Site-directed mutagenesis in hemoglobin: Test of functional homology of the F9 amino acid residues of hemoglobin α and β chains

Abstract

The cysteine residue at F9(93) of the human hemoglobin (Hb A) β chain, conserved in mammalian and avian hemoglobins, is located near the functionally important α1-β2 interface and C-terminal region of the β chain and is reactive to sulfhydryl reagents. The functional roles of this residue are still unclear, although regulation of local blood flow through allosteric S-nitrosylation of this residue is proposed. To clarify the role of this residue and its functional homology to F9(88) of the a chain, we measured oxygen equilibrium curves, UV-region derivative spectra, Soret-band absorption spectra, the number of titratable -SH groups with p-mercuribenzoate and the rate of reaction of these groups with 4,4'-dipyridine disulfide for three recombinant mutant Hbs with single amino acid substitutions: Ala→Cys at 88α (rHb A88αC), Cys→Ala at 93β (rHb C93βA) and Cys→Thr at 93β (rHb C93βT). These Hbs showed increased oxygen affinities and impaired allosteric effects. The spectral data indicated that the R to T transition upon deoxygenation was partially restricted in these Hbs. The number of titratable -SH groups of liganded form was 3.2-3.5 for rHb A88αC compared with 2.2 for Hb A, whereas those for rHb C93βA and rub C93βT were negligibly small. The reduction of rate of reaction with 4,4'-dipyridine disulfide upon deoxygenation in rub A88aC was smaller than that in Hb A. Our experimental data have shown that the residues at 88α and 93β have definite roles but they have no functional homology. Structure-function relationships in our mutant Hbs are discussed.