Crystallographic, Molecular Modeling, and Biophysical Characterization of the Valineβ67 (E11) → Threonine Variant of Hemoglobin,

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Abstract
The crystal structure of the mutant deoxyhemoglobin in which the β-globin Val67(E11) has been replaced with threonine [Fronticelli et al. (1993) Biochemistry32, 1235−1242] has been determined at 2.2 Å resolution. Prior to the crystal structure determination, molecular modeling indicated that the Thr67(E11) side chain hydroxyl group in the distal β-heme pocket forms a hydrogen bond with the backbone carbonyl of His63(E7) and is within hydrogen-bonding distance of the Nδ of His63(E7). The mutant crystal structure indicates only small changes in conformation in the vicinity of the E11 mutation confirming the molecular modeling predictions. Comparison of the structures of the mutant β-subunits and recombinant porcine myoglobin with the identical mutation [Cameron et al. (1993) Biochemistry32, 13061−13070] indicates similar conformations of residues in the distal heme pocket, but there is no water molecule associated with either of the threonines of the β-subunits. The introduction of threonine into the distal heme pocket, despite having only small perturbations in the local structure, has a marked affect on the interaction with ligands. In the oxy derivative there is a 2-fold decrease in O2 affinity [Fronticelli et al. (1993) Biochemistry32, 1235−1242], and the rate of autoxidation is increased by 2 orders of magnitude. In the CO derivative the IR spectrum shows modifications with respect to that of normal human hemoglobin, suggesting the presence of multiple CO conformers. In the nitrosyl derivative an interaction with the Oγ atom of Thr67(E11) is probably responsible for the 10-fold increase in the rate of NO release from the β-subunits. In the aquomet derivative there is a 6-fold decrease in the rate of hemin dissociation suggesting an interaction of the Fe-coordinated water with the Oγ of Thr67(E11).