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Molecular & Cell Biology Hemoglobin Hemoglobin Hemoglobin (Hb) is an iron-containing metalloprotein that functions to transport oxygen in the red blood cells of vertebrates. The protein, which accounts for 97% of the RBC’s dry content is responsible for delivering oxygen from the lungs to all other tissues in the body where it is taken up for cell use. Learning Objective After interacting with this Learning Object, the learner will be able to, Explain the structure of Hemoglobin, oxygen binding & structural changes. Describe factors affecting oxygen binding & Hemoglobin disorders. Compare the studies on Hemoglobin & Myoglobin. Molecular & Cell Biology Hemoglobin Structure of hemoglobin Hemoglobin is a heterotetramer composed of two alpha and two beta chains. The alpha globin gene locus resides on chromosome 16 with each gene contributing to the synthesis of the alpha globin chain. The beta globin gene locus resides on chromosome 11 and consists of all genes that are expressed from the time of embryonic development of Hb to that of the adult beta globin gene. The globin chains are synthesized by ribosomes in the cytosol. Molecular & Cell Biology Hemoglobin Structure of hemoglobin Every subunit of hemoglobin is bound to a prosthetic group known as heme. This consists of a central iron atom in its ferrous state surrounded by a complex organic ring structure known as protoporphyrin. The heme group is essential for the oxygen binding property of hemoglobin. The iron atom forms six coordination bonds, four of which are to the nitrogen atom of porphyrin rings, one to a His side chain in the globin subunit and the other being the binding site for oxygen. Iron in its Fe3+ state does not bind to oxygen. Molecular & Cell Biology Hemoglobin Oxygen binding & structural changes Hemoglobin binds four molecules of oxygen per tetramer, one per heme, in a cooperative manner. The first model for cooperative binding, proposed by Monod, Wyman and Changeux, also known as the MWC model, assumes that each subunit can exist in at least two conformational states with all subunits making the transition from one state to the other simultaneously. Each subunit of a cooperatively binding protein is functionally identical and binding of ligand can occur in either conformation but with varying degrees of affinity for each. Binding of ligand to a low affinity state makes the transition to the high affinity state more likely. Molecular & Cell Biology Hemoglobin Oxygen binding & structural changes The second model for cooperative binding of oxygen to hemoglobin, proposed by Daniel Koshland in 1966, postulates that binding of a ligand to one subunit can induce conformational changes in the other subunits. However, unlike the concerted model, it does not state that all subunits must transition from one state to the other simultaneously. Any conformational change brought about in one subunit enhances the likelihood of a similar change as well as binding of a ligand in the neighboring subunit. A larger number of intermediate states are therefore more likely in the sequential model. Molecular & Cell Biology Hemoglobin Oxygen binding & structural changes Two major conformations of Hb have been deduced by X-ray analysis – the T state which is stabilized in the absence of oxygen and the R state which is relatively more stable in the presence of oxygen. The pair of identical dimers of Hb are linked by several ion pairs at the interface that stabilize it in its deoxy state. Many of these interactions are disrupted upon binding to oxygen and new ones are formed instead. The porphyrin ring in the T state is found to be slightly puckered, thereby causing the iron atom to lie out of plane and protrude onto the proximal histidine. Molecular & Cell Biology Hemoglobin Oxygen binding & structural changes Upon binding to oxygen, one pair of subunits rotates with respect to the other by around 15o. Several interactions at the interaface are therefore disrupted and new ones formed. As Max Perutz rightly postulated, there are changes in the position of many key amino acid residues upon oxygenation. Binding of oxygen causes the iron atom to move back into the plane of the porphyrin ring thereby pulling the proximal histidine along with it. Once the transition from T state to R state takes place in one subunit, the remaining subunits also undergo the same transition more readily, thereby favouring the cooperative