Site-directed mutagenesis in hemoglobin: test of functional homology of the F9 amino acid residues of hemoglobin alpha and beta chains

The cysteine residue at F9(93) of the human hemoglobin (Hb A) beta chain, c onserved in mammalian and avian hemoglobins, is located near the functional ly important alpha 1-beta 2 interface and C-terminal region of the beta cha in and is reactive to sulfhydryl reagents, The functional roles of this res idue are still unclear, although regulation of local blood flow through all osteric S-nitrosylation of this residue is proposed. To clarify the role of this residue and its functional homology to F9(88) of the alpha chain, we measured oxygen equilibrium curves, UV-region derivative spectra, Soret-ban d absorption spectra, the number of titratable -SH groups with p-mercuriben zoate and the rate of reaction of these groups with 4,4'-dipyridine disulfi de for three recombinant mutant Hbs with single amino acid substitutions: A la-->Cys at 88 alpha (rHb A88 alpha C), Cys-->Ala at 93 beta (rHb C93 beta A) and Cys-->Thr at 93 beta (rHb C93 beta T). These Hbs showed increased ox ygen affinities and impaired allosteric effects, The spectral data indicate d 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 A88 alpha C compared with 2.2 for Hb A, whereas those for rub C93 beta A and rub C93 beta T 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 resid ues at 88 alpha and 93 beta have definite roles but they have no functional homology. Structure-function relationships in our mutant Hbs are discussed .