Module 3: Myoglobin and hemoglobin Flashcards
Myoglobin and Hemoglobin Structure
KEY CONCEPTS
Myoglobin is a protein consisting of a single subunit that contains a heme group with an iron atom bound. The iron atom binds one molecule of oxygen.
The tertiary structure of myoglobin is almost identical to each of the globin subunits of hemoglobin, though their amino acid sequences are quite different.
Hemoglobin is a tetramer protein containing two alpha and two beta globin subunits. As such, hemoglobin has quaternary structure, while myoglobin has only tertiary structure because it has only one subunit.
Each subunit contains a heme molecule and an iron atom, allowing each hemoglobin protein to bind four molecules of oxygen.
Hemoglobin structure is exquisitely sensitive to oxygen binding, and the entire conformation of the protein changes as a result of oxygen binding or release.
The oxygenated form is referred to as the relaxed or “R” state, while the deoxygenated form is know as the tense or “T” state of the protein.
Sickle cell anemia is an inherited disease that results from changes in hemoglobin structure.
Myoglobin is an Oxygen-Binding Protein Monomer
Myoglobin is a small protein inside vertebrate muscle cells. Its structure consists of a monomer (single polypeptide chain with no quaternary structure) with eight alpha helices that are arranged to form a globular protein (Fig. 3-1).
Myoglobin Contains a Heme Group
Myoglobin contains a single heme group (Figure 3-2). The heme is tightly wedged in a hydrophobic pocket between two alpha helices (Figure 3-1). The heme is a heterocyclic (meaning it contains both carbon and nitrogen) ring system known as a porphyrin. These types of molecules are named for their absorption of light, which leads to their bright colors (“porphyrin” comes from the Greek word “porphyra” which means purple). These types of systems, in which every other bond is a double bond, are known as conjugated systems and absorb colored light well.
Hemoglobin Is a Tetramer with Two Conformations
Hemoglobin, the protein inside red blood cells that give them their color, is one of the best-characterized proteins and was one of the first proteins to be associated with a specific physiological function (oxygen transport). Mammalian hemoglobin is an α2β2 tetramer, meaning it has two α (alpha) and two β (beta) subunits. (Note that these alpha/beta designations are in reference to the subunits, not the secondary structures inside these subunits.). The α and β subunits are structurally related to each other and to myoglobin; there are only small differences between the monomer of myoglobin and the α/β subunits of hemoglobin.
Sickle Cell Anemia: A Problem with Hemoglobin Structure
Mutations in the DNA sequence for the genes that encode the alpha and beta chains of hemoglobin produce hemoglobin proteins with altered amino acid sequences. In some cases, the mutation is benign and the hemoglobin molecules function more or less normally. But in other cases, the mutation results in serious physical complications for the individual, as the ability of the mutant hemoglobin to deliver oxygen to cells is compromised. A well-known hemoglobin variant is sickle cell hemoglobin (known as hemoglobin S or Hb S). Individuals with two copies of the defective gene develop sickle cell anemia, a debilitating disease that affects predominantly those of African descent. It’s estimated that 1 in 5,000 Americans has sickle cell anemia, and that over 2 million Americans carry the Hb S gene.
Sickle cell anemia is caused by the mutation of the beta hemoglobin gene that changes the charged amino acid glutamate (Glu) located at position 6 in the primary structure to the hydrophobic amino acid valine (Val). This amino acid is positioned on the outer surface of the protein, so the mutation to Val results in a hydrophobic amino acid anxious for opportunities to make hydrophobic interactions to escape the water that surrounds it. In normal hemoglobin, the switch from the oxy to the deoxy conformation exposes a hydrophobic patch on the protein surface between two of the alpha helices. The hydrophobic Val residues on hemoglobin S are optimally positioned to bind to this patch. This association between the Val of one Hb S and the hydrophobic patch of another Hb S leads to the rapid aggregation of hemoglobin S molecules to form long, rigid fibers.
Myoglobin Function
KEY CONCEPTS
Myoglobin binds oxygen reversibly in response to oxygen concentration.
Oxygen Binds Reversibly to Myoglobin
Myoglobin is found in the muscles of most mammals, and its function is to bind and store oxygen for the muscles to use when blood oxygen is low. For example, if you suddenly sprint to catch a bus, it’s likely that your breathing will not keep up with the oxygen demand of your muscles (you will feel “out of breath”), but you are able to sustain the sprint for a short while because the myoglobin in your muscles is able to deliver the oxygen needed to keep them moving. The same is true if you hold your breath: your muscles will use the oxygen stored in myoglobin to continue to function for a short time. Mammals that are diving animals, such as whales and dolphins, have high amounts of myoglobin in their muscles which enables them to hold their breath for an extended period of time.
