5.1 - protein function / myoglobin and hemoglobin: oxygen-binding proteins

ligand

(1) A small molecule that binds to a larger molecule. (2) A molecule or ion bound to a metal ion.

carbon monoxide poisoning

-carbon monoxide binds with hemoglobin and reduces oxygen supply to tissues The affinity of hemoglobin for carbon monoxide is about 250 times higher than its affinity for oxygen. However, the concentration of CO in the atmosphere is only about 0.1 ppm (parts per million by volume), compared to an O2 concentration of about 200,000 ppm. Normally, only about 1% of the hemoglobin molecules in an individual are in the carboxyhemoglobin (Hb · CO) form, probably as a result of endogenous production of CO in the body (CO acts as a signaling molecule, although its physiological role is not well understood). Danger arises when the fraction of carboxyhemoglobin rises, which can occur when individuals are exposed to high levels of environmental CO. For example, the incomplete combustion of fuels, as occurs in gas-burning appliances and vehicle engines, releases CO. The concentration of CO can rise to about 10 ppm in these situations and to as high as 100 ppm in highly polluted urban areas. The concentration of carboxyhemoglobin may reach 15% in some heavy smokers, although the symptoms of CO poisoning are usually not apparent. CO toxicity, which occurs when the concentration of carboxyhemoglobin rises above about 25%, causes neurological impairment, usually dizziness and confusion. High doses of CO, which cause carboxyhemoglobin levels to rise above 50%, can trigger coma and death. When CO is bound to some of the heme groups of hemoglobin, O2 is not able to bind to those sites because its low affinity means that it cannot displace the bound CO. In addition, the carboxyhemoglobin molecule remains in a high-affinity conformation, so that even if O2 does bind to some of the hemoglobin heme groups in the lungs, O2 release to the tissues is impaired. The effects of mild CO poisoning are largely reversible through the administration of O2. But because the CO remains bound to hemoglobin with a half-life of several hours, recovery is slow.

conservative substitution

A change of an amino acid residue in a protein to one with similar properties (e.g., Leu to Ile or Asp to Glu).

anemia

A condition caused by the insufficient production of or loss of red blood cells.

prosthetic group

A non-protein, but organic, molecule (such as vitamin) that is covalently bound to an enzyme as part of the active site. An organic group (such as a coenzyme) that is permanently associated with a protein.

variable residue

A position in a polypeptide that is occupied by different residues in evolutionarily related proteins; its substitution has little or no effect on protein function.

allosteric proteins

A protein in which the binding of ligand at one site affects the binding of other ligands at other sites. See also cooperative Binding.

heme

A protein prosthetic group that binds O2 (in myoglobin and hemoglobin) or undergoes redox reactions (in cytochromes).

invariant residues

A residue in a protein that is the same in all evolutionarily related proteins.

cooperative binding

A situation in which the binding of a ligand at one site on a macromolecule affects the affinity of other sites for the same ligand. See also allosteric protein.

In other words, the amount of O2 bound to myoglobin (Y) is a function of the oxygen concentration (pO2) and the affinity of myoglobin for O2 (K). A plot of fractional saturation (Y) versus pO2 yields a hyperbola (Fig. 5.3).

As the O2 concentration increases, more and more O2 molecules bind to the heme groups of myoglobin molecules until, at very high O2 concentrations, virtually all the myoglobin molecules have bound O2. Myoglobin is then said to be saturated with oxygen. The oxygen concentration at which myoglobin is half-saturated—that is, the concentration of O2 at which Y is half-maximal—is equivalent to K. For convenience, K is usually called p50, the oxygen pressure at 50% saturation. For human myoglobin, p50 is 2.8 torr

Red blood cells use one additional mechanism to fine-tune hemoglobin function. These cells contain a three-carbon compound, 2,3-bisphosphoglycerate (BPG):

BPG binds in the central cavity of hemoglobin, but only in the T (deoxy) state. The five negative charges in BPG interact with positively charged groups in deoxyhemoglobin; in oxyhemoglobin, these cationic groups have moved and the central cavity is too narrow to accommodate BPG. Thus, the presence of BPG stabilizes the deoxy conformation of hemoglobin. Without BPG, hemoglobin would bind O2 too tightly to release it to cells. In fact, hemoglobin stripped of its BPG in vitro exhibits very strong O2 affinity, even at low pO2

Therefore, increasing the pH of a solution of hemoglobin (decreasing [H+]) favors O2 binding by "pushing" the reaction to the right, as written above. Decreasing the pH (increasing [H+]) favors O2 dissociation by "pushing" the reaction to the left. The reduction of hemoglobin's oxygen-binding affinity when the pH decreases is known as the Bohr effect.

