Hemoglobin & Myoglobin Lecture Notes PDF

Hemoglobin & Myoglobin (Oxygen Binding Proteins) Dr. Sebnem GARIP USTAOGLU Ligand --- a molecule bound reversibly by a protein Binding site --- the site on protein to which a ligand binds complementary to the ligand in; size shape charge hydrophobic or hydrophilic character The bi...

Hemoglobin & Myoglobin (Oxygen Binding Proteins) Dr. Sebnem GARIP USTAOGLU Ligand --- a molecule bound reversibly by a protein Binding site --- the site on protein to which a ligand binds complementary to the ligand in; size shape charge hydrophobic or hydrophilic character The binding of a protein and ligand is often coupled to a conformational change in the protein that makes the binding site more complementary to the ligand, permitting tighter binding. Induced fit --- the structural adaptation that occurs between protein and ligand Reversible binding of a protein to a ligand: Oxygen-binding proteins Oxygen is poorly soluble in aqueous solutions and cannot be carried to tissues in sufficient quantity if it is simply dissolved in blood serum. Certain transition metals, including iron and copper have a strong tendency to bind oxygen. Free iron leads the formation of highly reactive oxygen species such as hydroxyl radicals which can damage DNA and other macromolecules.  Hemoglobin and Myoglobin Heme proteins-myoglobin and hemoglobin –maintain a supply of oxygen essential for oxidative metabolism Myoglobin and beta subunits of Hb share almost identical secondary and tertiary structures.  HEME The prosthetic group – heme – is the O2 binding moiety common to Myoglobin and hemoglobin Heme is a cyclic tetrapyrole consisting of 4 molecules of pyrole linked by a-methylene bridges (protoporphyrin) This planar network of conjugated double bonds absorbs visible light and gives its deep red color One atom of ferrous ion (Fe2+) resides at the center of planar tetrapyrole Heme consists of a complex organic ring structure, protoporphyrin, to which is bound a single iron atom in its ferrous (Fe2+) state. protoporphyrin The iron atom has six coordination bonds, four to nitrogen atoms that are part of the flat porphyrin ring system and two perpendicular to the porphyrin. The coordinated nitrogen atoms have an electron-donating character and help to prevent conversion of the heme iron to the ferric (Fe3+) state. Iron in the Fe2+ state binds oxygen reversibly; in the Fe3+ state it does not bind oxygen.  WHAT HAPPENS DURING O2 BINDING? When O2 occupies the 6th coordination position, conformational change occurs: The heme iron moves towards the plane of the heme from a position of 0.04 nm beyond it. The bond between the first O2 and Fe2+ is perpendicular to the plane of heme ring.  Myoglobin Sperm Whale Myoglobin was the first protein to have its 3-dimensional structure determined – John Kendrew(1958) – Shared the 1962 Nobel in chemistry “Solving the structure wasn’t hard, but getting the samples was a real achievement…” Kendrew, JC; Bodo, G; Dintzis, HM; Parrish, RG; Wyckoff, H; Phillips, DC (1958). "A three- dimensional model of the myoglobin molecule obtained by x-ray analysis". Nature 181 (4610): 662–666. A monomeric protein of red muscle Stores oxygen as a reserve against oxygen deprivation and facilitates oxygen diffusion rapidly in contracting muscle for mitochondrial synthesis of ATP Single polypeptide chain of 153 AA residues with one heme molecule Polypeptide is made of 8 α helices (7-20 residue) Compact, dense, hydrophobic core Oxygen binding curve is hyperbolic → relatively insensitive to small changes in the concentration of dissolved O2  Hemoglobin (Hb) First oligomeric protein (quaternery structure), a tetramer or a dimer of αβ protomers 4 times as large as myoglobin Transports O2 to the tissues and returns CO2 (15-20%) and protons (40%) from peripheral tissues to lungs and kidneys for excretion 4 polypeptide chains + 4 heme prosthetic groups  Hemoglobin structure Koolman, Color Atlas of Biochemistry, 2nd edition © 2005 Thieme  Protein portion is globin with 2 alpha chains (141 residues each) and 2 beta chains (148 residues each) More sensitive to small differences in O2 concentration between the tissues and lungs → Hb has a sigmoid binding curve for O2 → diagnostic of cooperative binding. O2 partial pressure (mmHg)  Myoglobin has only one subunit, relatively insensitive to small changes in the concentration of dissolved oxygen, functions well as an oxygen-storage protein Hemoglobin, with its multiple subunits and O2-binding sites, highly sensitive to small changes in ligand concentration, is better suited to oxygen transport. Exercise Tissues Lungs  Why CO is highly toxic to aerobic organisms? Some small molecules, such as carbon monoxide (CO) bind to heme iron with greater affinity than does O2. When a molecule of CO is bound to heme, O2 is excluded, which is why CO is highly toxic to aerobic organisms. CO binds to heme 200 times better than does O2.  Hb undergoes a structural change on binding oxygen two major conformations of hemoglobin: the R state and the T state. Although oxygen binds to hemoglobin in either state, it has a significantly higher affinity for hemoglobin in the R state. Oxygen binding stabilizes the R state. T and R originally denoted “tense” and “relaxed,” respectively  Salt bridges between the carboxyl terminal residues of all 4 subunits rupture progressivelly as oxygen is added → Transition from low affinity T (tense) state to high affinity R (relaxed) state occurs. T state is the predominat conformation of deoxy Hb, O2 binding stabilizes the R state.  Hb binds oxygen cooperatively A protein that bound O2 with high affinity would bind it efficiently in the lungs but would not release much of it in the tissues. If the protein bound oxygen with a sufficiently low affinity to release it in the tissues, it would not pick up much oxygen in the lungs.  