High and low oxygen affinity conformations of T state hemoglobin

Abstract

To understand the interplay between tertiary and quaternary transitions associated with hemoglobin function and regulation, oxygen binding curves were obtained for hemoglobin A fixed in the T quaternary state by encapsulation in wet porous silica gels. At pH 7.0 and 15°C, the oxygen pressure at half saturation (p50) was measured to be 12.4 ± 0.2 and 139 ± 4 torr for hemoglobin gels prepared in the absence and presence of the strong allosteric effectors inositol hexaphosphate and bezafibrate, respectively. Both values are in excellent agreement with those found for the binding of the first oxygen to hemoglobin in solution under similar experimental conditions. The corresponding Hill coefficients of hemoglobin gels were 0.94 ± 0.02 and 0.93 ± 0.03, indicating, in the frame of the Monod, Wyman, and Changeux model, that high and low oxygen-affinity tertiary T-state conformations have been isolated in a pure form. The values, slightly lower than unity, reflect the different oxygen affinity of ␣-and ␤-hemes. Significantly, hemoglobin encapsulated in the presence of the weak effector phosphate led to gels that show intermediate oxygen affinity and Hill coefficients of 0.7 to 0.8. The heterogeneous oxygen binding results from the presence of a mixture of the high and low oxygen-affinity T states. The Bohr effect was measured for hemoglobin gels containing the pure conformations and found to be more pronounced for the high-affinity T state and almost absent for the low-affinity T state. These findings indicate that the functional properties of the T quaternary state result from the contribution of two distinct, interconverting conformations, characterized by a 10-fold difference in oxygen affinity and a different extent of tertiary Bohr effect. The very small degree of T-state cooperativity observed in solution and in the crystalline state might arise from a ligand-induced perturbation of the distribution between the high-and low-affinity T-state conformations.Hemoglobin is an ␣ 2 ␤ 2 -tetramer that binds ligands cooperatively via the modulation of equilibria between tertiary and quaternary conformations. X-ray crystallographic studies have shown that hemoglobin exists in two quaternary states, T and R, characterized by the presence and absence of salt bridges, respectively, and by a different pattern of ␣ 1 ␤ 2 -and ␣ 2 ␤ 1 -interactions (Perutz 1970;Baldwin and Chothia 1979). Binding of ligands triggers a series of tertiary conformational changes that break salt bridges, destabilizing the T state and leading to the liganded R state (Perutz 1970;Antonini and Brunori 1971;Perutz et al. 1998). Oxygen binding curves have been extremely useful in evaluating the effect of allosteric effectors on the functional properties of quaternary states (Imai 1982). In particular, the oxygen affinity and the degree of cooperativity of T-state hemoglobin have been investigated on mixed metal hybrids, for which iron at either the ␣-or ␤-heme was substituted with metals that do not bind oxygen (Shibayama

Nitrite Reductase Activity of Sol-Gel-encapsulated Deoxyhemoglobin

“…These decreases include the reactivity of the ␤93 cysteines (50), the on-rates for CO binding (51,52), T-state oxygen affinity (17,31,43,45,(51)(52)(53)(54)56), and the rate of T-state tertiary relaxation upon ligand binding (26). These results have been interpreted in terms of effector-induced damping of the amplitudes of those conformational dynamics that (i) transiently expose the sulfhydryl groups of Cys-␤93, (ii) transiently enhance ligand access to the heme iron (57), and (iii) facilitate ligand binding induced conformational relaxation (21).…”

“…A summary of the MEM analyses of the solvent phase from several kinetic traces is given in Table 2. The MEM peaks associated with the solvent phase are assigned as high affinity T (HT), low affinity T (LT), and R based both on the lifetime value of the midpoints of each peak, as previously published (21), and on the previous accounts of high and low affinity forms of the T-state (26,43,45). The LT populations are further subdivided, albeit somewhat arbitrarily, based on the appearance and behavior of the different distinct LT peaks.…”

Immobilization of Proteins in Silica Gel: Biochemical and Biophysical Properties

“…However, the most striking result about Hb encapsulated in silica gels is the perfect conservation of equilibrium oxygen binding properties observed in solution under comparable conditions [54,57,58,82,100,101] (Fig. 3) [218,219], which legitimates the exploitation of this system in kinetic experiments aiming to test theoretical models of Hb allosteric regulation (see Section 4).…”

“…This transition can be significantly slowed down by encapsulating Hb in silica gels [82]. The low affinity T-state and the high affinity Rstate of Hb can be trapped by encapsulating Hb either as fully deoxygenated or fully oxygenated molecules, respectively [57,82,83,100]. Indeed, non-cooperative oxygen binding is observed both when Hb is encapsulated in the high-affinity R state or in the lowaffinity T state [57,58,82,83,85,100] (Fig.…”

Quadrupole splitting temperature dependence of high and low affinity deoxyhemoglobin encapsulated in wet silica gel

“…This clearly shows that the cooperative behaviour is coming from the change of the quaternary structure. In addition, two distinct affinity conformations [3] in T state have been found: a low affinity (LA) and a high affinity (HA) [4]. Separation of tertiary from quaternary conformational changes seems the way to understand this allosteric behaviour.…”