.

Unoxygenated hemoglobin is said to be in the “T” (i.e., tense) state, whereas oxygenation leads to the “R” (i.e., relaxed) state. In T-state hemoglobin, the iron atom of each heme group is pulled out of the plane of the tetrapyrrole and toward helix F by its interac­tion with the side chain of each proximal histidine. Binding of oxygen draws the iron atom into the plane, pulling the histidine side chain toward the heme group and straightening the asymmetric tilt (see above); hence, a small change in tertiary structure occurs.

Now, since each globin chain is bonded to neighboring subunits, changes taking place in one subunit will in­duce changes in another by altering the nature of the subunit associations. In particular, during oxygena­tion, ionic bonds linking subunits together are broken, leading to the R state. The changes in tertiary and quaternary structure that accompany the oxygena­tion and deoxygenation of hemoglobin have been sus­pected for some time, for it has long been known that crystals formed by oxy-hemoglobin are quite different from those formed by deoxyhemoglobin.

The binding of the first oxygen molecule to un­-oxygenated hemoglobin involves one of the alpha subunits; while this occurs slowly, the conformational change in this subunit induces a change in the associated subunit (i.e., the beta member of the asymmetric dimer).

The result is a more rapid binding of the second oxygen molecule by the beta subunit. The changes occurring in one subunit induce changes in the other subunit (i.e., the other asymmetric di­mer), including a 15° rotation of the two dimers with respect to one another. Although this movement accel­erates the binding of the third oxygen molecule, the fourth oxygen is bound less rapidly.

Effects in which conformational changes in one or more subunits of a protein alter the structure of other subunits in such a way as to modify the protein’s be­havior are called cooperative effects. In the case of hemoglobin, it may now be understood why the oxy­gen association/dissociation curve is sigmoid and not hyperbolic, for the individual alpha and beta subunits of the tetramer do not operate independently of one another in the binding of oxygen but instead influence each other.