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HemoglobinA classic example of allostery

• Hemoglobin and myoglobin are oxygen transport and storage proteins

• Compare the oxygen binding curves for hemoglobin and myoglobin

• Myoglobin is monomeric; hemoglobin is tetrameric

• Mb: 153 aa, 17,200 MW • Hb: two alphas of 141 residues, 2 betas of

146

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O2 -binding curves for hemoglobin and myoglobin.

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The myoglobin and hemoglobin molecules. Myoglobin (sperm whale): one polypeptide chain of 153 amino acid residues (mass = 17.2 kD) has one heme (mass = 652 D) and binds one O2 . Hemoglobin (human): four polypeptide chains, two of 141 amino acid residues (α) and two of 146 residues (β); mass = 64.45 kD. Each polypeptide has a heme; the Hb tetramer binds four O2 . (Illustration: Irving Geis Rights owned by Howard Hughes Medical Institute. Not to be reproduced without permission)

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Detailed structure of the myoglobin molecule. The myoglobin polypeptide chain consists of eight helical segments, designated by the letters A through H, counting from the N- terminus. These helices, ranging in length from 7 to 26 residues, are linked by short, unordered regions that are named for the helices they connect, as in the AB region or the EF region. The individual amino acids in the polypeptide are indicated according to their position within the various segments, as in His F8, the eighth residue in helix F, or Phe CD1, the first amino acid in the interhelical CD region. Occasionally, amino acids are specified in the conventional way, that is, by the relative position in the chain, as in Gly153. The heme group is cradled within the folded polypeptide chain. (Illustration: Irving Geis Rights owned by Howard Hughes Medical Institute. Not to be reproduced without permission)

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Hemoglobin Function Hb must bind oxygen in lungs and

release it in capillaries• When a first oxygen binds to Fe in heme of

Hb, the heme Fe is drawn into the plane of the porphyrin ring

• This initiates a series of conformational changes that are transmitted to adjacent subunits

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Hemoglobin Function Hb must bind oxygen in lungs and release it

in capillaries

• Adjacent subunits' affinity for oxygen increases

• This is called positive cooperativity

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Myoglobin StructureMb is a monomeric heme protein

• Mb polypeptide "cradles" the heme group • Fe in Mb is Fe2+ - ferrous iron - the form that

binds oxygen • Oxidation of Fe yields 3+ charge - ferric iron -

metmyoglobin does not bind oxygen • Oxygen binds as the sixth ligand to Fe • See Figure 15.25 and discussion of CO

binding

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The six liganding positions of an iron ion. Four ligands lie in the same plane; the remaining two are, respectively, above and below this plane. In myoglobin, His F8 is the fifth ligand; in oxymyoglobin, O2 becomes the sixth.

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Oxygen and carbon monoxide binding to the heme group of myoglobin.

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The Conformation Change

• Oxygen binding changes the Mb conformation • Without oxygen bound, Fe is out of heme plane • Oxygen binding pulls the Fe into the heme plane • Fe pulls its His F8 ligand along with it • The F helix moves when oxygen binds • Total movement of Fe is 0.029 nm - 0.29 A • This change means little to Mb, but lots to Hb!

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The displacement of the Fe ion of the heme of deoxymyoglobin from the plane of the porphyrin ring system by the pull of His F8. In oxymyoglobin, the bound O2 counteracts this effect.

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Binding of Oxygen by HbThe Physiological Significance

• Hb must be able to bind oxygen in the lungs • Hb must be able to release oxygen in

capillaries • If Hb behaved like Mb, very little oxygen

would be released in capillaries - see Figure 15.21!

• The sigmoid, cooperative oxygen binding curve of Hb makes this possible!

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Conformational drawings of the α

- and β- chains of Hb and the myoglobin chain. (Illustration: Irving Geis Rights owned by Howard Hughes Medical Institute. Not to be reproduced without permission)

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The arrangement of subunits in horse methemoglobin, the first hemoglobin whose structure was determined by X-ray diffraction. The iron atoms on metHb are in the oxidized, ferric (Fe3+) state. (Illustration: Irving Geis Rights owned by Howard Hughes Medical Institute. Not to be reproduced without permission)

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Side view of one of the two αβ

dimers in Hb, with packing contacts indicated in blue. The sliding contacts made with the other dimer are shown in yellow. The changes in these sliding contacts are shown in Figure 15.30. (Illustration: Irving Geis Rights owned by Howard Hughes Medical Institute. Not to be reproduced without permission)

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Oxygen Binding by HbA Quaternary Structure Change

• When deoxy-Hb crystals are exposed to oxygen, they shatter! Evidence of a structural change!