binding to oxygen. Molecular & Cell Biology Hemoglobin Oxygen binding & structural changes Cooperative binding of oxygen to hemoglobin allows efficient transfer of oxygen from lungs to tissues. The high partial pressure of oxygen in lungs allows Hb to be saturated upto 98% due to the cooperative nature while saturation level in tissues drops to 32% due to lower partial pressure. This allows 66% of the oxygen taken up to be released. In case of proteins showing no cooperativity, however, the maximum amount of oxygen that can be delivered under similar conditions is only around 38% due to less uptake and relatively less release. This cooperative binding nature leads to a sigmoidal binding curve for Hb allowing it to deliver 1.7 times more oxygen compared to non-cooperative proteins. Molecular & Cell Biology Hemoglobin Factors affecting oxygen binding The effect of pH and CO2 concentration on the binding and release of oxygen by hemoglobin is called the Bohr effect. Lowering the pH and raising the partial pressure of carbon dioxide results in the release of O2 from oxyhemoglobin. In peripheral tissues, CO2 released from Krebs cycle and other cellular processes combines with water to form carbonic acid, which dissociates into protons and bicarbonate ions. Hb which has just released its bound oxygen into the tissues acts as a buffer by binding protons and delivering them to the lungs. In the lungs, the uptake of oxygen by hemoglobin releases protons that combine with bicarbonate ion, forming carbonic acid, which when dehydrated by carbonic anhydrase becomes carbon dioxide, which is then exhaled. Molecular & Cell Biology Hemoglobin Factors affecting oxygen binding The chemical basis for the effect of pH has been found to lie in the interactions of the various side chains in hemoglobin. In deoxyHb, the -His 146 forms a salt bridge with a lysine residue in the subunit. This interaction is however not possible when the His residue is deprotonated, which occurs at high pH. Upon lowering the pH, protonation of the His residue brings about salt bridge formation, thereby favouring the deoxy form and leading to release of bound O2. CO2 reacts with the terminal amino groups forming negatively charged carbamate groups, thereby stabilizing the deoxyHb form. Molecular & Cell Biology Hemoglobin Factors affecting oxygen binding 2,3-bisphosphoglycerate is a highly anionic compound that is present in RBCs at around the same concentration as Hb. 2,3-BPG binds to a central pocket of the T form of Hb tetramer and stabilizes it by interacting with three positively charged amino acids on each β-chain. Transition from T to R state requires bonds to be broken between Hb and 2,3-BPG. This enhances the oxygen releasing capacity of Hb, without which it would be an extremely inefficient transporter molecule. Molecular & Cell Biology Hemoglobin Hemoglobin disorders A large number of mutations have been described in the globin genes. The mutation causing sickle cell anemia is a single nucleotide substitution of A to T in the codon for amino acid 6 of the chain. This change converts a glutamic acid residue to valine in the corresponding amino acid sequence. Replacement of the Glu residue by Val creates a “sticky” hydrophobic contact point at position 6 of the β chain. These sticky spots cause deoxyHbS molecules to associate abnormally with each other leading to clumping of the cells. Their oxygen carrying capacity is greatly reduced and these patients require frequent transfusions. Molecular & Cell Biology Hemoglobin Hemoglobin disorders Thalassemia are the result of abnormalities in hemoglobin synthesis. Deficiencies in βglobin synthesis result in the βthalassemias . Mutation of a single base from G to A in an intron of the β-globin gene generates a new splice site. The resulting mRNA contains a stop codon further upstream and leads to premature translation termination thereby producing aberrant protein. Deficiencies in α-globin synthesis due to inactivation of one or all the four α-globin genes results in the αthalassemias. Molecular & Cell Biology Hemoglobin Comparative study of hemoglobin & myoglobin X-ray crystallography is a very useful visualization technique that facilitates the determination of three-dimensional coordinates of atoms in a protein. Myoglobin was the first protein whose structure was determined by X-ray crystallography studies. When a beam of X-ray was passed through the crystals of myoglobin, some part of the beam was