Site-directed mutations of human hemoglobin at residue 35 beta: A residue at the intersection of the alpha 1 beta 1, alpha 1 beta 2, and at alpha 1 alpha 2 interfaces

Because Tyr35 beta is located at the convergence of the alpha1 beta1, alpha 1 beta2, and alpha1 alpha2 interfaces in deoxyhemoglobin, it can be argued that mutations at this position may result in large changes in the function al properties of hemoglobin. However, only small mutation-induced changes i n functional and structural properties are found for the recombinant hemogl obins beta Y35F and beta Y35A. Oxygen equilibrium-binding studies in soluti on, which measure the overall oxygen affinity (the p50) and the overall coo perativity (the Hill coefficient) of a hemoglobin solution, show that remov ing the phenolic hydroxyl group of Tyr35 beta results in small decreases in oxygen affinity and cooperativity. In contrast, removing the entire phenol ic ring results in a fourfold increase in oxygen affinity and no significan t change in cooperativity. The kinetics of carbon monoxide (CO) combination in solution and the oxygen-binding properties of these variants in deoxy c rystals, which measure the oxygen affinity and cooperativity of just the T quaternary structure, show that the ligand affinity of the T quaternary str ucture decreases in beta Y35F and increases in beta Y35A. The kinetics of C O rebinding following flash photolysis, which provides a measure of the dis sociation of the liganded hemoglobin tetramer, indicates that the stability of the liganded hemoglobin tetramer is not altered in beta Y35F or beta Y3 5A. X-ray crystal structures of deoxy beta Y35F and beta Y35A are highly is omorphous with the structure of wild-type deoxyhemoglobin. The beta Y35F mu tation repositions the carboxyl group of Asp 126 alpha1 so that it may form a more favorable interaction with the guanidinium group of Arg141 alpha2. The beta Y35A mutation results in increased mobility of the Arg141 alpha si de chain, implying that the interactions between Asp126 alpha1 and Arg141 a lpha2 are weakened. Therefore, the changes in the functional properties of these 35 beta mutants appear to correlate with subtle structural difference s at the C terminus of the a-subunit.