Hemoglobin PDF

Hemoglobin Presented by Dr. Zahi Damuni Professor of Biochemistry Reading: Lippincott Reviews in Biochemistry, 8th Ed., Chapter 3 1 Office hours: Typically, online via https://teach.webex.com/meet/zahi.damuni and will be announced in advance Email for questions or to set up a one-on-one meeting: z...

Hemoglobin Presented by Dr. Zahi Damuni Professor of Biochemistry Reading: Lippincott Reviews in Biochemistry, 8th Ed., Chapter 3 1 Office hours: Typically, online via https://teach.webex.com/meet/zahi.damuni and will be announced in advance Email for questions or to set up a one-on-one meeting: [email protected] 1 Learning Objectives 1. 2. 3. 4. 5. 6. 7. 8. Understand myoglobin and hemoglobin with respect to α-helical structure, the role of nonpolar interactions for the tertiary structure, and the roles of non-covalent interactions, proximal histidine and distal histidine for heme binding to the apoprotein. Understand the subunit structure of major adult hemoglobin and fetal hemoglobin and specify the interactions between subunits. Illustrate the reversible, coordinate covalent nature of oxygen binding to the heme iron and the importance of the iron being in the ferrous state. Understand the binding of 2,3-bisphosphoglycerate to adult hemoglobin and to fetal hemoglobin. Understand the combined effects of carbon dioxide, hydrogen ions and 2,3bisphosphoglycerate on oxygenation of hemoglobin in the lungs and in the tissues. Identify changes in protein structure due to mutations and deletions of globin genes that lead to disease. Understand the competitive interaction between carbon monoxide and oxygen in carbon monoxide poisoning and name the therapeutic approach. State the chemical differences between hemoglobin and methemoglobin and main causes of methemoglobin formation. 2 2 Hemoproteins • This is a group of specialized proteins that contain heme as a tightly bound prosthetic group. • Role of the heme group is dictated by the environment created by the 3-dimensional structure of the protein. 1. The heme group functions as an electron carrier in cytochromes. 2. The heme group is a part of the active site of catalase which catalyzes the breakdown of H2O2. 3. The heme group serves to bind oxygen reversibly in hemoglobin and myoglobin. 3 3 Heme • • • Heme consists of a planar porphyrin ring with an iron (Fe2+) in the center Methyl (1C) and vinyl or propionate (3C) side chains Fe2+ can form 6 covalent bonds: • 2 covalent and 2 coordinate bonds with N’s in the porphyrin ring • 1 coordinate bond with a His-residue in the globin chain and another 1 with oxygen 4 A coordinate covalent bond, also known as a dative bond or coordinate bond is a kind of 2-center, 2-electron covalent bond in which the two electrons derive from the same atom. 4 Myoglobin • A monomeric heme protein found in skeletal and heart muscle • Has 153 amino acid residues (Human Mb) • 80% of the polypeptide chain is folded into 8 stretches of -helix (labeled A to H) • Interior is composed almost entirely of non-polar amino acids • Stabilized by hydrophobic interactions • heme Charged amino acids are located mostly on the surface • Can form hydrogen bonds with water “proximal” histidine “distal” histidine 5 5 Myoglobin Forms a crevice that almost completely encloses a heme group o Hydrophobic interactions between the porphyrin ring of heme and the amino acid R-groups on the interior of the protein cleft stabilize the heme-protein conjugate o Cleft contains 2 histidine residues:  Proximal histidine (His F8) – binds directly to the iron in heme  Distal histidine (His E7) – function to assist O2-binding o A Phe residue is also at the surface of the cleft helping to hold the heme in place 6 6 Oxygenation of myoglobin is accompanied by movement of the iron atom, and consequently movement of His F8 and residues covalently linked to His F8, toward the plane of the ring. Oxygenation brings about a new conformation. 