A single mutation (Thr72-->Ile) at the subunit interface is crucial for the functional properties of the homodimeric co-operative haemoglobin from Scapharca inaequivalvis.
The in vivo expression and the functional and spectroscopic properties are reported for a mutant of the homodimeric haemoglobin of the mollusc Scapharca inaequivalvis (HbI), where residue threonine 72 (position 9 in the E helix) at the subunit interface has been substituted by isoleucine. The aim of this study is to test the hypothesis that increasing the hydrophobicity character of the subunit interface may modulate oxygen affinity and co-operativity of this haemoglobin. In fact, X-ray crystal structure studies have shown that the subunit interface, formed by the E and F helices of the two chains, changes its character from hydrophilic to hydrophobic upon oxygenation. This is primarily due to extrusion of Phe97 side-chain from the haem pocket toward the interface, 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 HbI-DNA and it displays a approximately 40-fold enhancement of oxygen affinity 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 indeed the increase of the hydrophobicity at the interface upon mutation stabilizes the "high affinity" conformation of the protein, suggesting that extrusion of Phe97 toward the interface should be facilitated even in the unliganded form.
Gambacurta
A
A
Department of Experimental Medicine and Biochemical Sciences, University Tor Vergata, Rome, Italy.
Piro
M C
MC
Coletta
M
M
Clementi
M E
ME
Polizio
F
F
Desideri
A
A
Santucci
R
R
Ascoli
F
F
D000818:Animals; D001483:Base Sequence; D049872:Bivalvia; D002248:Carbon Monoxide / Q000378:metabolism; D002942:Circular Dichroism; D004247:DNA / Q000737:chemistry; D006454:Hemoglobins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008024:Ligands; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010100:Oxygen / Q000378:metabolism; D017354:Point Mutation; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014867:Water / Q000378:metabolism
D006454:Hemoglobins; D008024:Ligands; D011994:Recombinant Proteins; D014867:Water; C083082:dimeric hemoglobin, Scapharca inaequivalvis; D002248:Carbon Monoxide; D004247:DNA; D010100:Oxygen