Transcript Document

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
A + B
<==> AB
equilibrium
CO2 + H2O <==> H2CO3
reaction
Hb + O2
binding
<==> HbO2
Binding is a reaction: that O2 is in a very special relationship!
Environment controls the reaction;
Macromolecular structure provides exquisitely adjusted environments!
Unfolded protein:
Folded protein:
Chemistry
Biology
Biochemistry
Role of the globins in oxygen
transport and storage
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 and
evolution.
Figure 7-1 Mathews, van Holde,Ahern, 2001
Summary: Role of the globins in oxygen transport
and storage
•
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.
Figure 7-1
Mathews, van Holde,
Ahern, 2001
QuickTime™ and a
GIF decompressor
are needed to see this picture.
Hyperbolic binding curves for transport proteins -relative to the storage protein myoglobin
A. Efficient in bindingQuickTime™
but and a B. Efficient in
GIF decompressor
not in unloading are needed to see this picture. unloading but not in
binding
Figure 7-8A,B Mathews, van Holde, Ahern, 2001
Sigmoidal binding curve for transport
proteins: Hemoglobin
cooperatively
binds O2
QuickTime™
and a
GIF decompressor
are needed to see this picture.
C. Efficient in both
binding and unloading
Figure 7-8C,D Mathews, van Holde, Ahern, 2001
D. Switch from
weak- to strongbinding state
Comparison of myoglobin and hemoglobin
QuickTime™ and a
GIF decompressor
are needed to see this picture.
Myoglobin (153 a.a.) (Mb)
(Monomer)
Hemoglobin 4 x (146 a.a.) (Hb)
(Tetramer)
Sequences of Mb and Hb are overall very similar
Letters A-H indicate a-helical regions of polypeptide chains
Hemoglobin contains two a- and two ß -chains (tetramer)
Figure 7-3 Mathews, van Holde, Ahern, 2001
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.
QuickTime™ and a
GIF decompressor
are needed to see this picture.
Figure 7-16 Mathews, van Holde, Ahern, 2001
Bohr Effect and CO2 transport
(a) 
f O2
– lower O2 affinity of Hb
– HbO2 + H+  HbH+ + O2
(b)  O2 
f O2
(c)  PG
f 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.
QuickTime™ and a
GIF decompressor
are needed to see this picture.
(a) 
Bohr Effect
- Mechanism
fO
2
– 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 a-chains.
QuickTime™ and a
GIF decompressor
are needed to see this picture.
Effect of
BPG
( a.k.a. DPG )
Mechanism of the T (low oxygen affinity) to R
transition (high oxygen affinity) in hemoglobin
• 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
a and -chains are broken.
• One a pair rotates and slides with respect to the
other
QuickTime™ and a
GIF decompressor
are needed to see this picture.
(a) Deoxyhemoglobin
(T state)
(b) Transition
Figure 7-13 Mathews, van Holde, Ahern, 2001
(c) Oxyhemoglobin
(R state)
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
Coordinates from Protein Data Bank:
http://www.rcsb.org
2DN1.pdb OxyHbA
2DN2.pdb DeoxyHbA
2DN3.pdb CO-HbA
Hydrogens added in MolProbity:
http://molprobity.biochem.duke.edu
Electron density maps from the Electron Density Server:
http://eds.bmc.uu.se
…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