7 7 Hemoglobin: A Tetrameric Protein Found Mainly in RBC Hemoglobin is a tetramer composed of two non-identical subunits. Each subunit has a heme prosthetic group identical to that described for myoglobin Types of Hb Embryonic/Fetal/Adult Adult: HbA α2β2 – 96% HbA2 α2δ2 – 3% HbF α2g2 – 1% 8 8 Hemoglobin at Various Stages of Development 9 9 • • -chains have 141 amino acid residues • -chains have 146 amino acid residues Even though the amino acid sequences differ, the 3-D structures of the - and chains of hemoglobin are similar to each other and to the single polypeptide chain of myoglobin Myoglobin and the  subunits of hemoglobin have almost identical secondary and tertiary structures 10 10 Hemoglobin • Functionally composed of 2  dimers • Subunits in the tetramer are held together by: 1. Inter-chain hydrophobic interactions 2. Ionic interactions 3. Hydrogen bonds •  dimers can move with respect to each other •  dimers occupy different relative positions in deoxyhemoglobin compared to oxyhemoglobin • This movement enables hemoglobin to exist in 2 stable states 11 11 Hemoglobin States 1. A stable inactive “taut” or “tense” (T) state • Low oxygen affinity • Resists the binding of oxygen • Deoxygenated 2. A stable active “relaxed” (R) state • High oxygen affinity • Binding of oxygen is facilitated • Oxygenated 12 12 13 13 The 11 and 22 interfaces are the strongest • 35 residues form the interface between 11 and 22 • 19 residues form the interface between 12 and 21 Interactions between 1 and 1 and between 2 and 2 are dominant & change little in the Tto-R conformational change. The major shifts are at the interfaces between 1 and 2 and 2 and 1. 14 14 Oxygenation of Hemoglobin is Accompanied by Conformational Changes Near the Heme Group 15 15 The conformational change at histidine F8 is transmitted throughout the peptide backbone resulting in a significant change in tertiary structure of the entire subunit. pink is the changed (new) 16 t a Ibecomes relaxed 16 Distal Histidine (E7) • Highly conserved throughout evolution • Prevents oxidation of Fe2+ to Fe3+ • Reduces binding of CO in place of O2 17 On the side of the heme group opposite the proximal histidine, where oxygen reversibly binds, is another histidine residue called the distal histidine. This residue serves two very important functions in the polypeptide. First, it prevents oxidation of the iron ion to a higher oxidation state by any number of possible nearby oxidizing agents. At a higher oxidation state, the iron (Fe+++) is unable to bond to oxygen. Secondly, this histidine residue acts to reduce carbon monoxide (CO) binding to the heme. If the distal histidine was absent, even low levels of CO would greatly interfere with oxygen binding. The heme group alone (in the absence of the surrounding protein of myoglobin or hemoglobin) has a much greater bonding affinity for carbon monoxide than for oxygen. 17 Binding of Oxygen to One Heme of Hemoglobin Leads to: • Changes in conformation at the subunit surface • A new set of binding interactions between adjacent subunits which are associated with • Disruption of salt bridges and the formation of new salt bridges • • • The formation of new hydrogen bonds and new hydrophobic interactions Alterations in the quaternary structure (T to R) Increased affinity of the hemoglobin molecule for subsequent oxygen molecules 18 18 Each pair of  and  subunits stay relatively in the same position to each other under both oxygenated and deoxygenated states. • Deoxy -  and  subunit pairs twist relative to other pair • Oxy -  and  subunits twist back to reform salt bridges Looking down from above the 2 alpha subunits 2,3BPG Deoxyhe moglobin Oxyhemo globin Looking from side with the 2 alpha subunits above beta subunits 19 19 The Difference In Function Between Myoglobin & Hemoglobin Stems From Their