Biochemistry Sixth Edition Berg • Tymoczko • Stryer Chapter 7: Hemoglobin: Portrait of a Protein in Action Copyright © 2007 by W. H. Freeman and Company

Erythrocytes (Red cells)

Hemoglobin and Myoglobin These are conjugated proteins. A simple protein has only a polypeptide chain. A conjugated protein has a non-protein part in addition to a polypeptide component. Both myoglobin and hemoglobin contain heme. Myoglobin - 17000 daltons (monomeric) 153 amino acids Hemoglobin - 64500 daltons ( tetrameric) a-chain has 141 amino acids b-chain has 146 amino acids

Hemoglobin O2 carrying capability Erythrocytes/ml blood: 5 billion ( 5 x 109 ) Hemoglobin/red cell: 280 million ( 2.8 x 108 ) O2 molecules/hemoglobin: 4 O2 ml blood: (5 x 109)(2.8 x 108)(4) = (5.6 x 1018) or (5.6 x 1020) molecules of O2/100 ml blood

A single subunit of Hemoglobin, an a2b2 tetramer

Myoglobin, monomeric

3o structure overlap: myoglobin, a-globin and b-globin a-Globin (blue) b-Globin (violet) Myoglobin (green)

Aromatic Heme

Iron in Hemoglobin binding O2

Iron in Myoglobin binding O2

Resonance in Iron binding O2

Hemoglobin, a2b2 tetramer

O2 binding: Hemoglobin & Myoglobin P50 = 2 torr P50 = 26 torr

O2 transport capability, a comparison

Resting state vs exercise

O2 Binding Changes 4o Structure

Allosteric Proteins There are two limiting models of allosterism: Monod, Wyman & Changeux: Two State, concerted Koshland, Nemethy & Filmer: One State, sequential Allosteric effectors (modulators) bind to a protein at a site separate from the functional binding site (modulators may be activators or inhibitors) Oxygen binding and release from Hb are regulated by allosteric interactions Hemoglobin cooperativity behaves as a mix of the above two models.

Concerted, two state model Monod, Wyman & Changeux

R-state vs T-state Binding

Sequential, one state model Koshland, Nemethy & Filmer

Decreasing O2 affinity 2,3-bisphospho-glycerate (2,3-BPG) Lowers the affinity of oxygen for Hemoglobin

2,3-bisphosphoglycerate (2,3-BPG) The binding pocket for BPG contains 4 His and 2 Lys

Binding of bisphosphoglycerate

The Bohr Effect Bohr Effect: Lowering the pH decreases the affinity of oxygen for Hb

Loss of O2 from Hemoglobin Carbamate: CO2 combines with NH2 at the N-terminus of globins

Carbamate formation Covalent binding at the N-terminus of each subunit

Combined Effects CO2 , BPG and pH are all allosteric effectors of hemoglobin.

CO2 & Acid from Muscle

CO2 & Hemoglobin Blood Buffering Metabolic oxidation in cells uses oxygen and produces CO2 . The pO2 drops to ~20 torr and oxygen is released from incoming HbO2-. HbO2- <===> Hb- + O2 Release is facilitated by CO2 reacting with the N- terminus of each hemoglobin subunit, by non-covalent binding of BPG and the Bohr effect.

Events at Cell sites The localized increase in CO2 results in formation of carbonic acid which ionizes to give bicarbonate and H+. CO2 + HOH <===> H2CO3 carbonic anhydrase H2CO3 <===> HCO3- + H+ pKa = 6.3 The increase in [H+] promotes protonation of Hb-. HHb <===> Hb- + H+ pKa = 8.2

Events at Cell sites The predominant species in this equilibrium at pH 7.2 is HHb. So, O2 remains at the cell site, HHb carries a proton back to the lungs and bicarbonate carries CO2 . Charge stability of the erythrocyte is maintained via a chloride shift, Cl- <==> HCO3- .

Events at Lung sites Breathing air into the lungs increases the partial pressure of O2 to ~100 torr. This results in O2 uptake by HHb to form HHbO2. HHb + O2 <===> HHbO2 Ionization of HHbO2 then occurs and HbO2- carries O2 away from the lungs. HHbO2 <===> HbO2- + H+ pKa = 6.6 So, the predominant species at pH (7.4) is HbO2-.

Events at Lung sites The localized increase in [H+] from hemoglobin ionization serves to protonate HCO3- . H2CO3 <===> HCO3- + H+ pKa = 6.3 H2CO3 <===> CO2 + HOH carbonic anhydrase The resulting H2CO3 decomposes in presence of carbonic anhydrase and CO2 is released in the lungs. Charge stability of the erythrocyte is maintained again via a chloride shift, HCO3- <==> Cl-.

Sickle Cell due to Glu 6  Val 6

Binding relationships The binding of O2 to myoglobin can be shown by the equilibriuim: Mb + O2 <===> MbO2 (1) The dissociation constant for the loss of O2 is: [Mb][O2] Keq = KD = -------------- (2) [MbO2] Define the fraction of sites, Y, occupied by O2 as: [MbO2] sites bound Y = --------------------- = ----------------- (3) [Mb] + [MbO2] total sites

Binding relationships Substituting from equation (2) into (3): [MbO2] 1 Y = ---------------------------- = ------------ K [MbO2] K ---------- + [MbO2] ---- + 1 O2 O2 or: [O2] pO2 pO2 Y = ---------- = ----------- = ------------ K + O2 K + pO2 p50 + pO2 Evaluating K at Y = 0.5 gives K = p50 for O2

Biochemistry Sixth Edition Berg • Tymoczko • Stryer End of Chapter 7 Copyright © 2007 by W. H. Freeman and Company