Cysteines β93 and β112 as Probes of Conformational and Functional Events at the Human Hemoglobin Subunit Interfaces

Three variants of tetrameric human hemoglobin, with changes at the α1β2/α2β1-interface, at the α1β1/α2β2-interface, and at both interfaces, have been constructed. At α1β2/α2β1-interface the β93 cysteine was replaced by alanine (βC93A), and at the α1β1/α2β2-interface the β112 cysteine was replaced by glycine (βC112G). The α1β2 interface variant, βC93A, and the α1β1/α1β2 double mutant, β(C93A+C112G), were crystallized in the T-state, and the structures determined at 2.0 and 1.8 Å resolution, respectively. A comparison of the structures with that of natural hemoglobin A shows the absence of detectable changes in the tertiary folding of the protein or in the T-state quaternary assembly. At the β112 site, the void left by the removal of the cysteine side chain is filled by a water molecule, and the functional characteristics of βC112G are essentially those of human hemoglobin A. At the β93 site, water molecules do not replace the cysteine side chain, and the alanine substitution increases the conformational freedom of β146His, weakening the important interaction of this residue with β94Asp. As a result, when Cl− is present in the solution, at a concentration 100 mM, the Bohr effect of the two mutants carrying the β93Cys→Ala substitution, βC93A and β(C93A+C112G), is significantly modified being practically absent below pH 7.4. Based on the crystallographic data, we attribute these effects to the competition between β94Asp and Cl− in the salt link with β146His in T-state hemoglobin. These results point to an interplay between the βHis146-βAsp94 salt bridge and the Cl− in solution regulated by the Cys present at position β93, indicating yet another role of β93 Cys in the regulation of hemoglobin function.