Difference In Structure • Myoglobin – can bind only 1 molecule of oxygen because it contains only 1 heme group • Hemoglobin – can bind 1 oxygen molecule (O2) at each of its 4 heme groups • Oxygen Binding Curve (also termed Oxygen Dissociation Curve, Oxygen Saturation Curve) • A plot of the degree of saturation (Y) of the oxygen binding sites measured at different partial pressures of oxygen (pO2) 20 20 • Function of Myoglobin • Binds oxygen released by hemoglobin at the low pO2 found in muscle • Releases oxygen within muscle cell in response to oxygen demand • Function of Hemoglobin • Transports O2 from the lung to peripheral tissues • Transports carbon dioxide and protons from peripheral tissues to the lung for subsequent excretion 21 21 Myoglobin Oxygen Saturation Curve is hyperbolic in shape • pO2 in lung capillary bed ~ 100 mm Hg • pO2 of venous blood ~ 40 mm Hg • pO2 of active muscle ~ 20 mm Hg 1. pO2 of muscle tissue during oxygen deprivation that accompanies strenuous physical exercise ~ 5 mm Hg 2. At 5 mm Hg, myoglobin readily releases its bound oxygen for oxidative synthesis of ATP by muscle mitochondria 3. Since myoglobin cannot deliver a large fraction of its bound oxygen even at 20 mm Hg, it cannot serve as an effective vehicle for delivery of oxygen from lungs to peripheral tissues 22 22 Oxygen Binding Curve for Hemoglobin Demonstrates Cooperativity • • • The steep slope over the range of oxygen concentrations that occur between the lungs and the tissues permits hemoglobin to carry and deliver oxygen efficiently from sites of high pO2 to sites of low pO2 Cooperative binding allows hemoglobin to deliver more oxygen to the tissues in response to relatively small changes in the partial pressure of oxygen pO2 in lung capillary bed ~ 100 mm Hg pO2 of venous blood ~ 40 mm Hg pO2 of active muscle ~ 20 mm Hg Lower P50 = higher affinity for oxygen 23 23 Binding of Ligands That Affect Hemoglobin’s Oxygen Binding Examples: • H+ • CO2 • 2,3-bisphosphoglycerate H+, CO2 and 2,3-BPG bind Hb in the T form, i.e., the deoxy form, shifting the equilibrium between the R and T states toward the T, i.e., the low affinity form. This shifts the oxygen binding curve to the right i.e., hemoglobin is less saturated at a given partial pressure of oxygen. 24 Torr is a unit of pressure equivalent to 1 mm of mercury in a barometer and equal to 133.32 pascals. 24 2,3-Bisphosphoglycerate • Synthesized from 1,3bisphosphoglycerate (an intermediate of the glycolytic pathway) • Present in small amounts in all cells • Most abundant organic phosphate in Red Blood Cells 25 25 2,3-Bisphosphoglycerate • Binds to hemoglobin in central “donut” cavity • Cavity is of sufficient size only when hemoglobin is in the T form • Its oxygen atoms form salt bridges with positivelycharged side chains in the cavity 26 26 2,3-Bisphosphoglycerate Important for the normal oxygen transport function of hemoglobin Permits greater unloading of oxygen in the capillaries of tissues Hemoglobin deficient in 2,3bisphosphoglycerate acts as an “oxygen trap” rather than an oxygen transport system. 27 27 2,3-Bisphosphoglycerate 2,3-Bisphosphoglycerate concentration increases in response to: • Chronic hypoxia, e.g., high altitude • Chronic anemia e.g., when fewer than normal red blood cells are available to supply the body’s oxygen needs 28 28 2,3 BPG and Smoking • Blood of smokers displays reduced levels of 2,3 BPG relative to non-smokers • Thus, oxygen release from hemoglobin to the peripheral tissues may be reduced 29 29 Carbon Monoxide Poisoning • Carbon monoxide (CO) • Binds tightly but reversibly to the hemoglobin iron forming carbon monoxyhemoglobin (HbCO) • Shifts hemoglobin to relaxed conformation causing the remaining heme sites to bind oxygen with high affinity 30 30 Carbon Monoxide Poisoning Carbon Monoxide (CO) • Shifts the oxygen binding curve to the left • Changes the