Divyan Bavan
Introduction
Structural biology revolves around the premise that a molecule’s function can be explained by its structure. This has been exemplified through the characterization of various protein structures. The first examples of this were hemoglobin and myoglobin—two proteins which share highly similar structures. Both proteins contain heme—a porphyrin ring with an iron ion—along with a globin fold. Due to this similar composition and geometry, these proteins are both able to bind oxygen. However, slight differences in structure lead to very different profiles in the body. These functional specializations are the consequences of various properties such as cooperativity and allosteric regulation. The presence, or absence, of these traits in hemoglobin and myoglobin are responsible for defining the protein’s character.
Oxygen Binding
Both hemoglobin and myoglobin can bind O2. This property is the result of several structural features which both proteins share. For example, myoglobin and hemoglobin monomers share a similar globin fold. Although the α subunit of hemoglobin lacks a D helix, this fold is considered to consist of eight helices. The positions of these helices were determined by Kendrew and Perutz. Kendrew first elucidated the high-resolution structure of sperm whale myoglobin in 1959, with Perutz finding the structure for horse methemoglobin in 1963. Both scientists used X-ray crystallography, more specifically with isomorphous replacement, to discover them. Solving the structures for these proteins greatly help explain some of their properties. For example, the E and F helices—along with smaller contacts from other residues as well—form a hydrophobic pocket in the protein. This is the heme-binding pocket, a key step in establishing oxygen binding (Voet and Voet, 2011).
As expected, the heme-binding pocket is occupied by a heme molecule. This is a combination of a protoporphyrin XI ring and Fe2+ ion. The protoporphyrin ring consists of four pyrrole rings linked by methene bridges. These pyrrole rings contain nitrogen atoms which coordinate the Fe2+ ion. Once the heme is bound to the hemoglobin subunit or myoglobin, it forms a fifth bond. This is with the proximal histidine—His F8 in myoglobin—which also has a coordinating nitrogen atom. Thus, the iron ion within the heme-protein complex is pentacoordinate (Voet and Voet, 2011).
Both hemoglobin and myoglobin are now able to bind oxygen. O2 forms a sixth coordinate bond to Fe2+, stabilizing its binding to hemoglobin and myoglobin. This bond is further stabilized by the distal histidine residue: His E6 in the latter. Unlike the proximal histidine, this residue does not bind directly with the iron. Instead, it forms a bond to O2, increasing its affinity with the protein. Although O2 is the intended molecule to be bound, others can bind as well. A famous example is CO, which binds with more affinity than O2. This is possible as CO can also form a coordinate bond with the heme iron. This is another example of how the behaviour of a protein can be explained by its structure. This mechanism is present in both myoglobin and hemoglobin. However, this is where the two proteins diverge and start to display functional specializations (Voet and Voet, 2011).
Cooperativity in Oxygen Binding
When oxygen binds to myoglobin, there are a few movements in side chains and Fe2+. However, these changes do not alter the properties of myoglobin to a great extent. The same cannot be said for hemoglobin. As mentioned previously, this protein is a tetramer consisting of two α and two β subunits. These subunits form four interfaces; each α subunit forms an interface with two β subunits, and vice versa. However, the α1 subunit binds stronger to the β1 interface. The same is true for α2 and β2. As such, changes in the structure of hemoglobin often change the orientation of these two groups (Voet and Voet, 2011).
When oxygen binds to hemoglobin, it must bind to one of the four subunits. In this subunit, oxygen binding induces a change in the position of Fe2+ with respect to the porphyrin ring. In deoxyhemoglobin, Fe2+ is situated about 0.6 angstroms out of the porphyrin ring. However, upon oxygen binding, it moves into the ring’s plane. This has two effects. First O2 is better able to bind iron because its bond length is stretched due to steric repulsions. This means that oxygen has a higher affinity for this state. Second, the proximal histidine residue shifts to accommodate for the change in the iron’s position. This causes the entire F helix to shift, changing the interface it is a part of. More specifically, oxygen binding leads to a 15° rotation of the α1β1 dimer relative to α2β2. This conformational change leads to Fe2+ in the other three subunits also moving into the porphyrin ring, increasing its affinity for oxygen. This is the R state of hemoglobin, whereas the low-affinity form is the T state. (Voet and Voet, 2011).
