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Site‐directed mutations of human hemoglobin at residue 35β: A residue at the intersection of the α1β1, α1β2, and α1α2 interfaces
Author(s) -
Kavanaugh Jeffrey S.,
Weydert Jamie A.,
Rogers Paul H.,
Ar Arthur,
Hui Hilda L.,
Wierzba Anita M.,
Kwiatkowski Laura D.,
Paily Paul,
Noble Robert W.,
Bruno Stefano,
Mozzarelli Andrea
Publication year - 2001
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.16401
Subject(s) - tetramer , cooperativity , chemistry , hemoglobin , stereochemistry , crystallography , oxygen , oxygen transport , protein quaternary structure , cooperative binding , binding site , biochemistry , organic chemistry , protein subunit , enzyme , gene
Because Tyr35β is located at the convergence of the α1β1, α1β2, and α1α2 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 βY35F and βY35A. 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 Tyr35β 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 βY35F and increases in βY35A. 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 βY35F or βY35A. X‐ray crystal structures of deoxy βY35F and βY35A are highly isomorphous with the structure of wild‐type deoxyhemoglobin. The βY35F mutation repositions the carboxyl group of Asp126α1 so that it may form a more favorable interaction with the guanidinium group of Arg141α2. The βY35A mutation results in increased mobility of the Arg141α side chain, implying that the interactions between Asp126α1 and Arg141α2 are weakened. Therefore, the changes in the functional properties of these 35β mutants appear to correlate with subtle structural differences at the C terminus of the α‐subunit.