Section 23.4.6.3.  Cooperativity in dimeric haemoglobin

The X-ray crystal structures of liganded and unliganded dimeric haemoglobin from Scapharca inaequivalvis have revealed that water molecules at the dimer interface form an integral part of the cooperativity mechanism in this system (Condon & Royer, 1994; Royer, 1994). The binding of oxygen to one of the monomers causes little rearrangement of quaternary structure. It does, instead, displace the side chain of Phe97 which, in the low-affinity deoxy form, packs in the haem pocket (Royer et al., 1990). Phe97 in the deoxy form lowers the oxygen affinity by restricting movement of the iron atom into the haem plane (Royer, 1994). Upon oxygen binding, Phe97 flips to the dimer interface, removing six out of the 17 water molecules that are found in the deoxy form (Fig. 23.4.6.1). The resultant destabilization of the water clusters found between the two subunits facilitates the flipping of Phe97 in the other subunit, with a concomitant increase in oxygen affinity of the haem in the second subunit (Pardanani et al., 1997; Royer et al., 1997).

Figure 23.4.6.1| top | pdf | Scapharca HbI interface water molecules. (a) Deoxy-HbI at 1.6 Å resolution (PDB code 3SDH) and (b) HbI-CO at 1.4 Å resolution (PDB code 4SDH). Included is a ribbon diagram showing the tertiary structure of each subunit, bond representations for the haem group and Phe97 side chain, and spheres representing the approximate van der Waals radii of oxygen atoms for core interface water molecules. Note the cluster of 17 ordered water molecules in the interface of deoxy-HbI for which Phe97 is packed in the haem pocket. Upon ligation, by either CO or O2, Phe97 is extruded into the interface and disrupts this water cluster, expelling six water molecules from the interface. These plots were produced with the program MOLSCRIPT (Kraulis, 1991). Reprinted with permission from Royer et al. (1997). Copyright (1997) The American Society for Biochemistry & Molecular Biology.

In each of the monomeric subunits, Thr72 is positioned to form a hydrogen bond with a water molecule at the periphery of the deoxy dimer interface (not shown in Fig. 23.4.6.1). In effect, this interaction caps the water cluster on either side of the interface, presumably helping to stabilize these well ordered water molecules. The isosteric mutation Thr72 to Val was designed to test the importance of this interaction to the stability of the water cluster in the low-affinity haemoglobin dimer and the resultant effect on ligand affinity and cooperativity (Royer et al., 1996). The crystal structure of the T72V mutant was solved to 1.6 Å resolution. This crystal structure reveals that the only significant difference between the mutant and wild-type proteins is the loss of the two water molecules that directly hydrogen-bond to Thr72 in each of the wild-type subunits. Furthermore, there is a significant increase in both activity and cooperativity resulting from the mutation (Royer et al., 1996). The authors conclude that, as a result of the mutation, the loss of two water molecules in the interface cluster is sufficient to alter the balance between the low- and high-affinity forms of the protein. This result demonstrates that water molecules are key mediators of information transfer between the haems in the two subunits in dimeric haemoglobin and that their precise positioning and interactions with protein atoms are crucial in maintaining the chemical balance required for biological function.