A transition from a low- affinity state (the T state) to a high-affinity state (the R state) as more O2 molecules are bound. As a result, hemoglobin has a hybrid S shaped, or sigmoid, binding curve for oxygen Figure A sigmoid (cooperative) binding curve  Cooperativity of Hb-O2 binding The first molecule of O2 that interacts with deoxyhemoglobin binds weakly, because it binds to a subunit in the T state. Its binding leads to conformational changes that are communicated to adjacent subunits, making it easier for additional molecules of O2 to bind. In effect, the T to R transition occurs more readily in the second subunit once O2 is bound to the first subunit. The last (fourth) O2 molecule binds to a heme in a subunit that is already in the R state, and hence it binds with much higher affinity than the first molecule. Allosteric protein – a protein in which the binding of a ligand to one site affects the binding properties of another site on the same protein allos --- other stereos --- solid or shape Homotropic interaction --- ligand = modulator Heterotropic interaction --- ligand = modulator O2 --- as both a normal ligand and an activating homotropic modulator for Hb  Hemoglobin Also Transports H+ and CO2 Hemoglobin carries two end products of cellular respiration—H+ and CO2— from the tissues to the lungs and the kidneys, where they are excreted. The CO2, produced by oxidation of organic fuels in mitochondria, is hydrated to form bicarbonate: CO2 + H2O H+ + HCO3- carbonic anhydrase Bohr Effect; The effect of pH and CO2 concentration on the binding and release of oxygen by hemoglobin O2 binding to Hb is regulated by 2,3-bisphosphoglycerate (BPG) HbBPG + O2 HbO2 +BPG 4 1 heterotropic allosteric modulation inverse relationship 2,3 BPG reduces the affinity of Hb to O2 by stabilizing T state. [BPG] during hypoxia BPG binds at a site distant from O2 binding site and regulates O2 lowered oxygenation of binding affinity of Hb in relation to peripheral tissues due to pO2 in the lungs. inadequate functioning of the lungs or circulatory system The site for BPG binding to ++ Hb is the cavity between the β subunits in T state. This cavity is lined with (+) ly charged AA residues that interact with T (-) ly charged groups of BPG O2 BPG is negatively charged R T state  BPG is important in the physiological adaptation to the lower pO2 at high altitudes For a healthy human at the ocean level, the amount of O2 delivered to the tissues is equivalent to nearly 40% of the maximum that could be carried by the blood. This person is quickly transported to a mountainside at an altitude of 4,500 meters, where the pO2 is considerably lower, the delivery of O2 to the tissues is now reduced. However, after just a few hours at the higher altitude, the BPG concentration in the blood has begun to rise, leading to a decrease in the affinity of hemoglobin for oxygen. This adjustment in the BPG level has only a small effect on the binding of O2 in the lungs but a considerable effect on the release of O2 in the tissues. As a result, the delivery of oxygen to the tissues is restored to nearly 40% of that which can be transported by the blood. The situation is reversed when the person returns to sea level.  The role of BPG in fetal development Regulation of O2 binding to Hb by BPG has an important role in fetal development Fetal Hb must have a greater affinity than material Hb for O2 because fetus must extract O2 from mother’s blood.  The role of BPG in fetal development Because a fetus must extract oxygen from its mother’s blood, fetal hemoglobin must have greater affinity than the maternal hemoglobin for O2. The fetus synthesizes γ subunits rather than β subunits, forming α2γ2 hemoglobin. This tetramer has a much lower affinity for BPG than normal adult hemoglobin, and a correspondingly higher affinity for O2. Ask participants to open joinmyquiz.com  SUMMARY A molecule bound by a protein is called a ligand, and the site to which it binds is called the binding site. Proteins may undergo conformational changes when a ligand binds, a process called induced fit. In a multi-subunit protein, the binding of a ligand to one subunit may affect ligand binding to other subunits. Ligand binding can be regulated. Hemoglobin and myoglobin contains a heme prosthetic group, which binds oxygen. Heme consists of a single atom of Fe2+ coordinated within a porphyrin.  SUMMARY Oxygen binds to hemoglobin and myoglobin reversibly; this simple reversible binding can be described by an association constant Ka or a dissociation constant Kd. For a monomeric protein such as myoglobin, the fraction of binding sites occupied by a ligand is a hyperbolic function and for a multisubunit protein such as hemoglobin, it is a sigmoidal function of ligand concentration. Normal adult hemoglobin has four hemecontaining subunits, two α and two β, similar in structure to each other and to myoglobin. Hemoglobin exists in two interchangeable structural states, T and R. The T state is most stable when oxygen is not bound. Oxygen binding promotes transition to the R state.  SUMMARY Oxygen binding to hemoglobin is both allosteric and cooperative. As O2 binds to one binding site, the hemoglobin undergoes conformational changes that affect the other binding sites —an example of allosteric behavior. Conformational changes between the T and R states, mediated by subunit-subunit interactions, result in cooperative binding; this is described by a sigmoid binding curve and can be analyzed by a Hill plot. Two major models have been proposed to explain the cooperative binding of ligands to multisubunit proteins: the concerted model and the sequential model.  SUMMARY Hemoglobin also binds H+ and CO2, resulting in the formation of ion pairs that stabilize the T state and lessen the protein’s affinity for O2 (the Bohr effect). Oxygen binding to hemoglobin is also modulated by 2,3- bisphosphoglycerate, which binds to and stabilizes the T state. THANK YOU Carbon monoxide binds to free heme molecules more than 20,000 times better than does O2. the Kd for CO binding to free heme is more than 20,000 times lower than that for O2. CO binds only about 200 times better when the heme is bound in hemoglobin. steric hindrance (structural limitation) 1: 20,000 1: 200  CO Poisoning Autopsy photo showing skin discoloration