Kevin Author: Schooler Date: January 2001 Hemoglobin Myoglobin: 1) Single polypeptide chain 2) Binds oxygen in a hyperbolic fashion. Each oxygen binds independently. Hemoglobin 1) 4 polypeptide chains (2 alpha and 2 beta) in a quaternary structure. 2) Each polypeptide has a heme and can bind O2 (4 molecules total). 3) Binds oxygen in a sigmoid fashion. Each oxygen binds in a cooperative fashion. Heme: a prosthetic group made of a hydrophobic porphyrin ring. Fe2+ is complexed in the porphyrin ring by two main chain His residues. Oxygen Binding 1) 4 N residues in heme bind Fe2+ a) Ferrous iron (Fe2+) binds O2 reversibly. b) Ferric iron (Fe3+) does not bind O2 reversibly. c) Heme buries Fe2+ in the protein and prevents oxidation from Fe2+ to Fe3+. 2) The proximal His, on the F8 helix, is always bound to Fe2+. 3) The distal His, on helix E7, binds O2 and the O2 binds Fe2+. 4) ValEll pushes molecules like C0 out of the binding plane (not perpendicular to the porphyrin ring, and destabilizes its binding. -CO still binds better than O2 but ValE11 makes the binding reversible. 5) Packing Contacts: Bonds that join alpha1/Beta1 and the bonds that join alpha2/Beta2. These bonds are fixed and do not change with the state of Hb oxygenation. 6) Sliding Contacts: Bonds that join alpha1/Beta2 and the bonds that join alpha2/Beta1. These bonds are not fixed and do change with the state of Hb oxygenation. DeoxyHb 1) The T form (Tense) of Hb, called deoxyHb, is stabilized by salt links between subunits. The chain ends are not free. a) COO- terminal (Arg141) of one alpha subunit binds Asp 126 of the other alpha chain. b) COO- terminal of each beta1 subunit interacts with Lys40 of alpha2. (Same interaction betweeen beta2 and alpha1) c) COO- terminus of arg141(alpha1) and alphaNH2 of alpha2. d) Asp94 of Beta2 to His 146 of Beta 2. (Same interaction o Beta1) 2) The proximal His is 7-8 degrees out of perpendicular with heme plane. a) Because proximal His is not perpendicular, the Fe2+ is also not perpendicular. The iron is pulled 0.6 A farther away from the distal His. b) This increased distance prevents Oxygen from binding between Fe2+ and the distal His. OxyHb 1) O2 binding to the distal His pulls Fe2+ back towards perpendicular. 2) Because Fe2+ is bound to the proximal His, and the proximal His is part of helix F8, helix F8 is also pulled back towards perpendicular.
3) Moving F8 helix back to perpendicular moves the E and G helices. 4) Combined movement of the helices moves the sliding surfaces (alpha1/B2 and alpha2/Beta1) 15 degrees with respect to each other. This movement breaks the salt bridges securing the subunits (sliding contacts) and shifts Hb into the R form (relaxed). R versus T equilibrium 1) The T form of Hb, lots of stabilizing bonds, does not bind O2 well which means it doesn't want to pick up oxygen in the lungs and that it releases O2 well in the tissues. T unloades oxygen efficiently. 2) The R form of Hb, less stabilizing bonds, binds O2 well which means it wants to be oxygenated in the lungs but also wants to be oxygenated in the tissues. R does not unload oxygen efficiently. 3) The balance of T versus R depends on number of O2 molecules bound. Each O2 molecule bound increases the proportion of Hb in R form so it increases the liklihood that another O2 will bind. In other words, everytime another O2 binds, it becomes easier to bind the next O2 until 4 O2 are bound for every Hb molecule and the Hb is considered saturated. Allosteric Effectors of Hb. **Increasing the number of contacts (salt bridges) increases the stability of T form Hb which improves the efficiency of oxygen unloading in the tissues. BPG (2,3 bisphosphoglycerate) 1) 2,3 BPG is formed as a glycolysis intermediate from 1,3 BPG 2) BPG decreases the affinity of Hb for O2. It shifts the Hb equilibrium to the T form and Hb unloads oxygen efficietly. 3) 1 BPG binds for every Hb molecule by binding in a central pocket. 4) BPG can only bind the deoxy form of Hb because in OxyHb, the central pocket is depressed and BPG no longer "fits". 5) 5 negative charges on BPG are bound to 8 positive charges in Hb Beta chains. a) alpha NH2 of Val1 (2, one from each beta chain) b) His2 (2, one from each beta chain). c) Lys82 (2, one from each beta chain). d) His146 (2, one from each beta chain). 6) Increasing the concentration of BPG in the blood stream decreases the O2 saturation of Hb because there is eficient unloading of O2 in the tissues. 7) Inosine is used to drive the formation of BPG in stored blood. If it is not used, the Hb would be primarily in the R form and would not unload in the tissues ---> decreased oxygenation of tissues. Protons 1) Carbonic Anhydrase catylyzes the formation of bicarbonate in the blood with the concomittant release of protons which decreases the pH (acidosis). CO2 + H2O ---> H2CO3 ---> HCO3- + H+ 2) Proton binding stabilizes the T form of Hb and is antagonistic to O2 binding. O2 bound Hb does not bind protons and proton carrying Hb does not bind O2. 3) Proton binding increases positive charges on Hb so that new salt bridges are formed, stabilizing T form. 4) Asp94 on Beta chain is in close proximity to His146 on the beta chains. a) The increased proximity of the negative charge on Asp94, withdraws the positive charge on His146 increasing its pKA. b) The increased pKA of His146 causes it to be protonated. c) Protonated His146 is positively charged so it binds (forms a salt bridge) the negative charge of Asp94 stabilizing T form of Hb. d) This accounts for 40% of the Bohr effect.