• One alpha-beta pair moves relative to the other by 15 degrees upon oxygen binding

• This massive change is induced by movement of Fe by 0.039 nm when oxygen binds

• See Figure 15.31

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Subunit motion in hemoglobin when the molecule goes from the (a) deoxy to the (b) oxy form. (Illustration: Irving Geis Rights owned by Howard Hughes Medical Institute. Not to be reproduced without permission)

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Changes in the position of the heme iron atom upon oxygenation lead to conformational changes in the hemoglobin molecule.

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Figure 15.32 Salt bridges between different subunits in hemoglobin. These noncovalent, electrostatic interactions are disrupted upon oxygenation. Argα141 and Hisβ146 are the C- termini of the α- and β-polypeptide chains. (a) The various intrachain and interchain salt links formed among the α

- and β-chains of deoxyhemoglobin. (b) A focus on those salt bridges and hydrogen bonds involving interactions between N-terminal and C-terminal residues in the α-chains. Note the Cl- ion, which bridges ionic interactions between the N-terminus of α2 and the R group of Argα141. (c) A focus on the salt bridges and hydrogen bonds in which the residues located at the C-termini of β-chains are involved. All of these links are abolished in the deoxy to oxy transition. (Illustration: Irving Geis Rights owned by Howard Hughes Medical Institute. Not to be reproduced without permission)

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The Bohr EffectCompetition between oxygen and H+

• Discovered by Christian Bohr • Binding of protons diminishes oxygen binding • Binding of oxygen diminishes proton binding • Important physiological significance • See Figure 15.33

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The oxygen saturation curves for myoglobin and for hemoglobin at five different pH values: 7.6, 7.4, 7.2, 7.0, and 6.8.

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Bohr Effect II

Carbon dioxide diminishes oxygen binding • Hydration of CO2 in tissues and extremities

leads to proton production • These protons are taken up by Hb as oxygen

dissociates • The reverse occurs in the lungs

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Oxygen-binding curves of blood and of hemoglobin in the absence and presence of CO2 and BPG. From left to right: stripped Hb, Hb + CO2 , Hb + BPG, Hb + BPG + CO2 , and whole blood.

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2,3-Bisphosphoglycerate

An Allosteric Effector of Hemoglobin• In the absence of 2,3-BPG, oxygen

binding to Hb follows a rectangular hyperbola!

• The sigmoid binding curve is only observed in the presence of 2,3-BPG

• Since 2,3-BPG binds at a site distant from the Fe where oxygen binds, it is called an allosteric effector

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Figure 15.35 The structure, in ionic form, of BPG or 2,3 -bisphosphoglycerate, an important allosteric effector for hemoglobin.

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2,3-BPG and HbThe "inside" story......

• Where does 2,3-BPG bind? – "Inside" – in the central cavity

• What is special about 2,3-BPG? – Negative charges interact with 2 Lys, 4 His,

2 N-termini • Fetal Hb - lower affinity for 2,3-BPG, higher

affinity for oxygen, so it can get oxygen from mother

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Figure 15.36 The ionic binding of BPG to the two β-subunits of Hb. (Illustration: Irving Geis Rights owned by Howard Hughes Medical Institute. Not to be reproduced without permission)

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Figure 15.37 The structures of inositol pentaphosphate and inositol hexaphosphate, the functional analogs of BPG in birds and reptiles.

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Figure 15.38 Comparison of the oxygen saturation curves of Hb A and Hb F under similar conditions of pH and [BPG].

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Figure 15.39 The polymerization of Hb S via the interactions between the hydrophobic Val side chains at position β6 and the hydrophobic pockets in the EF corners of β-chains in neighboring Hb molecules. The protruding “block” on Oxy S represents the Val hydrophobic protrusion. The complementary hydrophobic pocket in the EF corner of the β-chains is represented by a square-shaped indentation. (This indentation is probably present in Hb A also.) Only the β2 Val protrusions and the β1 EF pockets are shown. (The β1 Val protrusions and the β2 EF pockets are not involved, although they are present.)