Richardson Lecture, April 25, 2007 Hemoglobin: O 2 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: Look down in kinemage home page for: “Teaching: Course materials” … “CHEM22L” click on CHEM22L link --> …

Hemoglobin ---- Oxygen equilibria CO 2 + H 2 O H 2 CO 3 equilibrium reaction binding Hb + O 2 HbO 2 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: Chemistry Folded 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. O 2 diffusion through tissues thicker than 1 mm is too slow to support life: Myoglobin is the oxygen storage protein - High affinity for O 2 - 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 O 2 than myoglobin - Also for removing CO 2 from tissues - CO 2 is a major product of metabolite oxidation Illustrates regulation of protein function and evolution.

Summary: Role of the globins in oxygen transport and storage Figure 7-1 Mathews, van Holde, Ahern, 2001 Lungs: –Oxygenation favors the oxy Hb form, which stimulates the release of CO 2. Arteries and tissues –The lower pH and high CO 2 favor deoxy Hb Promote O 2 release and binding of CO 2 –CO 2 -- both in forming bicarbonate and in reacting with Hb -- causes the release of more protons, further stimulating O 2 release and CO 2 binding. –Hyperventilation: dizziness from breathing too rapidly and purging CO 2 from the tissues, which impairs the release of O 2 into the tissues. (Correct by breathing into a paper bag to bring CO 2 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 O 2 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 similar Letters A-H indicate  -helical regions of polypeptide chains Hemoglobin 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

Figure 7-16 Mathews, van Holde, Ahern, 2001 Bohr effect in hemoglobin is response to pH changes Allosteric effectors –CO 2 –protons –lactic acid Consequences: –In the capillaries, hydrogen ions promote the release of O 2 by driving the reaction to the right. –As venous blood enters the lungs, re-oxygenation reverses the effect, releasing the H + from HbnH + by shifting the equilibrium the left. –This in turn releases the CO 2 from the bicarbonate dissolved in the blood.

Bohr Effect and CO 2 transport (a)        f O 2  –lower O 2 affinity of Hb –HbO 2 + H +  HbH + + O 2 (b)   O 2       f O 2 (c)  PG      f O 2 A decrease in pH of only 0.8 units shifts the P 50 from 20 mm Hg to over 40 mm Hg, greatly increasing the amount of oxygen unloaded to myoglobin. The O 2 affinity of Hb increases with increasing pH.

Bohr Effect - Mechanism (a)        f O 2  –lower O 2 affinity –HbO 2 + H +  HbH + + O 2 Mechanism: The O 2 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 Figure 7-13 Mathews, van Holde, Ahern, 2001 (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: Hydrogens added in MolProbity: Electron density maps from the Electron Density Server: 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 view on the web in KiNG: 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