1. Chemical Biology 03 BLOOD Biomolecular Structure Myoglobin and Hemoglobin 9/28-30/09 www.optics.rochester.edu/.../image007.gif

2. Chemical Biology 03 BLOOD Biomolecular Structure Myoglobin and Hemoglobin Lecture 9 and 10: 9/28-30/09

3. The biochemistry of O 2 binding to Hb Each of the four subunits of Hb has a central helix which binds a heme with binds an iron ion (Fe(II) which in turn binds O 2.

4. O 2 exchanges from Hemoglobin (Hb) to Myoglobin (Mb) in the tissue Oxygen binding to Hb decreases as oxygen pressure decreases. Oxygen is released at the tissue, and myoglobin grabs it up. tissuelungs lungs pO 2 in air ~100 torrs

5. So, let’s first try to understand Mb!!! HEMOGLOBIN Crystal structure is very complicated. Hb protein is four subunits, four heme groups, and seems to behave differently when all together as compared with monomers. Oxygen saturation curve is “sigmoidal” complicated mathematical formula. MYOGLOBIN Crystal structure is very simple Mb protein is one subunit, one heme, and behaves simply Oxygen saturation curve is hyperbolic, which mathematically is quite simple y= x/(a+x) O 2 Exchanges from Hb to Mb O2O2

6. Myoglobin Structure: O 2 Binding Mb is the oxygen storage protein in muscle 153 amino acids 17,000 g/mole Protein has 8 helices A-H Heme Fe(II) is bound through side group N of His F8 (proximal his) His E7 is close to the other side of heme, but doesn’t coordinate (distal his)

7. Myoglobin Structure: O 2 Binding Fe(II), iron is bound to four N atoms within heme 5th coordination is N of Histidine amino acid, F8 of helix F. 6th coordination site is open for O 2 to bind. deoxyMb, deoxyHb, heme ring is puckered in absence of 6th ligand; Fe(II) out of plane. oxyMb, oxyHb ring is flat with sixth ligand bound to Fe(II), metal is in heme plane. Proximal His Distal His

8. Myoglobin Structure: O 2 Binding Heme group is a really special molecule made up of carbon, hydrogen, and nitrogen, with a big fat iron atom sitting in the center waiting to bind to the O 2

9. The biochemistry of oxygen binding Here’s a cartoon of what happens when the O 2 binds to the Fe in Myoglobin

10. Myoglobin Structure: O 2 Binding – Effect of sixth site coordination on the color of myoglobin. – O xygen does not bind straight on, the N from the distal His amino acid side group in the way. N from proximal His N from distal His

11. Myoglobin Structure: O 2 Binding Space filling model of myoglobin with His F8 coordinating and His E7 poised nearby See how little room oxygen has to snuggle in and bind to the Iron. Heme is bound in a hydrophobic crevice with propionic acid groups projecting into solution orienting the heme.

12. A Tale of Two Binding CurvesMyoglobin getting the dissociation constant from the saturation curveHemoglobin sigmoidal saturation curve two state model (T and R) O 2 binding is cooperative

13. Myoglobin : O 2 Binding What really happens in the binding (association) and unbinding (disassociation) of O 2 to Mb? Mb-O 2 ⇌ Mb + O 2 equilibrium described by the extent to which the dissociation occurs, measured by a K d We can write an equation for this equilibrium: K d = [Mb] free [O 2 ] / [Mb-O 2 ] K d = [Mb] free [pO 2 ]/[Mb-O 2 ] [Mb] free = [Mb] T - [Mb-O 2 ] K d = ([Mb] T - [Mb-O 2 ])(pO 2 ) [Mb-O 2 ]

14. Myoglobin : O 2 Binding Mb-O 2 ⇌ Mb + O 2 equilibrium K d = ([Mb] T - [Mb-O 2 ])[pO 2 ] [Mb-O 2 ] Now, just allow one more substitution and a rearrangement, and we’ll get someplace really great! Y = fractional saturation Y = [Mb-O 2 ] / [Mb] T Y = pO 2 / (K d + pO 2 ) WOW, that’s a lot simpler!

15. Let’s EXCEL together

16. Conclusions from excel about O 2 binding to Mb

17. Hemoglobin Hemoglobin : O 2 Binding Like Mb, Hb’s saturation decreases as pO 2 decreases. Unlike Mb, Hb at the same pO 2, say 10 torr, Hb is much less saturated. If O 2 was bound it would come off. Unlike Mb, Hb binding curve is not hyperbolic, but sigmoidal. Sigmoidal shape suggest two states for Hb (more on this in a minute) Y = O 2 n / (K d +O 2 n ) Y = O 2 / (K d + O 2 )

18. Let’s EXCEL together

19. Conclusions from excel about O 2 binding to Hb

20. Hb Hb Structure : (  ) 2 – Hemoglobin is a dimer of dimers,      dimer     – see http://www.umass.edu/microbio/chime/hemoglob/2frmcont.htm

21. Hemoglobin Hemoglobin : Two states for O 2 Binding R state relaxed high affinity for O 2 T state tense Low affinity for O 2 http://www.search.com/reference/Hemoglobin

22. Hemoglobin Hemoglobin : Two states for O 2 Binding R state relaxed high affinity for O 2 T state tense Low affinity for O 2 http://www.search.com/reference/Hemoglobin

23. Hb : Describing O 2 Binding Sigmoidal binding suggests Two state model: Hb can be in either high O 2 affinity (R state) low O 2 affinity (T state) Binding of O 2 to Hb is cooperative: binding of first ligand affects the affinity of the remaining sites for ligand.

24. Hemoglobin Hemoglobin Cooperativity R state oxy ALL T state deoxy NOTHING

25. Hemoglobin : ribbon structure