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5.3.2 Cooperative binding

A feature of some proteins comprising more than one subunit is that binding of a ligand to its binding site on one subunit, can increase the affinity of a neighbouring subunit for the same ligand, and hence enhance binding. The ligand-binding event on the first subunit is communicated, via conformational change, to the neighbouring subunit. This type of allosteric regulation is called cooperative binding.

Haemoglobin, as we have already discussed, is a tetramer consisting of two pairs of identical subunits (α and β) (Figure 20). Each subunit can bind an O2 molecule via its associated haem (Figure 24) and haemoglobin demonstrates cooperative binding of this ligand. Thus, binding of O2 by one subunit facilitates the binding of O2 molecules by the other subunits of the tetramer. This characteristic makes haemoglobin a more efficient O2 transporter than it would be if the O2 binding sites were independent of one another. As a result, O2 saturation of haemoglobin can be virtually complete in the lungs whilst, in the tissues, where the O2 concentration is low, O2 dissociates readily. At this point it is appropriate to consider the molecular basis for this effect.

The crystal structures of both the oxygenated form (oxy-Hb) and the deoxygenated form (deoxy-Hb) of haemoglobin have been determined (pdb files 1gzx and 1a3n respectively). Oxygenation of haemoglobin causes extensive changes in both the tertiary and quaternary structure of the protein. The quaternary structure of deoxy-Hb is referred to as the T state (for tense), whilst that of oxy-Hb is known as the R state (for relaxed). The R state has a higher affinity for O2 than does the T state. (Note that these terms are used to describe alternative structures of allosteric proteins in general, the T form being the one with the lower affinity for the ligand.) Individual globin subunits can adopt either the T or the R conformation and a change in the conformation of one subunit can affect the conformation of a neighbouring subunit, thereby altering its affinity for O2. This is the basis for cooperative binding of O2 by haemoglobin.

Max Perutz (1914–2002) proposed what is currently accepted as the molecular mechanism of haemoglobin oxygenation. Transition between the T and R states is triggered by stereochemical changes at the haem groups. In deoxy-Hb, the Fe2+ ion is about 0.6 Å (60 pm) out of the haem plane because of steric repulsion between the nearby histidine and the N atoms of the porphyrin ring. On binding of O2 to the haem, on the opposite side from the His residue, the Fe2+ ion is pulled into the plane of the haem, dragging the His residue with it (Figure 32a). These movements, in turn, affect the orientation and position of neighbouring side-chains and as a result, the tertiary structure of the subunit switches to the R form.

Figure 32 (a) Binding of O2 at the haem in haemoglobin causes the Fe2+ ion to move into the plane of the haem and causes conformational changes in the associated subunit; the oxygenated structure is shown in red and the deoxygenated structure is shown in blue. (b) Simplified schematic to show the relative rotation of the αβ dimers that switches haemoglobin from the T state (blue) to the R state (red).

How does the change in conformation of one subunit affect the conformation of a neighbouring subunit? The answer lies in the interactions at the interface between the subunits. The α1–β1 and α2–β2 contacts are equivalent. These interactions are extensive and stable, so that each of these dimers moves as one. In contrast, the α1–β2 (and the equivalent α2–β1) contact is weak. When O2 binds at one of the haems, the α1–β2 (and α2–β1) interaction is disrupted. As a result, one pair of αβ subunits rotates relative to the other pair by 15° (Figure 32b). In this way, the conformational change caused by O2 binding at one subunit is transmitted to ligand-free subunits. This conformational change effectively switches ligand-free subunits to the R state, increasing their affinity for O2.


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