found to pass straight through while the others were scattered in different directions. These scattered beams were detected by means of an X-ray film which, after several spot intensity calculations, provided an electron density map of the protein. The protein was found to consist of a single polypeptide chain having eight alpha helices along with a heme group in the centre similar to hemoglobin. Molecular & Cell Biology Hemoglobin Comparative study of hemoglobin & myoglobin Myoglobin, found largely in muscle tissues, has been found to be structurally similar to the alpha subunit of hemoglobin. The alpha helix arrangement of both proteins has been found to be the same with the recurring structure being known as the globin fold. The hemoglobin chain having 141 amino acids and myoglobin having 153 residues have also been found to have very high sequence homology. Despite the similarities, myoglobin functions largely as an oxygen binding protein that stores a reserve supply of oxygen in muscle tissues while hemoglobin serves to transport oxygen. Molecular & Cell Biology Hemoglobin Comparative study of hemoglobin & myoglobin Myoglobin binds strongly to oxygen and acts as an oxygen storage protein rather than a transporter. It shows 50% saturation at a pressure as low as 2 torr and gets saturated even under low oxygen pressure conditions that prevail in the muscle. Myoglobin can use only 7% of the oxygen carrying capacity as opposed to hemoglobin which can utilize nearly 90% of the carrying capacity. Unlike hemoglobin, which has a sigmoidal oxygen binding curve, myoglobin has a hyperbolic curve indicating that it binds to oxygen irrespective of the surrounding partial pressure of oxygen in the tissues. It is this property that allows sea mammals such as whales that have very high amounts of myoglobin in their muscle to remain underwater for long periods of time. Molecular & Cell Biology Hemoglobin Structure of Hemoglobin 1.Hemoglobin: Hemoglobin isthe predominat metalloprotein in the red blood cells that carries oxygen. Each hemoglobin molecule is made up of four heme groups surrounding a globin group. 2. , subunits: Subunits that occur in the functional organization of macromolecules, usually proteins. 3. Heme: Heme is a prosthetic group containing carbon, nitrogen and hydrogen atoms, with a single Fe2+ ion at the center. 4. Prosthetic group: A tightly bound non – protein chemical compound that is required for the activity of an enzyme. 5. Protoporphyrin IX: A metal-free porphyrin that combines with iron to form the heme of iron-containing proteins. It is made up of four pyrrole rings linked by methene bridges to form a tetrapyrrole ring. Four methyl groups, two vinyl groups, and two propionate side chains are attached. Molecular & Cell Biology Hemoglobin Oxygen binding and structural changes 6. Cooperative binding: It is a form of allosteric binding in which ligand binding to macromolecules having more than one binding site is carried out in a cooperative manner such that binding of ligand at one site increases the affinity of another site for the ligand. In tetrameric hemoglobin, binding of first oxygen molecule to one subunit brings about structural changes which in turn positively influences the binding of the remaining subunits to oxygen. 7. Oxyhemoglobin: When all subunits of hemoglobin are bound to oxygen, it is known as oxyhemoglobin and it transports oxygen to the various tissues of the body. 8. Deoxyhemoglobin: Hemoglobin in oxygen unloaded form is called deoxyhemoglobin. 9. T state: T stands for the tense state. It is one of the two quaternary forms of hemoglobin that predominates in absence of oxygen. It has a lower affinity for substrates and, hence, lower catalytic activity. 10. Models for oxygen binding to Hb: Two models have been proposed by different groups of scientists to explain the binding of oxygen to hemoglobin. These are the concerted and sequential models: Molecular & Cell Biology Hemoglobin Oxygen binding and structural changes a) Concerted model: The first model proposed by Monod, Wyman and Changeux, also known as the MWC model, assumes that each subunit can exist in at least two conformational states and hypothesizes that all subunits make the transition from one state to the other simultaneously. According to this model, each subunit of a cooperatively binding protein is functionally identical. Binding of ligand can occur with either conformation but with varying degrees of affinity for each. Binding of ligand to a low affinity state makes the transition to the high affinity state more likely. b) Sequential model: The second model, proposed by Daniel Koshland in 1966, postulates that binding of a ligand to one subunit can induce conformational changes in the other subunits. Unlike the concerted model, it does not state that all subunits must exhibit transition from one state to the other simultaneously, thereby making a larger number of intermediate states more likely. 