Structural basis of Hb co-operativity:

A mathematical model for co-operative behavior in proteins provides some insight into this very important biochemical process. The allosteric behavior that produces co-operativity requires action from a site distinct from the binding (or active) site. Binding of oxygen at the heme of one subunit in a hemoglobin molecule affects its binding at another site. Energy must be transmitted from this first binding event to the second site; which molecular contacts are important in transmitting this energy?

Useful observations:

a. -chains alone exhibit hyperbolic binding of oxygen (no co-operativity)

b. a tetramer of -chains (a form called Hb H) has no co-operative binding

(conclusion: a tetramer of two different polypeptide chains is essential)

There are two kinds of interactions possible between subunits in an ₂-₂ tetramer: ₁-₁ or ₁-₂. The latter is the important one in hemoglobin.

Two lines of evidence support this conclusion:

a. the amino acids found in the contact region between these two subunits are highly conserved during evolution.

b. the region around the ₁-₂ interface also shows greater changes between the X-ray structures of the oxy and deoxy forms of hemoglobin.

Mechanics of oxygen binding:

a. O₂ binding causes a 0.06 nm movement of Fe into the plane of porphyrin ring due to spin changes in the "d" electrons of the iron.

b. there is a movement of the proximal F8 His, pulled by the Fe

c. which moves the F helix in toward the heme

d. tyrosine-145 (HC-2) near the carboxy terminus is removed from pocket between F and H helices

e. interchain salt links (see below) are broken, as are H-bonds

The result of these changes is that the fourth molecule of O₂ binds 300 times as tightly as the first one.

The Connections:

Salt links

These are electrostatic interactions between charged amino acid side-chains.

When there are more salt links, hemoglobin behaves as the tense (T) form, and it has a lower affinity for oxygen. This is the deoxy form of Hb.

When the salt links are broken, the molecule is in the relaxed (R) form, and it has a higher affinity for oxygen. This is the oxy form of Hb.

The Bohr effect

H⁺ ions enhance the release of oxygen from Hb. This is quite useful, because muscles functioning under anaerobic conditions are acidic due to the build-up of carbon dioxide and lactate.

2 H⁺ (approx.) + Hb-(O₂)₄ <==> Hb-H⁺₂ + 4 O₂

The higher acidity in muscle favors deoxygenation based on LeChatelier's principle.

In order for this to occur, the pK_a of some amino acids side chain group(s) must be raised on going from oxy to deoxy forms.

To test this idea, biochemists prepared Hb devoid of His at 146 and noted that the Bohr effect was reduced to half of the original value.

Bisphosphoglycerate

Bisphosphoglycerate (BPG) has five negative charges and its levels in red blood cells changes as part of human adaptation to high altitude.

Hb + BPG <==> Hb-BPG

BPG binds to Lys and His residues on both -chains that face the central cavity of hemoglobin. This binding effectively "cross-links" hemoglobin and stabilizes the deoxy (or tense) form of the molecule (R + BPG ---> T-BPG).

Upon oxygenation of hemoglobin, BPG is extruded from the central cavity, which becomes smaller as the conformation change occurs.

The binding of the first molecule of oxygen to a hemoglobin molecule provides the energy to shift this equilibrium in the direction of R + BPG.