CHMI E.R. Gauthier, Ph.D. 1 CHMI 2227E Biochemistry I Proteins: - Quaternary structure

CHMI E.R. Gauthier, Ph.D. 2 Quaternary structure

CHMI E.R. Gauthier, Ph.D. 3 Quaternary structure Quaternary structure involves several polypeptides:  Oligomers  Heteromers These subunits interact with each other through the usual weak interaction forces (H bonds, Van der Waals, ionic interactions, hydrophobic interactions) and/or though disulfide bonds; For aquous proteins, frequently, but not always, the interface between two subunits is made of hydrophobic amino acids. For membrane-bound proteins, the amino acids at the interface between the subunits are usually hydrophilic; Porin: a trimeric membrane-bound protein hydrophobic hydrophilic

CHMI E.R. Gauthier, Ph.D. 4 Quaternary structure Hemoglobin Made up of 4 polypeptide chains:  2 copies of  -subunit (or HbA): yellow and blue;  2 copies of  -subunit (or HbB): red and pink Each subunit binds its own heme group: so each subunit can bind O 2 Each subunit is highly similar in structure to myoglobin; Both hemoglobin and myoglobin bind O 2 in a very similar fashion

CHMI E.R. Gauthier, Ph.D. 5 Quaternary structure Hemoglobin HbA vs myoglobin HbAMyoglobin

CHMI E.R. Gauthier, Ph.D. 6 Quaternary structure Oxygen binding by Hb and myoglobin 4 major residues surround the heme group:  Phe 43  His 64  Val 68  His 93 These amino acids create a hydrophobic environment while help hold the heme group in place; Also: His 93 binds the Fe 2+ atom;

CHMI E.R. Gauthier, Ph.D. 7 Quaternary structure Oxygen binding by Hb and myoHb O 2 binds the Fe 2+ atom of the heme group, and is held in place with His 64; Oxygen-bound myoglobin/Hb is called oxymyoglobin/oxyHb Oxygen-free myoglobin/Hb is called deoxymyoglobin/deoxyHb Now, if both Myo and Hb can bind O 2, why is it that Hb is a multimeric protein, while myoglobin is monomeric??? WHY????

CHMI E.R. Gauthier, Ph.D. 8 Quaternary structure Oxygen binding by Hb and myoHb A B O 2 binding to myoglobin shows a simple equilibrium where the amount of O 2 bound-myoglobin (y) directly depends on the concentration of O 2 present; However, O2 binding to Hb is more complex:  At low O 2 concentration, very little Hb binds O 2 even as the concentration of O 2 increases (part A of the Hb curve);  However, at a certain threshold of O 2 concentration, Hb becomes rapidly saturated with O 2 (part B of the Hb curve);

CHMI E.R. Gauthier, Ph.D. 9 Quaternary structure Oxygen binding by Hb and myoHb A B At high O 2 concentrations, both myoglobin and Hb are saturated, meaning there are no more O 2 -binding spots available. Interestingly: the affinity of myoglobin and Hb for oxygen varies by a factor of 10:  Only 2.8 Torr are required to get 50% of myoglobin saturated;  However, 26 Torr are required to half-saturate Hb.

CHMI E.R. Gauthier, Ph.D. 10 Quaternary structure O 2 binding changes the 3-D shape of Hb In the deoxyHb form, Fe 2+ is bonded to 5 ligands: His 93 and 4 amines from the heme group; When one subunit of Hb binds O 2, the Fe 2+ atom moves foward the plane of the heme group, pulling with it the His 93 and the  -helix; This causes a slight but significant change in the tertiary structure of all the other Hb subunits, even if they are in the deoxyHb form;

CHMI E.R. Gauthier, Ph.D. 11 Quaternary structure O 2 binding changes the 3-D shape of Hb The consequence of this slight change in conformation is an increase in the affinity of these other Hb subunits for O 2 ; This phenomenon, where a change in the shape in one subunit trigger similar changes in other subunits of the same molecule, is called cooperativity; Molecules exhibiting cooperativity are also called allosteric molecules;

CHMI E.R. Gauthier, Ph.D. 12 Quaternary structure Hb is an allosteric protein This phenomenon explains very well the behaviour of Hb in the presence of O 2 :  At low pO 2, all of the Hb subunits in the molecule are in the deoxy form with low affinity for O 2 : they bind O 2 very poorly;  At higher pO 2, one of the 4 subunits binds O 2, changes its conformation to the one with high affinity, and transmits this change in 3D structure to the other 3 subunits;  The other 3 subunits, now having high affinity for O 2, readily bind the molecule and rapidly become saturated. O2O2 O2O2 O2O2 O2O2 O2O2 O2O2 O2O2 O2O2 O2O2 O2O2 O2O2 Low affinity High affinity Saturated O2O2 O2O2 O2O2

CHMI E.R. Gauthier, Ph.D. 13 Quaternary structure Why Hb is allosteric, while Myoglobin is not? If Hb behaved like Myoglobin, then most of the Hb molecules would remained tightly bound to O 2 and would not unload O 2 in tissues; Conversely, if myoglobin behaved like Hb, it would readily let go of its O 2, drastically limiting our muscles ability to perform aerobic work;

CHMI E.R. Gauthier, Ph.D. 14 The Bohr effect The Bohr effect concerns the observed decrease in O 2 binding by hemoglobin when the pH is lowered; This effect explains why hemoglobin binds O 2 in the lungs, and releases it in the tissues;

CHMI E.R. Gauthier, Ph.D. 15 Gas exchange In the tissues Tissues Erythrocytes Glucose + O 2 ATP CO 2 H2OH2O Carbonic anhydrase H 2 CO 3 HCO 3 - H+H+ Plasma HCO 3 - (to lungs) Hb-4O 2 Hb-H + 4O 2 H 2 O + Cl -

CHMI E.R. Gauthier, Ph.D. 16 Gas exchange In the lungs Lungs Erythrocytes CO 2 H2OH2O Carbonic anhydrase H 2 CO 3 HCO 3 - H+H+ Plasma HCO 3 - Hb-4O 2 Hb-H + 4O 2 O2O2 H 2 O + Cl - CO 2 Air CO 2 O2O2