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

Abstract

Recent crystallographic studies on the mutant human hemoglobin Ypsilanti (beta 99 Asp-->Tyr) have revealed a previously unknown quaternary structure called "quaternary Y" and suggested that the new structure may represent an important intermediate in the cooperative oxygenation pathway of normal hemoglobin. Here we measure the oxygenation and subunit assembly properties of hemoglobin Ypsilanti and five additional beta 99 mutants (Asp beta 99-->Val, Gly, Asn, Ala, His) to test for consistency between their energetics and those of the intermediate species 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 the tetramer 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 to a 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 alpha 1 beta 2 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.

The X‐ray structure determination of bovine carbonmonoxy hemoglobin at 2.1 Å resoultion and its relationship to the quaternary structures of other hemoglobin crystal forms

“…In contrast, the heme, Fhelix, and the FG corner in the Y state are shifted further from the T state position (compared with the transition from the R state to the T state). This indicates that the Y state is even more relaxed than the R state, which is in accord with Ypsilanti Hb having an increased oxygen affinity Doyle et al 1992). Similar results are obtained when comparing the R2 state CO human Hb structure (instead of Y state) with the CO bovine, oxy human (R state), and dxy human (T state) Hb structures.…”

The oxygen-binding intermediates of human hemoglobin: evaluation of their contributions to cooperativity using zinc-containing hybrids

“…Equilibrium constants for dimer-tetramer assembly were evaluated as ratios of the respective association (kf) and dissociation (kr) rate constants. Association rates have previously been determined for normal hemoglobin over a wide range of conditions and for a large number of structurally altered hemoglobins, including mutant, chemically modified, and cobalt-substituted species (Pettigrew et al, 1982;Turner et al, 1992;Doyle et al, 1991). A value of 1.1(± 0.3) X 106 Ms-1 was found in all cases under solution conditions identical to the present study.…”

Properties of a recombinant human hemoglobin with aspartic acid 99 (β), an important intersubunit contact site, substituted by lysine

“…For example, whereas the dimer-tetramer dissociation constant for normal human HbA is about lo-' M in its oxygenated state and M in its deoxygenated form (Turner et al, 1981), the corresponding values for the natural mutants at Asp%(@) are increased by 3-5 orders of magnitude (Turner et al, 1981Doyle et al, 1992). We have measured the extent of dimer formation of the oxygenated D99K mutant Hb by several procedures including gel filtration, haptoglobin binding of ~$3 dimers determined by filtration of the haptoglobin-Hb dimer complex and by quenching of fluorescence, and by light scattering, as described below.…”