Preparation and Kinetic Characterization of a Series of βW37 Variants of Human Hemoglobin A:  Evidence for High-Affinity T Quaternary Structures

Abstract

Four variants of human beta globin in which the Trp at position 37 has been replaced with a Tyr, Ala, Gly, or Glu have been expressed in Escherichia coli. These globins have been combined with normal human alpha chains and heme to form tetrameric hemoglobin molecules. A technique for the preparation of alpha chain dimers, which are cross-linked between their alpha99 lysine residues, has been developed, and these alpha dimers were combined with two of the beta globins, betaW37G and betaW37E, to form the corresponding cross-linked variants. The kinetics of CO binding to the deoxygenated derivatives following rapid mixing and of CO rebinding following flash photolysis have been examined as functions of pH in the presence and absence of the organic phosphate inositol hexaphosphate, IHP. The kinetic measurements indicate that replacement of the tryptophan with other residues destabilizes the hemoglobin tetramer, resulting in considerable dissociation of even the deoxygenated hemoglobins into alphabeta dimers at micromolar protein concentrations. Substitutions at beta37 also alter the properties of the deoxygenated hemoglobin tetramer. The alteration of the functional properties of the T states of these variants as well as the tendency of the deoxygenated derivatives to dissociate into alphabeta dimers increases in the order HbA < betaW37Y < betaW37A < betaW37G < betaW37E. Stabilizing the betaW37G or betaW37E tetramers by addition of IHP or by cross-linking does not restore the normal functional properties of the T state. Measurements of the geminate rebinding of CO establish a kinetic difference between the normal R state tetramer and the alphabeta dimer consistent with quaternary enhancement, the greater affinity of oxygen for the R state tetramer than for the alphabeta dimer. Kinetics of geminate rebinding also suggest that quaternary enhancement may be altered by substitutions at the beta37 position.

Conformational and functional significance of the α140 side‐chain in HbA: a UV and visible resonance Raman study of three α140 mutants

“…thermal factors for 140Y and˛141R) in the hinge region as observed in the series of deoxy T state crystal structures. 8,32 The combination rates for the three˛140 mutants in the present study all show large increases in the CO combination rates, comparable to the increases observed for theˇ37 mutants showing the largest changes [in both (Fe-His) and CO combination rate]. 9,11 The results are completely consistent with the conclusion that these mutations have reduced proximal strain, resulting in enhanced CO binding for the deoxy T state.…”

“…Human hemoglobin was purified as described previously. 19 Mutant hemoglobins were prepared in R. Noble's laboratory as described previously 8,9 as part of a multi-institutional NIH-funded program project and provided as needed. Samples used for spectroscopic measurements were prepared in 50 mM bis-tris acetate, pH 6.5, at concentrations of 0.5 mM in heme.…”

“…Dimers show a reduced geminate yield. 8 Whereas the photoproduct wavenumbers of (Fe-His) for the˛140 mutants in the absence of effectors are very similar to the wavenumber from HbA at 230 cm 1 , the resulting wavenumbers in the presence of allosteric effectors are dramatically different. In all three cases, the decrease in wavenumber is substantially larger than for effector-induced decrease in HbA.…”

“…6,9 The hydrogen bonding network that couples these residues keeps the indole of Wˇ237 rigidly locked in its deoxy T state position. It has been shown that perturbations that loosen the packing of theˇ37 sidechain result in an increase in the wavenumber of (Fe-His) 10 and an increase in CO combination rates 5,8,11 and in oxygen affinity (overall and within the T state). 7 These effects were observed for a series of deoxyˇ37 Hb mutants in which the x-ray crystallographic data showed a progressive loosening of the hinge region within the T quaternary state.…”

Altered Ligand Rebinding Kinetics Due to Distal-side Effects in Hemoglobin Chico (Lysβ66(E10) → Thr)

“…The limitations of this simple view were indicated by studies showing that the last available subunit in R-state ␣ 2 ␤ 2 -tetramers binds ligands with higher affinity than ␣␤-dimers (51). This phenomenon, termed quaternary enhancement, was shown in recent studies to be evident in an increased geminate yield for fully liganded ␣ 2 ␤ 2 -tetramers relative to the corresponding dimers (52). Based on geminate rebinding data and Raman spectra for the photoproducts of tetrameric and dimeric forms of Hb A 0 , it was claimed that the quaternary enhancement effect arises from a proximal-heme environment in the R-state photodissociated ␣ 2 ␤ 2 -tetramer that favors rebinding relative to that for ␣␤-dimers (29).…”