Hemoglobin (Haemoglobin in many varieties of English) is a tetramer consisting of two dimers that binds to oxygen. Hemoglobin is the oxygen binding protein of red blood cells and is a globular protein with quaternary structure. Hemoglobin consists of four polypeptide subunits; 2 alpha chains and two beta chains. Hemoglobin transports oxygen in the blood from the lungs to the rest of the body. The three-dimensional structure of hemoglobin was solved using X-ray crystallography in 1959 by Max Perutz. The structure of hemoglobin is very similar to the single polypeptide chain in myoglobin despite the fact that their amino acid sequences differ at 83% of the residues. This highlights a relatively common theme in protein structure: that very different primary sequences can specify very similar three-dimensional structures.
There are two states in the hemoglobin, the T state (the tense state) and the R state (the relaxed state). The T state has a less of an affinity for oxygen than the R state. In the concerted mode of cooperativity, the hemoglobin must either be in its T state or R state. In the sequential mode of cooperativity, the conformation state of the monomer changes as it binds to oxygen. Actual experimental observation of hemoglobin shows that it is more complex than either of the models and is somewhere in between the two. The conformation of hemoglobin also changes as the oxygen binds to the iron, raising both the iron and the histidine residue bound to it. The oxygen binding changes the position of the iron ion by approximately 0.4 Å. Before oxygenation the iron ion lies slightly outside the plane of the porphyrin upon oxygenation it moves into the plane of the heme. The oxygen affinity of hemoglobin decreases as the pH decreases. This is useful because with a high affinity for oxygen in the lungs, hemoglobin can effectively bind to more oxygen. Once it reaches the muscle, where the pH is lower, the lowered affinity for oxygen allows hemoglobin to release its oxygen into the muscle tissues. When carbon dioxide diffuses into red blood cells, its dissociation also causes a decrease in pH.
The affinity of hemoglobin for oxygen is less than its structural analog myoglobin. Interestingly enough, however, this does not affect hemoglobin’s usefulness for the body; on the contrary, it allows hemoglobin to be a more efficient oxygen carrier than myoglobin. This is so because hemoglobin can release oxygen more easily than can myoglobin. While it is important for oxygen to be carried to different areas of the body, it is even more important for the oxygen to be released when needed. The higher the affinity of a given protein for oxygen, the harder it will be for that protein to release oxygen when the time comes. Thus, hemoglobin’s lower affinity for oxygen serves it well because it allows hemoglobin to release oxygen more easily in the body. Myoglobin, on the other hand, has a significantly higher affinity for oxygen and will, therefore, be much less inclined to release it once it is bound. Thus hemoglobin’s lower affinity for oxygen relative to myoglobin allows it to have a higher overall efficiency in binding and then releasing oxygen species. For this reason, the body tends to use hemoglobin more often for oxygen-distributing purposes, although myoglobin is used as well, particularly for carrying oxygen to muscle cells. More can be read on myoglobin in the appropriate section.
Also worth mentioning is the fact that fetal hemoglobin has a noticeably higher affinity for oxygen than does maternal hemoglobin. This is of crucial importance during pregnancy in human females (and presumably in other pregnant mammalian females) because it allows the fetus to obtain much-needed oxygen during development. Basically, the hemoglobin present in the fetus is able to strip oxygen species from the maternal hemoglobin when the mother’s blood comes into contact with fetal material. The portion of the mother’s blood that does not touch the fetus transfers oxygen as normal to the mother’s organ systems.
When oxygen is bound to hemoglobin, the color changes to crimson red. When oxygen is not bound, the color becomes a dark “rustic” shade of red  . Hemoglobin’s affinity to oxygen increases as more oxygen is bound to it. The disassociation curve represents how hemoglobin is cooperative to oxygen with its sigmoidal shape. - The left shift shows an increase in oxygen affinity. Hemoglobin has a better chance to hold onto oxygen. This normally occurs with a change in environmental factors such as low temperature, low metabolism rate, and high pH.
- The right shift shows a decrease in affinity. Hemoglobin is more likely to release Oxygen. This is due to high temperature, high metabolism, and low pH.