Due to its lack of a multimeric structure, myoglobin does not exhibit this mechanism. Instead, myoglobin is always in a high-affinity state for oxygen binding. This has important physiological consequences in the transport of oxygen. During gas exchange in the lungs, the partial pressure of oxygen is about 100Pa. For efficient transport, the protein carrying oxygen should saturate close to this value. However, a critical point is that it should also desaturate closer to the partial pressure of oxygen found in tissues. This is crucial as oxygen must be delivered to these tissues, not just carried around in the blood. Thus, the loading curve for this protein should be sigmoidal. These traits are fit by hemoglobin. Due to its T state, oxygen doesn’t cause much saturation at first. However, as its concentration increases, more hemoglobin shifts towards the R state. This causes a steady increase until it is fully saturated around 100Pa. Myoglobin on the other hand features rapid saturation even at low pO2. It reaches p50 at about 2.8 torr, much lower than physiological pO2 values. In sperm whales and other marine mammals, the role of myoglobin is to store oxygen within muscle tissue. If the oxygen gets very low in these tissues, oxymyoglobin releases its oxygen to keep the tissue alive. In other animals, however, myoglobin’s main role is to speed up transport of oxygen into muscle tissue. This is made possible by its very high affinity for oxygen (Voet and Voet, 2011).
Allosteric Regulation
Hemoglobin’s oxygen affinity is regulated by several allosteric factors. One of these is oxygen, as mentioned previously. Two other regulators are carbon dioxide and 2,3-BPG. The former is important in enhancing the sigmoidal curve already present in hemoglobin loading. This is done through the reaction of carbon dioxide with water, producing carbonic acid; after dissociation, this becomes an H+ ion and a bicarbonate ion. Thus, an increased carbon dioxide concentration will lead to a decrease in pH. The result of this is a stabilization of the T state. This is due to several factors. One of them is increased protonation of His146β. This induces the formation of a salt bridge which stabilizes the T state. This leads to a higher rate of oxygen release. The physiological consequence of this is increased oxygen release at tissues which produce more carbon dioxide, which makes sense. Furthermore, carbon dioxide release in the lungs increases the pH, thus increasing hemoglobin’s affinity for oxygen. This increases the efficiency of oxygen binding (Voet and Voet, 2011).
BPG also contributes to oxygen delivery. This molecule, which has similar effects to IHP and ATP in birds and fish, respectively, binds to the T state of hemoglobin. Thus, BPG stabilizes the T state. This is important for the release of oxygen, as the elimination of BPG from erythrocytes causes them to not release much oxygen into the capillaries. This can be shown experimentally by graphing the loading curve of hemoglobin in vitro in the absence of BPG. The result is that the curve shifts to the left, meaning that less O2 dissociates at the pO2 of tissues. Thus, BPG is critical for proper oxygen transport (Voet and Voet, 2011).
Diseases
As a short note, several diseases can affect hemoglobin. The most famous of these is sickle-cell disease, caused by a mutation of a hydrophilic glutamate to a hydrophobic valine. This causes polymerization of the deoxyhemoglobin mutants, causing sickle-shaped erythrocytes. The result of this is hemolytic anemia and blood flow blockages. Diseases can also affect other aspects of hemoglobin structure, such as polycythemia, which affects the α1-β2 interface (Voet and Voet, 2011).
Conclusion
Hemoglobin and myoglobin are excellent models for understanding how protein structure determines its function. The globin fold and heme pocket enable these proteins to both bind O2, enabling their functions within the body. However, differences in their structures lead to functional specializations. Myoglobin is a monomer which has a high affinity for oxygen; thus, it’s main role is to facilitate oxygen transport and store oxygen in aquatic mammals. Hemoglobin, on the other hand, exhibits positive cooperativity and effectively sequesters and releases oxygen in the lungs and tissues, respectively. This effect is made possible by the differences between its T and R states, a consequence of its tetrameric nature. It is also able to be regulated by chemicals such as CO2 and BPG. However, due to its highly coordinated structure, mutations in hemoglobin can cause various diseases. Thus, it is essential to continue studying hemoglobin’s biophysical profile to better understand these diseases.
Works Cited
Voet, Donald, and Judith G Voet. Biochemistry. 4th ed., Hoboken, N.J., John Wiley And Sons, 2011.