Globular Proteins - Myoglobin & Hemoglobin PDF

Globular Proteins Myoglobin and Hemoglobin E. Agyarko Learning Objectives At the end of the topic students should be able to; Describe the important structural similarities and differences between myoglobin and hemoglobin. Sketch binding curves for the oxygenation of myoglobin a...

Globular Proteins Myoglobin and Hemoglobin E. Agyarko Learning Objectives At the end of the topic students should be able to; Describe the important structural similarities and differences between myoglobin and hemoglobin. Sketch binding curves for the oxygenation of myoglobin and hemoglobin. Explain why the physiological function of hemoglobin requires that its O2-binding curve be sigmoidal rather than hyperbolic. Explain the role of 2,3-bisphosphoglycerate (BPG) and other allosteric effectors in oxygen binding and delivery. Globular Proteins Myoglobin Structure and Function Myoglobin is a hemeprotein present in heart and skeletal muscle It stores oxygen as a reserve against oxygen deprivation and increases the rate of oxygen transport within the muscle cell. Its polypeptide is structurally similar to the individual polypeptide chains of the tetrameric hemoglobin molecule. ~80% of its polypeptide chain folded into eight stretches of α-helix (designated A-H). The interior of the globular myoglobin molecule is composed almost entirely of nonpolar amino acids. The polar amino acids are located almost exclusively on the surface, where they can form hydrogen bonds with each other and with water. Structure of Myoglobin  Myoglobin Structure and Function The heme group of the myoglobin molecule is attached to a pocket, which is lined with nonpolar amino acids, except for two histidine residues. The first one, the proximal histidine (F8), binds directly to the Fe2+ of heme. The second, or distal histidine (E7), does not directly interact with the heme group but helps stabilize the binding of O2 to Fe2+. Thus, the protein, or globin, portion of myoglobin creates a special microenvironment for the heme that permits the reversible binding of one oxygen molecule (oxygenation).  Heme Myoglobin and hemoglobin contain heme. Heme is a cyclic tetrapyrrole consisting of four molecules of pyrrole linked by α-methylene bridges. One atom of ferrous iron (Fe2+) resides at the center of the planar tetrapyrrole. The iron of unoxygenated myoglobin lies 0.03 nm outside the plane of the heme ring, toward His F8. The heme therefore “puckers” slightly. When O2 is bound, the iron moves closer (within 0.01 nm) to the plane of the heme ring. Oxygenation of myoglobin thus is accompanied by motion of the iron, of His F8, and of the residues linked to His F8. Relative Position of Fe2+ in Heme  Oxygen binding to Myoglobin Myoglobin has a higher oxygen affinity at all PO2 values than does hemoglobin. The partial pressure of oxygen needed to achieve half saturation of the binding sites (P50) is ~1 mm Hg for myoglobin and 26 mm Hg for hemoglobin. Myoglobin loads O2 readily at the PO2 of the lung capillary bed (100 mm Hg) but it is an ineffective vehicle for delivery of O2. This is because myoglobin releases only a small fraction of its bound O2 at the PO2 values typically encountered in active muscle (20 mm Hg) or other tissues (40 mm Hg). However, when strenuous exercise lowers the PO2 of muscle tissue to about 5 mm Hg, myoglobin releases O2 for mitochondrial synthesis of ATP, permitting continued muscular activity. The oxygen binding curve for myoglobin is hyperbolic. Oxygen Dissociation Curve for Myoglobin Hemoglobin Structure and Function Hemoglobin is found exclusively in red blood cells (RBC), where its main function is to transport O2 from the lungs to the capillaries of the tissues. Hemoglobin is a tetramer, ie. it has four subunits. There are different types of the hemoglobin molecule based on the composition of the subunits (hemoglobin A (HbA), HbF, HbA2). Hemoglobin Structure and Function Hemoglobin A, the major hemoglobin in adults, is composed of four polypeptide chains (two α chains and two β chains) held together by noncovalent interactions. Each chain (subunit) has stretches of α-helical structure and a hydrophobic heme-binding pocket. The hemoglobin tetramer can be