e) In His146 OxyHb, is moved 0.3 A and can longer form contacts with Asp94 which promotes the antagonism between protons and O2. 5) The alpha NH2 amino group on 1 alpha chain is in close proximity to the COO- terminal of the other alpha chain. a) The inreased proximity of the COO- withdraws positive charge from thealpha NH2 group increasing its pKA. b) The increased pKA causes the alpha NH2 group to be protonated. c) Protonated form, NH3, is positively charged which allows it to form a salt bridge with Arg141(+) via a Cl- ion. d) Because more bonds are formed, the T form is more stable. e) Because Cl- is required in this bridge it is also a negative allosteric effector of Hb and stabilizes the T form. CO2 1) Although most CO2 in the blood stream is carried by bicarbonate, some is carried by Hb as carbamate. This reaction increases the number of protons in the blood stream and decreases the pH (acidosis) causing the effect seen above. CO2 is carried on alpha Amino groups in a covalent bond. R-NH2 + CO2 ---> R-NH-COO- + H+ 2) Although a carbamate can be formed with the alpha NH2 groups of either the Beta chains or the alpha chains, when the carbamate forms on the alpha chain alpha amino group, new salt bridges are formed which stabilize the T form of Hb. a) The negative charge of carbamate, R-NH-COO interacts with the Arg141 positive charge of the alpha chain. b) More bonds stabilize T. c) Arg141 is the same Arg141 described in the proton section above which linked to the alpha Amino NH3+ via a Cl- ion. Because Cl- is required in this bridge it is also a negative allosteric effector of Hb and stabilizes the T form. Hemeglobinopathies A) HbF (fetal Hb) 1) The gamma chain gives HbF a higher affinity for O2 than HbA (adult) which has Beta chains instead. 2) The Higher affinity of HbF for O2 is due to the fact that HbF has a low affinity for BPG. a) His 143 in HbA beta chains is an uncharged serine in HbF gamma chains and those chains no longer bind BPG. b) alpha NH2 of gamma chains are acetylated so they cannot bind BPG. 3) HbF has a stabilized R form of Hb giving it a high affinity for O2 B) Hb Titusville 1) Asp94 is converted to an Asn so His146 can no longer be protonated and form a salt bridge with with Asp 94. 2) Because less bonds are formed, Hb Titusville is stabilized a R form and no longer unloads O2 well in the tissues. 3) Saturation goes up but tissues are less oxygenated leading to cyanosis. C) Hb Helsinki 1) Beta chain lys 82 is converted to met. 2) Binding to BPG is reduced and Hb helsinki is stabilized as the R form so that it unloads O2 less well in the tissues. 3) Some compensation occurs by increasing the number of RBCs (polycythemia). D) HbM (methemoglobin)
1) Hb boston, proximal His is replaced by Tyr. Tyr cannot maintain ferric form of Fe2+ so ferrous forms decreasing the oxygen carrying capacity of Hb. 2) Hb lwate, distal his is replaced by Tyr so O2 can no longer be caarried. 3) Since these mutations decrease oxygenation by more than half (cooperative binding so decrease in half the binding sites dereases binding by more than half), they are always heterozygous. Homozygous would have no oxygen carrying capacity and be lethal. E) alpha thalassemia 1) alpha thalasemia occurs when the production of alpha chain protein is dereased. Balanced formation of 2 alpha chains and 2 beta chains per Hb molecule is no longer maintained. Hb formed by 4 beta chains has completely non-cooperative binding and is lethal. 2) There are 4 alpha chains on chromosome 16 and the phenotype changes based on the number that are mutant. a) 1 mutant: no phenotype b) 2 mutant: classical thallasemia, minor RBC abnormalities with no anemia, c) 3 mutant: HbH disease, only 25% Hb is made of 2 alpha chains and 2 beta chains. d) 4 mutant: lethal 3) A chain termination mutation can also cause a disease that looks like HbH. Extra amino acids are added to the alpha chains and they can no longer form tetramers. F) Beta Thalassemia 1) beta thalasemia occurs when the production of beta chain protein is dereased. Balanced formation of 2 alpha chains and 2 beta chains per Hb molecule is no longer maintained. Hb formed by 4 alpha chains precipitates damaging the RBC membrane and leads to premature RBC destruction. 2) Beta thalassemia major: homozygous mutatin, very little beta chain produced. a) Hydroxyurea treatment increases the level of HbF, and can partially compensate for the loss of Beta chains. 3) beta thalassemia minor: heterozygous mutation, less sever symptoms. G) Sickle Cell anemia 1) HbS is formed when a single Glu residue is replace by Val at Beta6 placing a hydrophobic residue on the protein surface. a) The hydrophobic residue provides a nucleating site for protein protein interactions ("sticky patch"). Higher hydrophobicity increases tendancy to aggregate lowerin Hb concentration required for aggregation to occur. b) Although it is present in both oxy and deoxy forms, proteins only appear to aggregate at low oxygen tension. c) Agreggation of Hb molecules increases viscosity, lowers solubility and aggregates precipitate decreasing RBC lifespan. 2) Drugs that increas Hb oxygenation are anti-sickling agents. a) Cyanate irreversibly carbamoylates Hb alpha amino groups (see CO2 as allosteric efector above). The c=0 cannot form salt bridges so bond number is reduced and R form is stabilized. b) Hydroxyurea treatment increases the level of HbF, increasing the level of oxygenation and decreasing sickling. 3) Elevated K+ transport in sickled erythrocytes may contribute to malaria resistance.