Abstract
The subunit interface of the homodimeric hemoglobin from Scapharca inaequivalvis, HbI, is stabilized by a network of interactions that involve several hydrogen-bonded structural water molecules, a hydrophobic patch, and a single, symmetrical salt bridge between residues Lys-30 and Asp-89. Upon mutation of Lys-30 to Asp, the interface is destabilized markedly. Sedimentation equilibrium and velocity experiments allowed the estimate of the dimerization constants for the unliganded (K(1,2D) = 8 x 10(4) M(-1)) and for the CO-bound (K(1,2L) = 1 x 10(3) m(-1)) and oxygenated (K(1,2L) = 70 m(-1)) derivatives. For the oxygenated derivative, the destabilization of the subunit interface with respect to native HbI corresponds to about 8 kcal/mol, an unexpectedly high figure. In the K30D mutant, at variance with the native protein, oxygen affinity and cooperativity are strongly dependent on protein concentration. At low protein concentrations (e.g. 1.2 x 10(-5) m heme), at which the monomeric species becomes significant also in the unliganded derivative, oxygen affinity increases and cooperativity decreases. At protein concentrations where both derivatives are dimeric (e.g. 3.3 x 10(-3) m heme), both cooperativity and oxygen affinity decrease. Taken together, the experimental data indicate that in the K30D mutant, the mechanism of cooperativity is drastically altered and is driven by a ligand-linked monomer-dimer equilibrium rather than being based on a direct heme-heme communication as in native HbI.
Highlights
The homodimeric hemoglobin from Scapharca inaequivalvis (HbI) is characterized by an unusual assembly of the globin chains that entails the juxtaposition of the heme carrying E-F helices
Dimer stability is achieved through a hydrophobic patch, a network of hydrogen bonds that is primarily mediated by structural water molecules at the subunit interface and two symmetrically related salt bridges between residues Lys-30 and Asp-89, located at the extreme edges of the interface itself
Spectroscopic Characterization—The visible and circular dichroism spectra of the K30D mutant measured on the oxygenated and deoxygenated derivatives are superimposable to those reported for native HbI (Fig. 1) over a wide range of protein concentration (0.1–200 M)
Summary
The homodimeric hemoglobin from Scapharca inaequivalvis (HbI) is characterized by an unusual assembly of the globin chains that entails the juxtaposition of the heme carrying E-F helices. At low protein concentrations (e.g. 1.2 ؋ 10؊5 M heme), at which the monomeric species becomes significant in the unliganded derivative, oxygen affinity increases and cooperativity decreases.
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