39.3 OXYGEN TRANSPORT BY HEMOGLOBIN

Once O₂ is extracted from the environment and diffuses into the animal, it is transported to respiring cells. Oxygen is transported in the circulation by specialized body fluids—the blood of vertebrates or the hemolymph of invertebrates.

Blood plasma is the fluid portion of blood without the cells. It can hold only as much O₂ or CO₂ as can be dissolved in solution. How much O₂ goes into solution at a given partial pressure is a measure of its solubility. Because O₂ is about 30 times less soluble than CO₂, only about 0.2 ml of O₂ can be carried in 100 ml of blood. In both vertebrates and invertebrates, hemoglobin evolved as a specialized iron-containing, or heme, molecule for O₂ transport. By binding O₂ and removing it from solution, hemoglobin increases the amount of O₂ in the blood a hundredfold. Because of its greater solubility, CO₂ is transported in solution within the blood. Rather than remaining dissolved in solution as a gas, most of the CO₂ (about 95%) is converted to carbonic acid, which dissociates to form bicarbonate ions (HCO₃⁻) and protons (Chapter 6).

Whereas some invertebrate hemoglobins exist in solution in the hemolymph and others exist within cells carried by the hemolymph, all vertebrate hemoglobins are produced by and reside in red blood cells. Hemoglobin gives the cells and the blood their red appearance.

39.3.1 Hemoglobin is an ancient molecule with diverse roles related to oxygen binding and transport.

Hemoglobin is an ancient molecule that has been found in organisms representing all kingdoms of life. In bacteria, archaeons, and fungi, similar forms of hemoglobin evolved as a terminal electron acceptor in cellular respiration and may have also served a role in scavenging O₂ to avoid damage due to oxidative stress.

Plant hemoglobins are known that bind and transport O₂ within the cell. In soybeans, hemoglobin binds O₂ in root nodule cells to keep concentrations low enough to avoid inhibiting nitrogenase, the enzyme used by symbiotic rhizobial bacteria in the root nodules for nitrogen fixation (Chapter 29).

Invertebrate hemoglobins found in roundworms, annelid worms, and arthropods bind O₂ in the circulatory fluid, as discussed above. They share considerable gene and amino acid sequence similarity with vertebrate hemoglobins, indicating that a common ancestor to invertebrate and vertebrate animals evolved a shared form of hemoglobin more than 670 million years ago.

39.3.2 Hemoglobin reversibly binds oxygen.

Hemoglobin exists in large concentrations within red blood cells. It is a globular protein that consists of four polypeptide units (Fig. 39.11). Each of these four units surrounds a heme group that contains iron, which reversibly binds one O₂ molecule. After O₂ diffuses into the blood, it diffuses into the red blood cells and binds to the heme groups in hemoglobin. Hemoglobin’s binding of O₂ removes O₂ from solution, keeping the pO₂ of the red blood cell below that of the blood plasma so that O₂ continues to diffuse into the cell. The removal of O₂ from the plasma, in turn, keeps the pO₂ of the plasma below that of the lung alveolus, so O₂ continues to diffuse from the lungs into the blood.

FIG. 39.11: What is the molecular structure of hemoglobin and myoglobin?

BACKGROUND In the 1950s, the Austrian Max Perutz worked with John Kendrew in the Cavendish Laboratory of the University of Cambridge to determine the molecular structure of globular proteins. Perutz and Kendrew were interested in understanding how the structures of hemoglobin and myoglobin enabled binding and transport of O₂.

EXPERIMENT Perutz and Kendrew developed and applied the new technique of X-ray crystallography to determine the three-dimensional molecular structures of hemoglobin and myoglobin.

RESULTS Perutz and Kendrew showed that adult hemoglobin consists of four subunits, two α (alpha) and two β (beta) subunits. Each subunit contains a heme group that contains iron, which is the site of O₂ binding. By contrast, myoglobin consists of only a single subunit with one heme group. These differences in molecular structure underlie the O₂-binding and dissociation properties of the two O₂ transport proteins.

