Site-directed mutations of human hemoglobin at residue 35β: A residue at the intersection of the α1β1, α1β2, and α1α2 interfaces (original) (raw)

Because Tyr35␤ is located at the convergence of the ␣1␤1, ␣1␤2, and ␣1␣2 interfaces in deoxyhemoglobin, it can be argued that mutations at this position may result in large changes in the functional properties of hemoglobin. However, only small mutation-induced changes in functional and structural properties are found for the recombinant hemoglobins ␤Y35F and ␤Y35A. Oxygen equilibrium-binding studies in solution, which measure the overall oxygen affinity (the p50) and the overall cooperativity (the Hill coefficient) of a hemoglobin solution, show that removing the phenolic hydroxyl group of Tyr35␤ results in small decreases in oxygen affinity and cooperativity. In contrast, removing the entire phenolic ring results in a fourfold increase in oxygen affinity and no significant change in cooperativity. The kinetics of carbon monoxide (CO) combination in solution and the oxygen-binding properties of these variants in deoxy crystals, which measure the oxygen affinity and cooperativity of just the T quaternary structure, show that the ligand affinity of the T quaternary structure decreases in ␤Y35F and increases in ␤Y35A. The kinetics of CO rebinding following flash photolysis, which provides a measure of the dissociation of the liganded hemoglobin tetramer, indicates that the stability of the liganded hemoglobin tetramer is not altered in ␤Y35F or ␤Y35A. X-ray crystal structures of deoxy ␤Y35F and ␤Y35A are highly isomorphous with the structure of wild-type deoxyhemoglobin. The ␤Y35F mutation repositions the carboxyl group of Asp126␣1 so that it may form a more favorable interaction with the guanidinium group of Arg141␣2. The ␤Y35A mutation results in increased mobility of the Arg141␣ side chain, implying that the interactions between Asp126␣1 and Arg141␣2 are weakened. Therefore, the changes in the functional properties of these 35␤ mutants appear to correlate with subtle structural differences at the C terminus of the ␣-subunit. . Abbreviations: Bis-tris, bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane; Tris, tris(hydroxymethyl)aminomethane; EDTA, disodium ethylenediaminetetraacetate; PEG, polyethylene glycol; r.m.s., root mean square; IHP, inositol hexaphosphate; HbA, human hemoglobin major component; ␤V1M, recombinant hemoglobin with the Val 1␤ → Met mutation; ␤Y35F, recombinant hemoglobin with the Tyr 35␤ → Phe and Val 1␤ → Met mutations; ␤Y35A, recombinant hemoglobin with the Tyr 35␤ → Ala and Val 1␤ → Met mutations; met-hemoglobin, hemoglobin with iron oxidized to Fe(III). Article and publication are at