SINGLE MUTATION (THR72-]ILE) AT THE SUBUNIT INTERFACE IS CRUCIAL FOR THE FUNCTIONAL-PROPERTIES OF THE HOMODIMERIC COOPERATIVE HEMOGLOBIN FROM SCAPHARCA-INAEQUIVALVIS

The in vivo expression and the functional and spectroscopic properties are reported for a mutant of the homodimeric haemoglobin of the mollu sc Scapharca inaequivalvis (HbI), where residue threonine 72 (position 9 in the E helix) at the subunit interface has been substituted by is oleucine. The aim of this study is to test the hypothesis that increas ing the hydrophobicity character of the subunit interface may modulate oxygen affinity and co-operativity of this haemoglobin. In fact, X-ra y crystal structure studies have shown that the subunit interface, for med by the E and F helices of the two chains, changes its character fr om hydrophilic to hydrophobic upon oxygenation. This is primarily due to extrusion of Phe97 side-chain from the haem pocket toward the inter face, which disrupts a network of ordered water molecules and results in close van der Waals contacts between Phe97 and Thr72 of the partner subunit. Thr72-->Ile HbI was expressed in E, coli after mutation of H bI-DNA and it displays a similar to 40-fold enhancement of oxygen affi nity and a marked reduction of co-operativity in oxygen binding, with respect to native HbI. These functional properties and the kinetics of oxygen dissociation and carbon monoxide combination rates, as well as data from EPR and circular dichroism spectroscopy, indicate that inde ed the increase of the hydrophobicity at the interface upon mutation s tabilizes the ''high affinity'' conformation of the protein, suggestin g that extrusion of Phe97 toward the interface should be facilitated e ven in the unliganded form.