Bohr effect - The decrease in O2 binding affinity of hemoglobin in response to a decrease in pH.

H+ ions and bisphosphoglycerate regulate oxygen binding to hemoglobin in vivo

Decades of study have revealed the detailed chemistry behind hemoglobin's activity (and have also revealed how molecular defects can lead to disease). The conformational change that transforms deoxyhemoglobin to oxyhemoglobin alters the microenvironments of several ionizable groups in the protein, including the two N-terminal amino groups of the α subunits and the two histidine residues near the C-terminus of the β subunits. As a result, these groups become more acidic and release H+ when O2 binds to the protein:

The shift in conformation between the oxy and deoxy states primarily involves rotation of one αβ unit relative to the other. Oxygen binding decreases the size of the central cavity between the four subunits and alters some of the contacts between subunits. The two conformational states of hemoglobin are formally known as T (for "tense") and R (for "relaxed"). The T state corresponds to deoxyhemoglobin, and the R state corresponds to oxyhemoglobin.

Deoxyhemoglobin is reluctant to bind the first O2 molecule because the protein is in the deoxy (T) conformation, which is unfavorable for O2 binding (the Fe atom lies out of the heme plane). However, once O2 has bound, probably to the α chain in each αβ pair, the entire tetramer switches to the oxy (R) conformation as the Fe atom and the F helix move. An intermediate conformation is not possible, because the contacts between the αβ units do not allow it (Fig. 5.9). Molecular dynamics studies suggest that hemoglobin does not instantaneously snap from one conformation to the other but instead undergoes fluctuations in tertiary structure that precede the shift in quaternary structure.

Evolution of the globins

Duplication of a primordial globin gene allowed the separate evolution of myoglobin and a monomeric hemoglobin. Additional duplications among the hemoglobin genes gave rise to six different globin chains that combine to form tetrameric hemoglobin variants at various times during development.

The hemoglobin in red blood cells, like myoglobin, binds O2 reversibly, but it does not exhibit the simple behavior of myoglobin. A plot of fractional saturation (Y) versus pO2 for hemoglobin is sigmoidal (S-shaped) rather than hyperbolic (Fig. 5.7).

Furthermore, hemoglobin's overall oxygen affinity is lower than that of myoglobin: Hemoglobin is half-saturated at an oxygen pressure of 26 torr (p50 = 26 torr), whereas myoglobin is half-saturated at 2.8 torr.

Oxygen transport and the Bohr effect

Hemoglobin picks up O2 in the lungs. In the tissues, H+ derived from the metabolic production of CO2 decreases hemoglobin's affinity for O2, thereby promoting O2 release to the tissues. Back in the lungs, hemoglobin binds more O2, releasing the protons, which recombine with bicarbonate to re-form CO2.

Why is hemoglobin's binding curve sigmoidal? At low O2 concentrations, hemoglobin appears to be reluctant to bind the first O2, but as the pO2 increases, O2 binding increases sharply, until hemoglobin is almost fully saturated. A look at the binding curve in reverse shows that at high O2 concentrations, oxygenated hemoglobin is reluctant to give up its first O2, but as the pO2 decreases, all the O2 molecules are easily given up. This behavior suggests that the binding of the first O2 increases the affinity of the remaining O2-binding sites. Apparently, hemoglobin's four heme groups are not independent but communicate with each other in order to work in a unified fashion. This is known as cooperative binding behavior. In fact, the fourth O2 taken up by hemoglobin binds with about 100 times greater affinity than the first.

Hemoglobin's relatively low oxygen affinity and its cooperative binding behavior are the keys to its physiological function (see Fig. 5.7). In the lungs, where the pO2 is about 100 torr, hemoglobin is about 95% saturated with O2. In the tissues, where the pO2 is only about 20 to 40 torr, hemoglobin's oxygen affinity drops off rapidly (it is only about 55% saturated when the pO2 is 30 torr). Under these conditions, the O2 released from hemoglobin is readily taken up by myoglobin in muscle cells, since myoglobin's affinity for O2 is much higher, even at the lower oxygen pressure. Myoglobin can therefore relay O2 from red blood cells in the capillaries to the muscle cells' mitochondria, where it is consumed in the oxidative reactions that sustain muscle activity. Agents such as carbon monoxide, which interferes with O2 binding to hemoglobin, prevent the efficient delivery of O2 to cells (Box 5.A).

