Cysteines beta 93 and beta 112 as probes of conformational and functional events at the human hemoglobin subunit interfaces

Three variants of tetrameric human hemoglobin, with changes at the alpha(1) beta(2)/alpha(2)beta(1)-interface, at the alpha(1)beta(1)/alpha(2)beta(2)-i nterface, and at both interfaces, have been constructed. At alpha(1)beta(2) /alpha(2)beta(1)-interface the beta 93 cysteine was replaced by alanine (be ta C93A), and at the alpha(1)beta(1)/alpha(2)beta(2)-interface the beta 112 cysteine was replaced by glycine (beta C112G). The alpha(1)beta(2) interfa ce variant, beta C93A, and the alpha(1)beta(1)/alpha(1)beta(2) double mutan t, beta(C93A+C112G), were crystallized in the T-state, and the structures d etermined at 2.0 and 1.8 Angstrom resolution, respectively. A comparison of the structures with that of natural hemoglobin A shows the absence of dete ctable changes in the tertiary folding of the protein or in the T-state qua ternary assembly. At the beta 112 site, the void left by the removal of the cysteine side chain is filled by a water molecule, and the functional char acteristics of beta C112G are essentially those of human hemoglobin A. At t he beta 93 site, water molecules do not replace the cysteine side chain, an d the alanine substitution increases the conformational freedom of beta 146 His, weakening the important interaction of this residue with beta 94Asp. A s a result, when Cl- is present in the solution, at a concentration 100 mM, the Bohr effect of the two mutants carrying the beta 93Cys-->Ala substitut ion, beta C93A and beta(C93A+C112G), is significantly modified being practi cally absent below pH 7.4. Based on the crystallographic data, we attribute these effects to the competition between beta 94Asp and Cl- in the salt li nk with beta 146His in T-state hemoglobin. These results point to an interp lay between the beta His146-beta Asp94 salt bridge and the Cl- in solution regulated by the Cys present at position beta 93, indicating yet another ro le of beta 93 Cys in the regulation of hemoglobin function.