L13-Hemoglobin - Lecture notes 13 PDF

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notes on the specific interactions of hemoglobin...

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Chemistry 558

Fung

Structure - Functione Relationship Use two oxygen binding protein molecules, hemoglobin (Hb) & myoglobin (Mb), as examples to demonstrate the structure-function relationship in biomolecules.

red blood cell

I.

Why do we need proteins to bind oxygen? Can we just have oxygen in plasma (H2O)? Solubility of O2 in H2O Henry's Law [O2] in H2O = k x [Partial Pressure of O2]

At 100 mm Hg pressure (in lung) at 40E E C, k given in Handbook is 23.

[O2] = 23 x 100/760 = 3 mL O2 per liter of H2O

O2 in Hemoglobin

Hb + 4 O2

HbO2

1 mole of Hb carries 4 moles of O2 1 gm of Hb carries 4 x 22.4 / 64,500 = 1.35 ml O2 (22.4 L is the volume of one mole of an ideal gas at standard temperature and pressure.)

100 ml blood contains about 15 gm Hb (15 gm %), or 150 gm Hb/liter blood. 1 liter of blood carries 150 x 1.35 ml of O2 = 200 mL O2 So hemoglobin/blood is about 70 times more efficient than water in carrying oxygen. Icefish have no Hb,& rely on oxygen dissolved in their plasma, which can transport only10% as much oxygen as the Hb-filled blood of most fish. http://www.the-scientist.com/?articles.view/articleNo/34797/title/-White-Blooded--Icefish--1927/

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Chemistry 558

Fung

II. Since the HEME groups carry oxygen, how about just 4 heme groups and no "globin" (protein part)? In order to answer this question, we need to look at the structures of both heme groups and “globin” part. Heme Group A porphyrin ring - tetrapyrrole rings with Fe. Coordination # for Fe is 6, with 4 pyrrole N's, 1 proximal His N, & 1 ligand (O2).

Deoxy Hb Fe++ has 6 electrons with 4 unpaired electrons. S = 4/2 = 2. d orbitals dx2-y2 dz2 dxy, dxz, dyz

8 9 9 9 98

eg t2g

Oxy Hb (Fe++ with a ligand, O2) Fe++ also has 6 electrons, but due to ligand-field theory, are distributed differently, no unpaired electrons, with S = 0. dx2-y2 dz2

eg

.

dxy, dxz, dyz

98 98 98

t2g .

https://en.wikibooks.org/wiki/Introduction_to_Inorganic_Chemistry/Coordination_Chemistry_and_Crystal_Field_Theory

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Chemistry 558

Fung

Met Hb (Oxidized Hb) Fe+++ has 5 electrons, all unpaired, with S = 5/2; can't bind O2. dx2-y2 dz2

9 9

eg

dxy, dxz, dyz

9 9 9

t2g

Heme Fe++ in water ----> Fe+++ readily. Fe in hemes can not maintain +2 state in water. Heme in non-polar polystyrene matrix to exclude water is able to bind O2 reversibly. In general, about 60 interactions between atoms of globin chains coming to within 4 Å of atoms in heme, all but 3 are non-polar type. So the globin part around heme provide non-polar environment for hemes in a protein that is soluble in water. III. Why do we have one chain for myoglobin and 4 chains (2 chains and 2 -chains) for Hb? Let us look at the oxygen binding properties of Mb and Hb. Lung oxygen, arterial pressure: 100 torr (mm Hg); Tissue oxygen, venous pressure: 40 torr.

Hb releases its oxygen at low oxygen conc. environment easier than Mb. At 26 Torr, Hb releases half of the oxygen while Mb is still fully saturated. Mb can be oxygenated easier than Hb. At 1 torr, 50 % Mb is saturated. So Mb is for oxygen storage. 3

Chemistry 558

Fung

IV. How does Hb regulate O2 binding to give sigmoidal curve? Tertiary structure * 141 amino acid residues in -chain, and 146 amino acid residues in -chain. * Peptide chains form 8 major helical segments (A, B, C, D, E, F, G & H), with 7 non-helical segments (NA, AB, CD, EF, FG, GH & HC) in between. They are folded into a compact shallow box, ~ 45 x 35 x 25 Å per chain. * Heme groups at corner and buried between E helix and F helix. (F8 His is liganded to Fe.) * Non-polar residues are in the interior and are conserved in different species. They are important for functional purposes. * Polar residues at surface; often not conserved. Some His on surface are important. Salt bridge - β2 146 His and β2 94 asp form salt bridge in deoxy form, but not in oxy form. Different structure for oxy and deoxy states. Salt bridge (ion pair)

2 94 Asp

2 146 His

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Chemistry 558

Fung

Quaternary Structure About 65 x 55 x 50 Å 2 's and 2 's are held together by inter-chain interactions. * hydrogen bonds. * salt bridges (ion pairs). * no disulfide bonds. * 11 contact extensive, 34 side chains. * 12 contact only has 19 side chains in ontact. Upon oxygenation, quaternary structural changes, T ---> R VDW (4 Å) H-bonds Salt Bridges Hb(deoxy) 1 1 98 5 0 (T) 1 2 69 1 1 1 2 2 β1 β2 1 HbO2 α1 β1 110 5 (R) α1 β2 80 1 α1 α2 β1 β2 http://en.wikipedia.org/wiki/Hemoglobin

