richardson lecture, april 25, 2007 hemoglobin: o 2 binding equilibria and the t-r, deoxy-oxy...

Richardson Lecture, April 25, 2007

Hemoglobin: O2 binding equilibria and the T-R, Deoxy-Oxy Transition

• Powerpoint introduction

• High resolution crystal structures with electron density shown in KiNG

• Good-parts version of ProTour8.kin shown in Mage

Files available for downloading and on-line viewing at web-site:

http://Kinemage.biochem.duke.edu

Look down in kinemage home page for: “Teaching: Course materials” … “CHEM22L” click on CHEM22L link --> …

Hemoglobin ---- Oxygen equilibria

CO2 + H2O <==> H2CO3

equilibrium

reaction

bindingHb + O2 <==> HbO2

Binding is a reaction: that O2 is in a very special relationship!

A + B <==> AB

Environment controls the reaction;Macromolecular structure provides exquisitely adjusted environments!

Unfolded protein: ChemistryFolded protein: Biology Biochemistry

Role of the globins in oxygen transport and storage

Figure 7-1 Mathews, van Holde,Ahern, 2001

The transport rate of a diffusing substance varies inversely

with the square of the distance it must diffuse. O2 diffusion through tissues thicker than 1 mm is too slow to support life:

Myoglobin is the oxygen storage protein- High affinity for O2

- Major physiological role is to facilitate oxygen transport in rapidly respiring muscle.

Hemoglobin is used for transport of oxygen from the lungs, gills, or skin of an animal to its capillaries.

- Lower affinity for O2 than myoglobin- Also for removing CO2 from tissues- CO2 is a major product of metabolite

oxidation

Illustrates regulation of protein function andevolution.

Summary: Role of the globins in oxygen transport and storage

Figure 7-1Mathews, van Holde,Ahern, 2001

QuickTime™ and aGIF decompressor

are needed to see this picture.

• Lungs:– Oxygenation favors the oxy Hb form, which

stimulates the release of CO2.

• Arteries and tissues– The lower pH and high CO2 favor deoxy Hb

• Promote O2 release and binding of CO2

– CO2 -- both in forming bicarbonate and in reacting with Hb -- causes the release of more protons, further stimulating O2 release and CO2 binding.

– Hyperventilation: dizziness from breathing too rapidly and purging CO2 from the tissues, which impairs the release of O2 into the tissues. (Correct by breathing into a paper bag to bring CO2 back into the blood.

Hyperbolic binding curves for transport proteins --relative to the storage protein myoglobin

Figure 7-8A,B Mathews, van Holde, Ahern, 2001



A. Efficient in binding but not in unloading

B. Efficient in unloading but not in binding

Sigmoidal binding curve for transport proteins: Hemoglobin cooperatively binds O2

Figure 7-8C,D Mathews, van Holde, Ahern, 2001

C. Efficient in both binding and unloading

D. Switch from weak- to strong-binding state



Comparison of myoglobin and hemoglobin

Figure 7-3 Mathews, van Holde, Ahern, 2001



Myoglobin (153 a.a.) (Mb) Hemoglobin 4 x (146 a.a.) (Hb) (Monomer) (Tetramer)

Sequences of Mb and Hb are overall very similarLetters A-H indicate -helical regions of polypeptide chainsHemoglobin contains two - and two ß -chains (tetramer)

Sigmoidal Curve?

Low affinity and High affinity interconverting forms:

Multiple subunits interact with and control each other…

Deoxy-oxy states board drawing




Bohr effect in hemoglobin is response to pH changes

• Allosteric effectors– CO2 – protons– lactic acid

• Consequences:– In the capillaries, hydrogen

ions promote the release of O2 by driving the reaction to the right.

– As venous blood enters the lungs, re-oxygenation reverses the effect, releasing the H+ from Hb•nH+ by shifting the equilibrium the left.

– This in turn releases the CO2 from the bicarbonate dissolved in the blood.

Bohr Effect and CO2 transport

(a) f O2– lower O2 affinity of Hb– HbO2 + H+ HbH+ + O2

(b) O2f O2

(c) PGf O2

A decrease in pH of only 0.8 units shifts the P50 from 20 mm Hg to over 40 mm Hg, greatly increasing the amount of oxygen unloaded to myoglobin.

The O2 affinity of Hb increases with increasing pH.



Bohr Effect - Mechanism(a) f O2

– lower O2 affinity– HbO2 + H+ HbH+ + O2

Mechanism: The O2 affinity of Hb increases with increasing pH. Certain proton binding sites in deoxy Hb are of

higher affinity than in oxy Hb.(Example of details to be looked at…:)

• In the deoxy form, His146 at the C-terminus of a -chain can make a salt bridge with Asp 94, if the His is protonated.• The salt bridge stabilizes the proton against dissociation. • In the oxy form, the pKa of His146 falls to about 6.5. The salt bridge cannot be formed.

At blood pH (7.4) His 146 is largely unprotonated in oxyhemoglobin.

Other amino acid residues are involved too, like those at the N-terminal of -chains.



Effect of BPG

( a.k.a. DPG )

Mechanism of the T (low oxygen affinity) to R transition (high oxygen affinity) in hemoglobin




(a) Deoxyhemoglobin (T state)

(b) Transition (c) Oxyhemoglobin (R state)

• Fe moves (from ~0.6 A out of the heme plane) into the porphyrin plane toward His F8

• Salt bridges and H-bonds holding the C-termini in the and -chains are broken.

• One pair rotates and slides with respect to the other

Hemoglobin, the evidence… 1.25 Å Resolution Crystal Structures of Human Haemoglobin in

the Oxy, Deoxy and Carbonmonoxy Forms

Sam-Yong Park1⁎, Takeshi Yokoyama1, Naoya Shibayama2, Yoshitsugu Shiro3 and Jeremy R. H. Tame1⁎

J. Mol. Biol. (2006) 360, 690–701

2DN2.pdb DeoxyHbA

Coordinates from Protein Data Bank:http://www.rcsb.org

Hydrogens added in MolProbity:http://molprobity.biochem.duke.edu

Electron density maps from the Electron Density Server:http://eds.bmc.uu.se

2DN1.pdb OxyHbA

2DN3.pdb CO-HbA

…and now into the crystals…

Hemoglobin, the story…

"THE PROTEIN TOURIST #8 -

THE T-R, DEOXY-OXY TRANSITION IN HUMAN HEMOGLOBIN"

David Richardson, Celia Bonaventura, and Jane Richardson

Protein Science vol. 3, #10 electronic supplement, Oct. 1994.

view on the web in KiNG:

http://kinemage.biochem.duke.edu/teaching/chem22l/index.php

ProTour8.kin:

Kin.1- Hb tetramer: deoxy vs oxy transition animated

Kin.2- Hb T-R transition: alpha chain and heme closeup

Kin.3- The alpha1-beta2 allosteric interface

Kin.4- Alpha1-alpha2 salt bridges

Kin.5- Beta2 salt bridges

Good parts version: HbAllo.kin

richardson lecture, april 25, 2007 hemoglobin: o 2 binding equilibria and the t-r, deoxy-oxy...

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