Conformational changes in hemoglobin upon O2 binding

In deoxyhemoglobin (blue), the porphyrin ring is slightly bowed down toward His F8 (shown in ball-and-stick form). The remainder of the F helix is represented by its alpha carbon atoms. In oxyhemoglobin (purple), the heme group becomes planar, pulling His F8 and its attached F helix upward. The bound O2 is shown in red.

The planar heme is tightly wedged into a hydrophobic pocket between helices E and F of myoglobin

It is oriented so that its two nonpolar vinyl (─CH═CH2) groups are buried and its two polar propionate (─CH2─CH2─COO−) groups are exposed to the solvent. The central iron atom, with six possible coordination bonds, is liganded by four N atoms of the porphyrin ring system A fifth ligand is provided by a histidine residue of myoglobin known as His F8 (the eighth residue of helix F) Molecular oxygen (O2) can bind reversibly to form the sixth coordination bond. (This is what allows certain heme-containing proteins, such as myoglobin and hemoglobin, to function physiologically as oxygen carriers.) Residue His E7 (the seventh residue of helix E) forms a hydrogen bond to the O2 molecule (Fig. 5.2). By itself, heme is not an effective oxygen carrier because the central Fe(II) (or Fe2+) atom is easily oxidized to the ferric Fe(III) (or Fe3+) state, which cannot bind O2 Oxidation does not readily take place when the heme is part of a protein such as myoglobin or hemoglobin.

homologous proteins

Proteins that are related by evolution from a common ancestor.

Clearly, the globins are homologous proteins that have evolved from a common ancestor through genetic mutation (see Section 3.4). The α and β chains of human hemoglobin share a number of residues; some of these are also identical in human myoglobin. A few residues are found in all vertebrate hemoglobin and myoglobin chains. The invariant residues, those that are identical in all the globins, are essential for the structure and/or function of the proteins and cannot be replaced by other residues. Some positions are under less selective pressure to maintain a particular amino acid match and can be conservatively substituted by a similar amino acid (for example, isoleucine for leucine or serine for threonine). Still other positions are variable, meaning that they can accommodate a variety of residues, none of which is critical for the protein's structure or function. By looking at the similarities and differences in sequences among evolutionarily related proteins such as the globins, it is possible to deduce considerable information about elements of protein structure that are central to protein function.

Sequence analysis also provides a window on the course of globin evolution, since the number of sequence differences roughly corresponds to the time since the genes diverged. An estimated 1.1 billion years ago, a single globin gene was duplicated, possibly by aberrant genetic recombination, leaving two globin genes that then could evolve independently (Fig. 5.6). Over time, the gene sequences diverged by mutation. One gene became the myoglobin gene. The other coded for a monomeric hemoglobin, which is still found in some primitive vertebrates such as the lamprey (an organism that originated about 425 million years ago). Subsequent duplication of the hemoglobin gene and additional sequence changes yielded the α and β globins, which made possible the evolution of a tetrameric hemoglobin (whose structure is abbreviated α2β2). Additional gene duplications and mutations produced the ζ chain (from the α chain) and the γ and ϵ chains (from the β chain). In fetal mammals, hemoglobin has the composition α2γ2, and early human embryos synthesize a ζ2ϵ2 hemoglobin. In primates, a recent duplication of the β chain gene has yielded the δ chain. An α2δ2 hemoglobin occurs as a minor component (about 2%) of adult human hemoglobin. At present, the δ chain appears to have no unique biological function, but it may eventually evolve one.

The Bohr effect plays an important role in O2 transport in vivo. Tissues release CO2 as they consume O2 in respiration. The dissolved CO2 enters red blood cells, where it is rapidly converted to bicarbonate (HCO3−) by the action of the enzyme carbonic anhydrase (see Section 2.5):

The H+ released in this reaction induces hemoglobin to unload its O2 (Fig. 5.10). In the lungs, the high concentration of oxygen promotes O2 binding to hemoglobin. This causes the release of protons that can then combine with bicarbonate to re-form CO2, which is breathed out.