envisioned as composed of two identical dimers, (αβ)1 and (αβ)2. Often described as a dimer of dimers. Hemoglobin Structure and Function Hemoglobin Structure and Function Hemoglobin is described as a dimer of dimers Hemoglobin Structure and Function Each chain (subunit) has stretches of α-helical structure and a hydrophobic heme-binding pocket. Oxygen binding to Hemoglobin Hemoglobin can bind four molecules of O2, one at each of its four heme binding pockets. To bind oxygen, the iron (Fe) of hemoglobin must be in the ferrous (2+) state. Hemoglobin with oxygen bound is called oxyhemoglobin while that without oxygen is deoxyhemoglobin. The degree of saturation (Y) of the oxygen binding sites on all hemoglobin molecules can vary between zero (all sites are empty) and 100% (all sites are full). This phenomenon is used in pulse oximetry. In this method, the oxygen saturation of arterial blood is indirectly measured based on differences in light absorption by oxyhemoglobin and deoxyhemoglobin. Oxygen binding to Hemoglobin Haemoglobin may be considered to exist in two states: the taut/tense (T) state, and the relaxed (R) state. The T form corresponds to the quaternary structure of deoxyhaemoglobin, and R to that of oxyhaemoglobin. The binding of a molecule of oxygen to a haemoglobin subunit stabilizes the R state, and thereby increases the affinity of remaining deoxygenated subunits for oxygen. Oxygen binding to Hemoglobin Oxygen binding to Hemoglobin The binding of oxygen by haemoglobin is cooperative. Thus, as haemoglobin binds successive oxygen molecules, the oxygen affinity of the subunits increases. This behaviour results in an oxygen dissociation curve (a plot of saturation of oxygen-binding sites vs partial pressure of oxygen (PO2)) that is sigmoidal, rather than hyperbolic. Oxygen binding to Hemoglobin Myoglobin is designed to bind oxygen released by hemoglobin at the low pO2 found in muscle. It then releases oxygen within the muscle cell in response to oxygen demand The sigmoidal curve means that hemoglobin becomes highly saturated at high oxygen partial pressures, and releases a significant amount of oxygen at pressures which are fairly low. Hemoglobin is 50% saturated at 26mmHg, while 50% saturation of myoglobin occurs at only 1mmHg. The higher the oxygen affinity (that is, the more tightly oxygen binds), the lower the P50. Because of its cooperative binding mechanism, hemoglobin is more efficient at collecting oxygen where it is in high concentration, and supplying it where it is needed (ie. at low O2 partial pressures).  Primary Regulators of Affinity of Hb for O2 There are four primary regulators, each of which has a negative impact on affinity. These are: hydrogen ion (H+) CO2 chloride ion (Cl–) 2,3-bisphosphoglycerate (2,3-BPG or BPG). ✦ these regulators influence O2 binding independent of each other. These are collectively called allosteric (“other site”) effectors, because their interaction at one site on the hemoglobin molecule affects the binding of oxygen to heme groups at other sites on the molecule. NB: O2 is also an allosteric effector because its interaction at one site on the hemoglobin affects the binding of oxygen to heme groups at other sites. Except that O2 has a positive impact on affinity. Mutations can change hemoglobin’s O2-binding properties and cause disease. Effect of Hydrogen ion Concentration [H+] The binding of protons by hemoglobin lowers its affinity for oxygen. The pH of the blood decreases as it enters the tissues (and the proton concentration rises) Dissociation of carbonic acid produces protons (H+)that react with several amino acid residues in hemoglobin, causing conformational changes that promote the release of oxygen. This contributes to the Bohr Effect. The Bohr Effect The Bohr effect refers to the increase in O2 delivery when CO2 or H+ increases. The release of O2 from hemoglobin is enhanced: when the pH is lowered (ie. proton concentration [H+] is increased) or when the hemoglobin is in the presence of an increased pCO2. Both result in decreased oxygen affinity of hemoglobin  Effect of pH on O2 Saturation Curves As the pH decreases, the affinity of hemoglobin for oxygen decreases, producing the Bohr effect Mechanism of the Bohr Effect