While Hemoglobin has 4 subunits, Myoglobin has one subunit. It is the enzyme of oxygen storage within the cells (found in skeletal muscle cells). The reason muscles are red is because they contain large amount of myoglobin. Organisms such as diving mammals have very large amounts of myoglobin so that they can go for an extended period of time without breathing.
As mentioned above, hemoglobin exists in two distinct states: the T-state and the R-state. The T-state of hemoglobin is the more “Tense” of the two; this is the deoxy form of hemoglobin (meaning that it lacks an oxygen species) and is also known as "deoxyhemoglobin. The R-state of hemoglobin is more “Relaxed” and is the fully oxygenated form; it is also known as oxyhemoglobin…
One of the unique features of hemoglobin is that it exhibits cooperativity. This means that hemoglobin can transmit intramolecular messages to its various functional groups to help it attain a maximum affinity for the ligand of interest, which is oxygen in this case. When a monomer of hemoglobin binds to oxygen, it alerts other nearby hemoglobin monomers to start the binding process as well. This means that, as more and more oxygen is bound by hemoglobin monomers, the affinity of hemoglobin will increase more and more as well. In other words, the affinity of hemoglobin is proportional to the quantity of oxygen bound at a given time. This allows hemoglobin to increase its affinity for oxygen over time, a property that brands it as one of the most flexible proteins in the body. Because it can modify its affinity for oxygen, hemoglobin can exhibit a range of different affinities. As stated before, this makes it quite flexible in terms of how much oxygen it can bind and therefore how much it can release. This is one of the reasons that the body prefers to use hemoglobin, as opposed to myoglobin, for oxygen transport: hemoglobin can modify its own affinity for oxygen to suit the situation at hand, making it capable of handling a wider variety of chemical environments and organ systems while still being able to distribute oxygen effectively.There are two main models of cooperativity for hemoglobin. One of these is the concerted model of cooperativity. This model states that the hemoglobin molecule changes rapidly between its R- and T-states in order to maximize its affinity for oxygen. According to this model, hemoglobin is constantly “flipping” back and forth between states in an attempt to bind as much oxygen as possible. The other model is the sequential model of cooperativity. This model maintains that one strand of hemoglobin starts a sequence of conformational changes in hemoglobin that increase its affinity for oxygen. When one strand of hemoglobin binds oxygen, the hemoglobin rearranges in a manner that favors additional oxygen binding. When the next oxygen is bound, another conformational change occurs to further supplement binding; Thus, hemoglobin can sequentially increase its affinity for oxygen as more and more of its strands bind oxygen.
Experimental data obtained from kinetics experiments with hemoglobin reveals that neither the concerted nor the sequential model of cooperativity is heavily favored. If anything, the data suggests that hemoglobin’s behavior represents a hybrid of the two models; thus hemoglobin’s cooperativity is somewhere in between the concerted and sequential models.
It is known that hemoglobin undergoes several conformational changes upon binding with oxygen. First of all, as soon as the iron cation within hemoglobin begins to move, the Histidine residues and the alpha-helix of hemoglobin start moving as well to stabilize the changes caused by the movement of iron. Second, the carboxyl terminal end of the alpha-helix usually resides at the interface between the two alpha- and beta-dimers that make up hemoglobin. Finally, the positional changes of the carboxyl terminal end create favorable conditions for transitions between the T- and the R-states of hemoglobin.
The above description makes clear that the concerted and sequential models do not fully explain hemoglobin’s behavior, nor the behavior of related classes of proteins. To account for this discrepancy, more complex models have been devised that more accurately reflect the kinetic data gained from experiments with hemoglobin binding.Oxygen binding to iron in the heme group pulls part of the electron density from the ferrous ion to the oxygen molecule. It is important to leave the myoglobin in the dioxygen form rather than superoxide form when the oxygen is released because the superoxide can be generated by itself to have a new form that gives negative effect on many biological materials, and also the superoxide prevents the iron ion from binding to the oxygen in its ferric state (Metmyoglobin). Superoxide and superoxide-derived oxygen species are so reactive compared to the stable O2 molecule that they could have a destructive effect both within the cell and in its environment. A distal histidine residue in myoglobin regulates the reactivity of the heme group to make it more suitable for oxygen binding. It does this by H-bonding with the oxygen molecule; the additional electron density of the oxygen molecule makes the H-bond unusually strong and therefore even more effective as a stabilizing agent.