* * *

β1 β2 getting closer: β1 (Fe) - β2 (Fe) = 40 Å in deoxy 33 Å in oxy 1 1 minor changes 1 2 major changes, drastic movement in 1 2 subunit interface, "Dovetail" movement. 5

c

Chemistry 558

Fung

Changes in hydrogen bonds and salt bridges. Hydrogen bonds broken and a new set reformed. Some atoms moved about 6 Å . C-terminal residue salt bridges broken releasing terminal ends out of the globin part in oxy form. * 145 tyrosines are free to rotate. * the overall structure is from a more "tensed" (T) to a more "relaxed" (R) form. Functional properties * oxy form is the high affinity form * deoxy form is the low affinity form * oxygenation is cooperative - the binding of the first oxygen enhances the binding of the next oxygen. The binding of oxygen to one subunit, changes the electronic state of Fe and initiates a series of changes in intermolecular interaction to change the structure of hemoglobin and consequently changes the functional properties of the neighboring subunit. Bohr Effect H+ Hb

+

H+ + CO2

O2

+

HbO2

CO2 Deoxy

HbO2

Oxy

+

H+

H+ + CO2

CO2

Oxy

Hb Deoxy

Increase [H+] or [CO2], reduce O2 affinity. 6

+

O2

Chemistry 558

Fung

Increase pH (decrease H+), increase O2 affinity. In contracting muscle, H+ and CO2 are released, HbO2 will pick up H+ and CO2 and release O2.

So, where does H+ bind in Hb? Binding sites of H+ Hb- H+ + O2 HA Deoxy Hb

HbO2 + A+ Oxy Hb

H+ H+

[A-]/[HA] = 10 pH - pKa = 10 7.4 - pKa = [Oxy Hb]/[Deoxy Hb] At pH ~ 7.4, if we want mainly [A-] (oxy Hb), pKa < 7.4 if we want mainly [HA] (deoxy Hb), pKa > 7.4 pKa of the binding site has to change from pKa > 7.4 in deoxy to pKa < 7.4 in oxy. The pKa of 146 terminal His in oxy is about 7.1; in deoxy is about 8.0 since 94 Asp (- charge) is near 146 in deoxy, so high pKa.

The most important physiological function of Hb is to transport oxygen from the lungs to the tissues. Hemoglobin is also the predominant buffer in the red cells and thereby plays an important role in the maintenance of the intracellular pH. The only known physiological function of carbonic anhydrase is to catalyze the hydration of CO2 or the dehydration of HCO3-. 7

Chemistry 558

Fung

In addition to its respiratory role of facilitating the transport of metabolic CO2, carbonic anhydrase is involved in the transfer and accumulation of H and HCO3- in various tissues. BPG Effect (2,3-biphosphoglycerate) (DPG = 2,3 diphosphoglycerate; old name, wrong name)

BPG is negatively charged. It binds to a cavity that is largely positively charged (His and Lys) in deoxy state. BPG is ~ 9 C & fits in the deoxy cavity (~11 C), but not in the oxy cavity (~5 C). It stabilizes the "tense" deoxy state and allosterically promotes the release of the remaining oxygen molecules bound to the Hb. BPG is allosteric effector. It affects the oxygen affinity of Hb. Without BPG, deoxy Hb is more like oxy Hb. So by varying BPG level, we vary the oxygen affinity of the blood. At high attitude, BPG level increases; low oxygen affinity of Hb, more O2 released from blood to tissue. Water Effect (Protein Solvation in Allosteric Regulation: A water Effect on Hemoglobin, M.F. Colombo, D.C. Rau, V.A. Parsegian, Science 256, 655-659, 1992.)

“For hemoglobin, equilibrium between R and T conformations, and consequently its oxygen affinity, is modulated by the binding of several small molecules and ions, such as H+, CO2, phosphate, and Cl-. The structures of the two limiting conformations show that several direct contacts between subunits are broken and exposed to solvent during the transition from deoxy T state to the fully oxygenated R state. This change in structure implies a difference in hydration or water binding 8

Chemistry 558

Fung

between the two conformations However, the energetic consequences of solvation regulating protein activity are usually neglected......These data show that 50 to 70 solute-excluding water molecules become part of the Hb tetramer in its transition from the deoxy to oxy form...... The ligated oxy protein exposes from 500 to 800 D2 more surface to the solvent than the unligated deoxy form...... It is doubtful that the R state would even exist without the stabilization promoted by hydration of the extra surface. We are forced to recognize the importance of solvation in protein regulation.”

Hb F (fetus Hb) transports O2 efficiently in a low oxygen environment. Hb A (adult Hb) = “normal Hb”; HbS = sickle Hb Hb Kansas; Hb D-Punjab; Hb O-Arab; Hb G-Philadelphia; Hb Hasharon; Hb Lepore; Hb M Milwaukee; Hb Chicago;....

Structure-function relationship!!

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