Chemical Biology 03 BLOOD

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

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

The biochemistry of O2 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 O2.

lungs pO2 in air ~100 torrs O2 exchanges from Hemoglobin (Hb) to Myoglobin (Mb) in the tissue tissue lungs • Oxygen binding to Hb decreases as oxygen pressure decreases. • Oxygen is released at the tissue, and myoglobin grabs it up.

O2 Exchanges from Hb to Mb 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) O2

Myoglobin Structure: O2 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)

Myoglobin Structure: O2 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 O2 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

Myoglobin Structure: O2 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 O2

The biochemistry of oxygen binding Here’s a cartoon of what happens when the O2 binds to the Fe in Myoglobin

Myoglobin Structure: O2 Binding • Effect of sixth site coordination on the color of myoglobin. • Oxygen does not bind straight on, the N from the distal His amino acid side group in the way. N from distal His N from proximal His

Myoglobin Structure: O2 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.

A Tale of Two Binding Curves Myoglobin getting the dissociation constant from the saturation curve Hemoglobin sigmoidal saturation curve two state model (T and R) O2 binding is cooperative

Myoglobin: O2 Binding • What really happens in the binding (association) and unbinding (disassociation) of O2 to Mb? Mb-O2⇌ Mb + O2 equilibrium described by the extent to which the dissociation occurs, measured by a Kd • We can write an equation for this equilibrium: Kd = [Mb]free [O2] / [Mb-O2] Kd=[Mb]free [pO2]/[Mb-O2] [Mb]free = [Mb]T - [Mb-O2] Kd= ([Mb]T - [Mb-O2])(pO2) [Mb-O2]

Myoglobin:O2 Binding Mb-O2⇌ Mb + O2 equilibrium Kd= ([Mb]T - [Mb-O2])[pO2] [Mb-O2] Now, just allow one more substitution and a rearrangement, and we’ll get someplace really great! Y = fractional saturation Y = [Mb-O2] / [Mb]T Y = pO2 / (Kd+ pO2) WOW, that’s a lot simpler!

Let’s EXCEL together

Conclusions from excel about O2 binding to Mb

Hemoglobin: O2 Binding • Like Mb, Hb’s saturation decreases as pO2 decreases. • Unlike Mb, Hb at the same pO2, say 10 torr, Hb is much less saturated. If O2 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 = O2/ (Kd+ O2) Y = O2n/ (Kd+O2n)

Let’s EXCEL together

Conclusions from excel about O2 binding to Hb

Hb Structure: (ab)2 • Hemoglobin is a dimer of dimers,a1 b1dimera2 b2 • see http://www.umass.edu/microbio/chime/hemoglob/2frmcont.htm

Hemoglobin: Two states for O2 Binding http://www.search.com/reference/Hemoglobin T state tense Low affinity for O2 R state relaxed high affinity for O2

Hb: Describing O2 Binding • Sigmoidal binding suggests • Two state model: Hb can be in either • high O2 affinity (R state) • low O2 affinity (T state) • Binding of O2 to Hb is cooperative: binding of first ligand affects the affinity of the remaining sites for ligand.

Hemoglobin Cooperativity R state oxy ALL T state deoxy NOTHING

Hemoglobin: ribbon structure

Chemical Biology 03 BLOOD