An oxygen-binding curve is a plot that shows fractional saturation versus the concentration of oxygen. By definition, fractional saturation indicates the presence of binding sites that have oxygen. Fractional saturation can range from zero (all sites are empty) to one (all sites are filled). The concentration of oxygen is determined by partial pressure.
Hemoglobin’s oxygen affinity is relatively weak compared to myoglobin’s affinity for oxygen. Hemoglobin’s oxygen-binding curve forms in the shape of a sigmoidal curve. This is due to the cooperativity of the hemoglobin. As hemoglobin travels from the lungs to the tissues, the pH value of its surroundings decrease, and the amount of CO2 that it reacts with increases. Both these changes causes the hemoglobin to lose its affinity for oxygen, therefore making it drop the oxygen into the tissues. This causes the sigmoidal curve for hemoglobin in the oxygen-binding curve and proves its cooperativity.
In red blood cells, the oxygen-binding curve for hemoglobin displays an “S” shaped called a sigmoidal curve. A sigmoidal curve shows that oxygen binding is cooperative; that is, when one site binds oxygen, the probability that the remaining unoccupied sites that will bind to oxygen will increase.
The importance of cooperative behavior is that it allows hemoglobin to be more efficient in transporting oxygen. For example, in the lungs, the hemoglobin is at a saturation level of 98%. However, when hemoglobin is present in the tissues and releases oxygen, the saturation level drops to 32%; thus, 66% of the potential oxygen-binding sites are involved in the transportation of oxygen.
Purified hemoglobin binds much more tightly to the oxygen, making it less useful in oxygen transport. The difference in characteristics is due to the presence of 2,3-Bisphosphoglycerate(2,3-BPG) in human blood, which acts as an allosteric effector. An allosteric effector binds in one site and affects binding in another. 2,3-BPG binds to a pocket in the T-state of hemoglobin and is released as it forms the R-state. The presence of 2,3-BPG means that more oxygen must be bound to the hemoglobin before the transition to the R-form is possible.
Other regulation such as the Bohr effect in hemoglobin can also be depicted via an oxygen-binding curve. By analyzing the oxygen-binding curve, one can observe that there is a proportional relationship between affinity of oxygen and pH level. As the pH level decreases, the affinity of oxygen in hemoglobin also decreases. Thus, as hemoglobin approaches a region of low pH, more oxygen is released. The chemical basis for this Bohr effect is due to the formation of two salt bridges of the quaternary structure. One of the salt bridges is formed by the interaction between Beta Histidine 146 (the carboxylate terminal group) and Alpha Lysine 40. This connection will help to orient the histidine residue to also interact in another salt bridge formation with the negatively charged aspartate 94. The second bridge is form with the aid of an additional proton on the histidine residue.
As carbon dioxide diffuses into red blood cells, it reacts with water inside to form carbonic acid. Carbonic acid disociated leads to lower pH and stabilizes the T state.
An oxygen-binding curve can also show the effect of carbon dioxide presence in hemoglobin. The regulation effect by carbon dioxide is similar to Bohr effect. A comparison of the effect of the absence and presence of carbon dioxide in hemoglobin revealed that hemoglobin is more efficient at transporting oxygen from tissues to lungs when carbon dioxide is present. The reason for this efficiency is that carbon dioxide also decreases the affinity of hemoglobin for oxygen. The addition of carbon dioxide forces the pH to drop, which then causes the affinity of hemoglobin to oxygen to decrease. This is extremely evident in the tissues, where the carbon dioxide stored in the tissues are released into the blood stream, then undergoes a reaction that releases H+ into the blood stream, increasing acidity and dropping the pH level.