Somewhat surprisingly, the amino acid sequences of the three globin polypeptides are only 18% identical. Figure 5.5 shows the aligned sequences, with the necessary gaps (for example, the hemoglobin α chain has no D helix). The lack of striking sequence similarities among these proteins highlights an important principle of protein three-dimensional structure: Certain tertiary structures—for example, the backbone folding pattern of a globin polypeptide—can accommodate a variety of amino acid sequences. In fact, many proteins with completely unrelated sequences adopt similar structures.

The amino acid sequences of myoglobin and the hemoglobin α and β chains The sequence of human myoglobin (Mb) and the human hemoglobin (Hb) chains are written so that their helical segments (bars labeled A through H) are aligned. Residues that are identical in the α and β globins are shaded yellow; residues identical in myoglobin and the α and β globins are shaded blue, and residues that are invariant in all vertebrate myoglobin and hemoglobin chains are shaded purple. The one-letter abbreviations for amino acids are given in Figure

A conformational shift explains hemoglobin's cooperative behavior

The four heme groups of hemoglobin must be able to sense one another's oxygen-binding status so that they can bind or release their O2 in concert. But the four heme groups are 25 to 37 Å apart, too far for them to communicate via an electronic signal. Therefore, the signal must be mechanical. In a mechanism worked out by Max Perutz, the four globin subunits undergo conformational changes when they bind O2.

Some of the subunit interactions in hemoglobin

The interactions between the αβ units of hemoglobin include contacts between side chains. The relevant residues are shown in space-filling form. (a) In deoxyhemoglobin, a histidine residue on the β chain (blue, left) fits between a proline and a threonine residue on the α chain (green, right). (b) Upon oxygenation, the His residue moves between two Thr residues. An intermediate conformation (between the deoxy and oxy conformations) is disallowed in part because the highlighted side chains would experience strain.

Oxygen binding to myoglobin depends on the oxygen concentration

The muscles of diving mammals are especially rich in myoglobin. At one time, myoglobin was believed to be an oxygen-storage protein—which would be advantageous during a long dive—but it most likely just facilitates oxygen diffusion through muscle cells or binds other small molecules such as nitric oxide (NO).

A milliliter of human blood contains about 5 billion red blood cells, each of which is packed with about 300 million hemoglobin molecules. Consequently, blood can carry far more oxygen than a comparable volume of pure water.

The oxygen-carrying capacity of the blood can be quickly assessed by measuring the hematocrit (the percentage of the blood volume occupied by red blood cells, which ranges from about 40% (in women) to 45% (in men). Individuals with anemia, too few red blood cells, can sometimes be treated with the hormone erythropoietin to increase red blood cell (erythrocyte) production by bone marrow.

Oxygen binding to hemoglobin

The relationship between fractional saturation (Y) and oxygen concentration (pO2) is sigmoidal. The pO2 at which hemoglobin is half-saturated (p50) is 26 torr. For comparison, myoglobin's O2-binding curve is indicated by the dashed line. The difference in oxygen affinity between hemoglobin and myoglobin ensures that O2 bound to hemoglobin in the lungs is released to myoglobin in the muscles. This oxygen-delivery system is efficient because the tissue pO2 corresponds to the part of the hemoglobin binding curve where the O2 affinity falls off most sharply.

The proportion of the total myoglobin molecules that have bound O2 is called the fractional saturation and is abbreviated Y:

fractional saturation The fraction of a protein's ligand-binding sites that are occupied by ligand. oxygen conc in terms of partial pressure

In deoxyhemoglobin (hemoglobin without any bound O2), the heme Fe ion has five ligands, so the porphyrin ring is somewhat dome-shaped and the Fe lies about 0.6 Å out of the plane of the porphyrin ring. As a result, the heme group is bowed slightly toward His F8 (Fig. 5.8). When O2 binds to produce oxyhemoglobin, the Fe—now with six ligands—moves into the center of the porphyrin plane. This movement of the Fe ion pulls His F8 farther toward the heme group, and this in turn drags the entire F helix so that it moves as much as 1 Å. The F helix cannot move in this manner unless the entire protein alters its conformation, culminating in the rotation of one αβ unit relative to the other. Consequently, hemoglobin has two quaternary structures, corresponding to the oxy and deoxy states.

rotates to be closer together as His F8 is pulled further towards heme group

with a dissociation constant, K:

where the square brackets indicate molar concentrations. (Note that biochemists tend to describe binding phenomena in terms of dissociation constants, sometimes given as Kd, which are the reciprocals of the association constants, Ka, used by chemists.)