normal sigmoidal shape toward a hyperbola • Affected hemoglobin releases less oxygen to the tissues • 50% CO-Hb is lethal but mainly because CO has poisoned cytochrome oxidase 31 31 Carbon Monoxide Poisoning Treatment is to administer pure O₂ and simultaneous 5% CO₂ which strongly stimulates respiratory center to increase alveolar ventilation and decrease alveolar CO. 32 32 Methemoglobin • Contains iron in the ferric (Fe3+) state • Does not bind oxygen (flat oxygen binding curve) • Contains water at the sixth coordinate position • Forms in response to certain drugs, H2O2, free radicals or mutations in the  or  chain • Normal, but occasional, formation is corrected by NADH-cyt b5 reductase present in erythrocytes 33 33 Methemoglobin In cyanide poisoning: 1. Vitamin B12 administration is the first line of treatment 2. Second line of treatment: Amyl nitrite is administered with other agents e.g., sodium thiosulfate and sodium nitrite 3. Hemoglobin is oxidized to produce methemoglobin 4. Methemoglobin sequesters the circulating cyanide 5. Inhibition of ETC by cyanide is reduced and prevented 34 34 Fetal Hemoglobin, Hb F • Has a higher affinity for O2 than maternal Hb A • Structure is 2g2 • Binds less strongly to 2,3-BPG than does Hb A since a serine in gamma globin replaces histidine 143 in beta globin 35 35 Mutations in Globin - Hemoglobinopathies Hemoglobinopathies: A fascinating array of familial diseases affecting many aspects of hemoglobin biology. Abnormal hemoglobin subunits are associated with hemoglobinopathies Thalassemia is underproduction of one type of hemoglobin subunit • Most mutations are in regulatory gene • α thalassemia – • One of 4 gene copies are not expressed • ↓ α leads to ↑ death • β subunits form tetramers and g subunits form tetramers • β thalassemia – (microcytic anemia) • One copy mutated or not expressed or incorrectly spliced • Anemia • Sickle cell anemia - point mutation in Hb → Hb S (Glu → Val at amino acid 6) • Chronic hemolytic anemia - Hb point mutation → Hb C or Hb E 36 Hereditary persistence of fetal hemoglobin (HPFH) is a benign condition in which significant fetal hemoglobin (hemoglobin F) production continues well into adulthood, disregarding the normal shutoff point after which only adult-type hemoglobin should be produced. Hemoglobin Gun Hill is an unstable mutant hemoglobin associated with mild compensated hemolysis. This abnormal protein has a deletion of five amino acids in the β-chains. Hb Barts (gamma 4) and HbH (beta 4) in alpha-thalassemia 36 Other Examples of Hemoglobinopathies HbE-Gower 1 ( ζ2ε2) and HbE Gower 2 (α2ε2) Hb Barts (g4) and HbH (4) in -thalassemia There are many others (- and -thalassemias, Hb Gun Hill , HPFHb etc.) 37 Hereditary persistence of fetal hemoglobin (HPFH) is a benign condition in which significant fetal hemoglobin (hemoglobin F) production continues well into adulthood, disregarding the normal shutoff point after which only adult-type hemoglobin should be produced. Hemoglobin Gun Hill is an unstable mutant hemoglobin associated with mild compensated hemolysis. This abnormal protein has a deletion of five amino acids in the β-chains. Hb Barts (gamma 4) and HbH (beta 4) in alpha-thalassemia 37 Diabetes and HbA1c • Blood glucose level is normally around 90-100 mg/dL (~ 5.6 mM) • At this concentration, some HbA gets glucosylated nonenzymatically to form HbA1c • In uncontrolled diabetes, blood glucose can increase more than twice normal – this generates more HbA1c than normal • HbA1c has a long half-life compared to insulin and thus its measurement can give information on how well a patient’s diabetes has been controlled over several months 38 38 teaser 39 39 Summary • • • • • • • Hemoproteins – Mb and Hb - similarities, differences and functions Co-operative binding of oxygen by Hb H+, CO2, 2,3- biphosphoglycerate CO poisoning Methemoglobin Fetal Hb HbA1c 40 40