title: The mutation K30D disrupts the only salt bridge at the subunit interface of the homodimeric hemoglobin from Scapharca inaequivalvis and changes the mechanism of cooperativity.
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.
authors.affiliation: Consiglio Nazionale delle Ricerche Center of Molecular Biology, Department of Biochemical Sciences, University La Sapienza, 00185 Rome, Italy.
authors.forename: Pierpaolo
authors.initials: P
authors.lastname: Ceci
authors.forename: Laura
authors.initials: L
authors.lastname: Giangiacomo
authors.forename: Alberto
authors.initials: A
authors.lastname: Boffi
authors.forename: Emilia
authors.initials: E
authors.lastname: Chiancone
mesh_terms: D000818:Animals; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D006418:Heme / Q000737:chemistry; D006454:Hemoglobins / Q000737:chemistry* / Q000235:genetics*; D006859:Hydrogen; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008024:Ligands; D008239:Lysine / Q000737:chemistry; D008974:Mollusca; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010100:Oxygen / Q000378:metabolism; D011485:Protein Binding; D012492:Salts / Q000737:chemistry / Q000378:metabolism*; D014461:Ultracentrifugation
chemical_list: D006454:Hemoglobins; D008024:Ligands; D012492:Salts; C083082:dimeric hemoglobin, Scapharca inaequivalvis; D006418:Heme; D006859:Hydrogen; D008239:Lysine; D010100:Oxygen