Distinct Domain Responses of R-state Human Hemoglobins A, C, and S to Anions

Anionic regulation of hemoglobin (Hb) is of increasing interest for the design of Hb-based oxygen carriers. Even “external” amino-acid substitutions can alter the nature and extent of anionic control. This was shown by evaluation of the anion sensitivities of liganded, R-state, forms of HbA, HbC (β6 Glu → Lys) and HbS (β6 Glu → Val). The β6 mutants differ in the anion-sensitivity of their central cavities, α1β2 interfaces, and heme and β93 Cys environments. The mutant Hbs also exhibit increased anion-dependent oxidation and surface denaturation. Moreover, differential chloride effects on oxygen binding by Hbs C, S compared to HbA occur after R-state stabilization by fluoresceination of β93 Cys. It is concluded that the “external” substitutions in the mutant Hbs have structural consequences that are propagated to varying extents to other domains as a result of anion binding, and that these anion-dependent changes may underlie mechanisms leading to the observed increase in oxidation propensity and surface denaturation.