Kinetics of azide binding to normal and mutant ferrihemoglobins as evidence for subunit interaction

Abstract

The reaction of azide ion with ferrihemoglobin is 30-fold slower than with ferrimyoglobin. The binding of azide ion to two mutant human ferrihemoglobins was investigated to determine whether the lower reaction rate of Hb A is due to subunit interactions. The abnormal hemoglobins studied were Hb M I, in which the proximal heme-linked histidine residue of each α-chain is replaced by tyrosine, and Hb M HP, in which the same change occurs in the β-chains. In these mutant hemoglobins, only the two normal subunits bind azide ion. Hb M I, like myoglobin, reacts rapidly with azide ion, while Hb M HP reacts at the slower rate characteristic of Hb A. In contrast, the four proteins have similar binding affinities for azide ion. The large difference in reaction rate between Hb M I and Hb A shows that the binding kinetics of the β-chain depend on whether the adjacent subunit is normal or mutant. Differences in absorption spectra in the Soret region of the azide derivatives of Hb M I and Hb M HP, as well as differences in their rates of reaction with azide ion, indicate that closely related structural changes in the α and β chains can have dissimilar effects.