Regulation of oxygen affinity by quaternary enhancement: Does hemoglobin ypsilanti represent an allosteric intermediate?

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Abstract
Recent crystallographic studies on the mutant human hemoglobin Ypsilanti (β99 Asp→xsTyr) have revealed a previously unknownquaternary structure called “quaternary Y” and suggested that the new structure may represent an important intermediate in the cooperative oxygenationpathway of normal hemoglobin.15 Here we measure the oxygenation and subunit assembly properties of hemoglobin Ypsilanti and five additionalβ99 mutants (Asp β99→Val, Gly, Asn, Ala, His) totest for consistency between their energetics and those of the intermediatespecies of normal hemoglobin. Overall regulation of oxygen affinity in hemoglobin Ypsilanti is found to originate entirely from 2.6 kcal of quaternary enhancement, such that thetetramer oxygenation affinity is 85-fold higher than for binding to the dissociated dimers. Equal partitioning of this regulatory energy among the four tetrameric binding steps (0.65 kcal per oxygen) leads toa noncooperative isotherm with extremely high affinity (pmedian = .14 torr). Temperature and pH studies of dimer-tetramer assembly and sulfhydryl reaction kinetics suggest that oxygenation-dependent structural changes in hemoglobin Ypsilanti are small. These properties are quite different from the recently characterized allosteric intermediate, which has two ligands bound on the same side of the α1β2 interface (see ref. 1 for review). The combined results do, however, support the view that quaternary Y may represent the intermediate cooperativity state of normal hemoglobin that binds the last oxygen.