11. R state: R stands for relaxed state. One of the two quaternary forms of hemoglobin that predominates when oxygen is bound. . It has a higher affinity for substrates and, hence, higher catalytic activity. 12. Distal histidine: Distal histidine in vertebrate hemoglobins plays an important role in oxygen binding and has been strongly conserved during evolution. It is considered to be important in finetuning the ligand affinities of hemoglobin. Molecular & Cell Biology Hemoglobin Oxygen binding and structural changes 13. Proximal histidine: The heme in hemoglobin is held in the cleft both by hydrophobic interactions and by a covalent bond between the iron and a nitrogen atom of a nearby histidine side chain. This histidine is referred to as the proximal histidine. 14. Sigmoidal plot: The binding of oxygen to hemoglobin displays marked sigmoidal behaviour, which is indicative of cooperation between subunits. The binding of first oxygen causes a conformational change in the other binding sites that makes it easier for oxygen to bind there. This explains the initial up-swing in the sigmoidal curve. The "leveling out" at the top of the curve is caused by saturation of the hemoglobin-oxygen binding sites. Molecular & Cell Biology Hemoglobin Factors effecting oxygen binding 15. Bohr Effect: The effect of pH and CO2 concentration on the binding and release of oxygen by hemoglobin is called the Bohr effect. Lowering the pH and raising the partial pressure of carbon dioxide results in the release of O2 from oxyhemoglobin. 16. Effect of 2,3-bisphosphoglycerate: 2,3bisphosphoglycerate is a highly anionic compound that is present in RBCs at around the same concentration as Hb. 2,3-BPG binds to a central pocket of the T form of Hb tetramer and stabilizes it by interacting with three positively charged amino acids on each β-chain. 17. Fractional saturation: It is the fraction of active sites that are bound to the substrate and is directly proportional to reaction velocity. 18. Oxygen partial pressure: It is the partial pressure of oxygen in the gas phase above the solution. Molecular & Cell Biology Hemoglobin Hemoglobin disorders 19. Sickle cell anemia: Sickle-cell anemia is a genetic disease in which an individual has inherited the allele for sickle-cell hemoglobin from both parents characterized by abnormal, rigid, sickle shape (HbS) as compared to the normal flexible biconcave disk shaped red blood cells (HbA). It results from a single amino acid substitution, a Val instead of a Glu residue at position 6 in the two β chains. As a result of this change, deoxyhemoglobin S has a hydrophobic patch on its surface, which causes the molecules to aggregate into strands that align to form insoluble fibres. Sickle-cell disease may lead to various acute and chronic complications, several of which are potentially lethal. 20. Thalassemia: This is another inherited genetic disorder due to deletions or mutations in the globin genes which results in abnormalities and in α-globin synthesis. Signs and symptoms of thalassemias are due to lack of oxygen in the bloodstream. This occurs because the body doesn't make enough healthy red blood cells and hemoglobin. The severity of symptoms depends on the severity of the disorder. deficiencies 21. Point mutation: A type of mutation resulting from substitution of a single nucleotide base with another. Molecular & Cell Biology Hemoglobin Comparative study of Hemoglobin and Myoglobin 22. Myoglobin (Mb): Myoglobin is a globular protein having a single polypeptide chain consisting of eight alpha helices linked by short polypeptide segments, with a total of 153 amino acid residues. It is found in muscle tissues of most mammals and plays a role in oxygen binding. Myoglobin resembles the alpha subunit of hemoglobin and like hemoglobin, it consists of a central heme group which enables it to bind oxygen. Myoglobin was the first protein to have its X-ray crystallography structure determined in 1959 by John Kendrew.