T State Hemoglobin Binds Oxygen Noncooperatively with Allosteric Effects of Protons, Inositol Hexaphosphate, and Chloride

Stefano Bettati,Andrea Mozzarelli

doi:10.1074/jbc.272.51.32050

Abstract

Hemoglobin is the paradigm of allosteric proteins. Over the years, cooperative oxygen binding has been explained by different models predicting that the T state of hemoglobin binds oxygen either noncooperatively or with some degree of cooperativity or with strong cooperativity. Therefore, a critical test that discriminates among models is to determine the oxygen binding by the T state of hemoglobin. Fixation of hemoglobin in the T state has been achieved either by crystallization from polyethylene glycol solutions or by encapsulation in wet porous silica gels. Hemoglobin crystals bind oxygen noncooperatively with reduced affinity compared with solution, with no Bohr effect and with no influence of other allosteric effectors. In this study, we have determined accurate oxygen-binding curves to the T state of hemoglobin in silica gels with the same microspectrophotometric apparatus and multiwavelengths analysis used in crystal experiments. The T state of hemoglobin in silica gels binds oxygen noncooperatively with an affinity and a Bohr effect similar to those observed in solution for the binding of the first oxygen molecule. Other allosteric effectors such as inositol hexaphosphate, bezafibrate, and chloride significantly affect oxygen affinity. Therefore, T state hemoglobins that are characterized by strikingly different functional properties share the absence of cooperativity in the binding of oxygen. These findings are fully consistent with the Monod, Wyman, and Changeux model and with most features of Perutz's stereochemical model, but they are not consistent with models of both Koshland and Ackers.

Highlights

Hemoglobin binds oxygen cooperatively with an affinity that is regulated by protons, organic phosphates, chloride, and carbon dioxide [1, 2]
To obtain the fractional saturation of the T state hemoglobin at a defined oxygen pressure without the small contribution arising from the T to R transition, the slow phase was back extrapolated to time 0 assuming a linear dependence (Fig. 1e)
When the data were fitted, assuming that (i) the ␣-subunits with broken salt bridges exhibit a p50 of 4.1 torr, the value determined for ␣(Fe2ϩ)2␤(Ni2ϩ)2 hybrid at pH 8.5 and corrected for temperature difference [51]; (ii) the ␣-subunits with intact salt bridges exhibit a p50 of 94 torr, the value determined in hemoglobin crystals [22]; and (iii) the ␣-␤ inequivalence is 2–5-fold, the fraction of sites with broken salt bridges was found to decrease from about 50% to 25% in the absence and presence of allosteric effectors, respectively. These results indicate that the T state of hemoglobin in silica gels binds oxygen without any degree of cooperativity, and in conjunction with the results obtained on hemoglobin crystals, limit the validity of most of the models so far proposed to explain the hemoglobin function

Summary

Introduction

Hemoglobin binds oxygen cooperatively with an affinity that is regulated by protons, organic phosphates, chloride, and carbon dioxide [1, 2] These properties are associated to the peculiar structure of the T quaternary state since the R quaternary state exhibits functional properties similar to those of isolated ␣-␤ dimers and isolated chains. On the basis of their findings, a symmetry rule that controls the relationship between the binding affinity and the T to R quaternary transition was proposed According to this model, the ligand binding within a dimeric half of a T state tetramer is strongly cooperative. Measurements were carried out with the same microspectrophotometric apparatus previously used in the determination of the oxygen binding by hemoglobin crystals (20 –22, 39 – 44)

Methods

Results

Conclusion