FOLLOW-UP WORK Work by Perutz in the 1960s showed how the molecular structure of hemoglobin changes when it binds O₂ versus when it is in an unbound state. Perutz also supervised Francis Crick as a doctoral student, who later worked with James Watson to resolve the structure of DNA. Perutz and Kendrew received the 1962 Nobel Prize in Chemistry for their work.

SOURCES Kendrew J. C., M. F. Perutz. 1948. “A Comparative X-Ray Study of Foetal and Adult Sheep Haemoglobins.” Proceedings of the Royal Society, London, Series A 194:375–98; Perutz, M. F. 1964. “The Hemoglobin Molecule.” Scientific American 211:64–76; Perutz, M. F. 1978. “Hemoglobin Structure and Respiratory Ttransport.” Scientific American 239:92–125.

When more O₂ is present in blood plasma, we expect more O₂ to become bound to hemoglobin, and that is what happens. But there is an interesting wrinkle that enables hemoglobin to readily bind O₂ leaving the lung. If we plot blood pO₂ against the percentage of O₂ bound to hemoglobin (the relation defined as “hemoglobin saturation”), we get the curve shown in Fig. 39.12a. As blood pO₂ increases, hemoglobin saturation rises slowly at first, then more steeply, and then more slowly again until it levels out. At 100% saturation, all hemoglobin molecules bind four O₂ molecules. The curve is called the hemoglobin’s oxygen dissociation curve, and it has a sigmoidal shape—that is, a shape like an “S.”

Figure 39.12: (a) Hemoglobin and (b) myoglobin dissociation curves.

The shape of this curve can be explained as a result of changes in the ability of hemoglobin to bind O₂ (a property called its binding affinity) at different O₂ partial pressures. At 25% saturation, each hemoglobin molecule binds on average one O₂ molecule. As pO₂ rises, hemoglobin binds O₂ with increasing binding affinity. This increase in affinity results from the interaction of adjacent heme groups in each hemoglobin molecule. After one heme group binds the first O₂ molecule, hemoglobin undergoes a conformational change that increases the binding affinity of the remaining heme groups for additional O₂. The increase in binding affinity with additional binding of O₂ is called cooperative binding, and it gives the O₂ dissociation curve for hemoglobin its sigmoidal shape.

The increasing affinity of hemoglobin for O₂ with increasing O₂ concentration has important physiological consequences. In the middle part of the O₂ dissociation curve, small increases in O₂ concentration lead to large increases in hemoglobin saturation. Therefore, under normal circumstances, hemoglobin in the blood leaving the lung is fully saturated with O₂ —each hemoglobin molecule is bound to four O₂ molecules.

The shape of the O₂ dissociation curve also helps to explain how O₂ is delivered to respiring cells. When the hemoglobin in red blood cells reaches tissues needing O₂ to supply their mitochondria, the O₂ is released from the hemoglobin. As active cells consume O₂, they reduce the local pO₂ of the cell and surrounding tissues to 40 mmHg or less. At these lower pO₂ values, the hemoglobin’s O₂ dissociation curve has a steep slope (Fig. 39.12a). The steepness of the slope indicates that for a relatively small decrease in pO₂, large amounts of O₂ can be released from hemoglobin to diffuse into the cell.

39.3.3 Myoglobin stores oxygen, enhancing delivery to muscle mitochondria.

Myoglobin is a specialized O₂ carrier within the cells of vertebrate muscles. In contrast to hemoglobin, myoglobin is a monomer that contains only a single heme group. Because there are no interacting subunits, the O₂ dissociation curve for myoglobin has a different shape from that of hemoglobin (Fig. 39.12b). In fact, over the physiological range of pO₂, myoglobin has a greater affinity for O₂ than hemoglobin does and binds O₂ more tightly. As a result, hemoglobin releases O₂ to exercising muscles. Once the exercising muscles consume the available O₂, the intracellular pO₂ drops, and the myoglobin releases its bound O₂ to the muscle cell’s mitochondria.