As we saw in the previous chapter, myoglobin is a protein with a single subunit containing both heme and iron. Oxygen can bind reversibly to the iron, meaning that it will bind to the iron when oxygen concentrations are relatively high and release from the iron when oxygen concentration are low (such as when you hold your breath). Thus, whether or not myoglobin binds oxygen will depend on the concentration of oxygen around it (Figure 3-8). A graph of myoglobin’s oxygen binding has a hyperbolic shape (increases rapidly and then levels off), indicating its sensitivity to low oxygen concentrations.
Hemoglobin Function
KEY CONCEPTS
Hemoglobin binds oxygen cooperatively due to structural communication between the four subunits. The cooperativity allows the protein to coordinate oxygen binding and release to support its function in oxygen delivery.
The Bohr effect describes the influence that pH has on oxygen binding by hemoglobin. At high pH hemoglobin has a high affinity for oxygen, while at low pH it has a low affinity for oxygen and tends to release it.
2,3-BPG binds to deoxygenated hemoglobin and stabilizes the T state of the protein.
Carbon monoxide binds to the iron atoms in hemoglobin and stabilizes the R state of the protein.
Oxygen Binds Cooperatively to Hemoglobin
A milliliter of human blood contains about 5 billion red blood cells, each of which is packed with ~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 to increase red blood cell production.
Like myoglobin, the hemoglobin in red blood cells binds O2 reversibly, but it does not exhibit the simple behavior of myoglobin. A plot of the fraction of hemoglobin with oxygen bound versus oxygen concentration is sigmoidal (S-shaped) rather than hyperbolic (Fig. 3-8). Furthermore, hemoglobin’s overall oxygen affinity is lower than that of myoglobin: hemoglobin is half-saturated (50% of all hemoglobin is full of oxygen) at an oxygen pressure of 26 torr, whereas myoglobin is half-saturated at 2.8 torr. Thus, it takes a much higher concentration of oxygen to fill hemoglobin than it does myoglobin, despite the structural similarity of their individual subunits.
Why is hemoglobin’s binding curve sigmoidal? At low O2 concentrations, hemoglobin appears to be reluctant to bind the first O2, but as the O2 concentration 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 O2 concentration 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. In fact, the fourth O2 taken up by hemoglobin binds with about 100 times greater affinity than the first. 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.
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 they can bind or release their O2 at the same time. The signal of one subunit to the next is provided by the conformational change that occurs when the subunit binds O2 and segments of the protein literally move. As we saw in the previous chapter, when oxygen binds to the iron atom in a heme group it causes the heme to change from a bent to a planar state. The change in the heme shape also “tugs” on the protein chain holding the heme group, causing a conformational shift in the protein subunit. Once one subunit has undergone the conformational shift, it influences the conformations of the neighboring subunits such that oxygen binding occurs more easily.
Recall that the two conformational states of hemoglobin quaternary structure are formally known as T (for “tense”) and R (for “relaxed”). The T state corresponds to deoxyhemoglobin, and the R state corresponds to oxyhemoglobin. The shift in conformation between the oxy and deoxy states primarily involves rotation of the subunits relative to one another.
The Bohr Effect Enhances Oxygen Transport
The conformational changes in hemoglobin that occur upon oxygen binding or release are also impacted by the pH surrounding the protein. For example, the T state of hemoglobin is favored by low pH and induces the release of oxygen, while the R state is favored by higher pH and stimulates hemoglobin to bind more O2 by increasing its affinity for oxygen.
The T-State is Favored by 2,3-BPG
Other molecules can also influence the conformation of hemoglobin and affect its oxygen delivery. For example, 2,3-bisphosphoglycerate (2,3-BPG) is a molecule produced by red blood cells that binds preferentially to deoxygenated hemoglobin and stabilizes the T state. This reduces hemoglobin’s affinity for O2 and promotes O2 release to the tissues, in much the same way as low pH. The amount of 2,3-BPG increases under conditions of low tissue O2 concentration, including high altitude conditions. Levels of 2,3-BPG also increase dramatically in the RBCs of pregnant women to facilitate greater transfer of O2 to fetal blood. Fetal hemoglobin, on the other hand, has a single amino acid difference (compared to adult hemoglobin) that prevents it from interacting efficiently with 2,3-BPG. As a result, fetal hemoglobin has a much higher affinity for O2 than adult hemoglobin. The combined effect of the lower O2 affinity of maternal hemoglobin and the increased O2 affinity of fetal hemoglobin allows O2 transfer to the fetus to happen quickly and efficiently throughout gestation.
Carbon Monoxide Poisoning: A Problem with Hemoglobin Function
Carbon monoxide (CO) is another molecule that influences the ability of hemoglobin to deliver O2. The affinity of hemoglobin for CO is over 200 times higher than its affinity for O2. 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 CO production in the body.
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 (R state), so that when O2 does bind to some of the hemoglobin heme groups in the lungs, O2 release to the tissues is impaired. In other words, carboxyhemoglobin never switches to the T state to allow for efficient oxygen delivery to the tissues. The binding of CO is sometimes said to shift the Bohr effect to the left (Figure 3-10) because it has the same effect as high pH—an increased affinity for O2 that leads to less O2 release. 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.