The Bohr effect reflects the fact that the deoxy form of hemoglobin has a greater affinity for protons than does oxyhemoglobin. This effect is caused by ionizable groups such as specific histidine side chains that have a higher pKa in deoxyhemoglobin than in oxyhemoglobin. Therefore, an increase in the concentration of protons (resulting in a decrease in pH) causes these groups to become protonated (charged) and able to form ionic bonds (salt bridges). These bonds preferentially stabilize the deoxy form of hemoglobin, producing a decrease in oxygen affinity. Thus: Mechanism of the Bohr Effect In the lungs, oxygen binds to hemoglobin, causing a release of protons, which combine with bicarbonate to form carbonic acid. This decrease of protons causes the pH of the blood to rise. Carbonic anhydrase cleaves the carbonic acid to H2O and CO2, and the CO2 is exhaled. Effect of Carbon Dioxide (CO2) In the tissues, most of the carbon dioxide released from cellular respiration is converted to carbonic acid, catalyzed by carbonic anhydrase. The carbonic acid dissociates into bicarbonate and protons, thus decreasing the pH of the extracellular fluid by increasing the [H+]. The protons bind to Hb, causing it to release oxygen to the tissues. Effect of Carbon Dioxide (CO2) Some of the CO2 is covalently bound to hemoglobin in the tissues. In this form, CO2 forms carbamate adducts with the N-terminal amino groups of deoxyhemoglobin and stabilizes the deoxy conformation In the lungs, where the pO2 is high, oxygen binds to hemoglobin and the bound CO2 is released. Carbamates change the charge on amino terminals from positive to negative, favoring polar bond formation between the α and β chains. Effect of Chloride Ion (Cl–) Chloride reduces the oxygen affinity of mammalian haemoglobin by acting as an allosteric effector that stabilizes the quaternary deoxy (T) structure. It does so by neutralizing electrostatic repulsion by an excess of positive charges in the cavity that runs through the centre of the molecule, but without binding to any specific site. Effect of 2,3-bisphosphoglycerate The compound 2,3-bisphosphoglycerate (2,3-BPG), is derived from the glycolytic intermediate 1,3-bisphosphoglycerate through the Rapoport-Luebering shunt: It is a potent allosteric effector on the oxygen binding properties of hemoglobin. Synthesis of 2,3-BPG represents a major reaction pathway for the consumption of glucose in erythrocytes. the synthesis of 2,3-BPG in erythrocytes is critical for controlling hemoglobin affinity for oxygen. Other cells do produce 2,3-BPG but in trace quantities. Effect of 2,3-bisphosphoglycerate In the deoxygenated T conformation, a cavity capable of binding 2,3- BPG forms in the center of the hemoglobin tetramer. A single molecule of 2,3-BPG can occupy this cavity; stabilizes the T state  Effect of 2,3-bisphosphoglycerate (2,3-BPG) When the oxygen pressure is high as in the alveoli of the lungs, the binding of a mole of O2: induces the T-to-R transition causes the 2,3-BPG binding pocket to collapse 2,3-BPG is expelled allowing for the rest of the monomers of globin protein to bind O2  Effect of 2,3-bisphosphoglycerate (2,3-BPG) Conversely, when 2,3-BPG is not bound in the central cavity, Hb can bind oxygen (forming HbO2) more readily. NOTE increased 2,3-BPG concentration (like increased hydrogen ion concentration), favors: conversion of R form of Hb to T form of Hb decreased amount of oxygen bound by Hb at any oxygen concentration. Different Responses to 2,3-BPG HbF (the fetal form of hemoglobin) is composed of two α-proteins and two γ (gamma) -proteins the γ-globin and β-globin proteins are 72% identical at the amino acid level one significant amino acid difference exists in the 2,3-BPG binding pocket. In the γ-globin protein, His 143 is replaced by a Ser in the 2,3-BPG- binding site. The effect of this amino acid substitution is the loss of two positive charges (one from each γ-subunit relative to the β-subunits). Different Responses to 2,3-BPG Result 2,3-BPG is bound much less avidly to HbF than to HbA Consequence HbF in fetuses binds oxygen with greater affinity than the mothers HbA, giving the fetus preferential access to oxygen carried by the mothers circulatory system.