Red muscle cells that depend mainly on aerobic respiration to produce ATP (Chapter 37) store large amounts of myoglobin. The myoglobin in these cells can release O₂ quickly at the onset of activity, before respiratory and circulatory systems have had time to increase the supply of O₂. Diving marine mammals, such as whales and seals, carry large amounts of myoglobin within their muscles. The myoglobin loads up with O₂ when the animals breathe at the surface before a dive. The O₂ bound to the myoglobin is then used to supply ATP during the dive, when the animal cannot breathe. These marine mammals can stay under water for 30 minutes or longer and dive to considerable depths (Fig. 39.13).

Figure 39.13: Myoglobin as an O₂ store for long-lasting dives. The graph tracks the dives of a beaked whale (Ziphius cavirostris) fitted with a tracking device.

39.3.4 Many factors affect hemoglobin-oxygen binding.

Obtaining enough O₂ is a special challenge in some circumstances. How does a mammalian fetus in its mother’s uterus take up O₂ from its mother’s blood? How do animals that live at high altitude obtain O₂ under conditions of low pO₂? In each case, the species adapts by evolving forms of hemoglobin with higher binding affinity.

In mammals, the mother’s respiratory and cardiovascular systems must provide the fetus with O₂ and remove CO₂, as well as supply nutrients and remove waste. Maternal and fetal blood do not mix. Rather, they exchange gases across the placenta. Yet the O₂ concentration gradient at this exchange point does not favor the movement of O₂ from the mother’s blood to the fetal blood. Fetal mammals solve the problem of extracting O₂ from their mother’s hemoglobin by producing a form of hemoglobin before birth that has a higher affinity for O₂ than their mother’s hemoglobin. The O₂ dissociation curve for fetal hemoglobin is shifted to the left of the curve for maternal hemoglobin (Fig. 39.14a), allowing the fetal hemoglobin to extract O₂ from its mother’s circulation. At birth, when the newborn begins to breathe, its red blood cells rapidly shift to producing the adult form of hemoglobin, maintaining this form throughout life.

Figure 39.14: Changes in hemoglobin’s O₂ dissociation curve.

Many animals live at high altitude. For example, birds have been observed flying over the highest peaks (taller than 9000 m) despite the fact that air at this altitude has less than one-third the O₂ content of air at sea level. Similarly, llamas inhabit the Andes at altitudes up to 5000 m, where at a pO₂ of approximately 80 mmHg the air has about one-half the O₂ content of air at sea level. Consequently, the lung pO₂ of a llama is only about 50 mmHg. How do highflying birds and llamas obtain enough O₂? Their hemoglobin has a higher affinity for O₂ and binds O₂ more readily than that of mammals living at sea level. Consequently, their O₂ dissociation curve is shifted to the left, favoring the binding of more O₂ at a given pO₂.

Tissue and blood pH also has an important physiological effect on the O₂ affinity of hemoglobin. When a decrease in pH (that is, an increase in H⁺ concentration) occurs during exercise, the affinity of hemoglobin for O₂ decreases, resulting in a rightward shift in the O₂ dissociation curve (Fig. 39.14b). This phenomenon is called the Bohr effect after Christian Bohr, the Danish physiologist who first described it in 1904. Decreases in pH occur when CO₂ is released from metabolizing cells, or when an inadequate supply of O₂ leads to the production of lactic acid (Chapter 7). Because hemoglobin’s affinity for O₂ is reduced, more O₂ is released and supplied to the cells for aerobic ATP synthesis.

Carbon dioxide also reacts with the amine (NH₂) groups of hemoglobin, reducing hemoglobin’s affinity for O₂. Thus, when released from respiring tissues, CO₂ promotes increased O₂ delivery both through its direct effect on hemoglobin and its contribution to a decrease in blood pH by the Bohr effect. While the production of CO₂ promotes O₂ release at the tissues, its elimination at the lung increases hemoglobin’s affinity for O₂, enhancing O₂ uptake.