Allosteric Effectors Influence the Tetramer Stability of Both R- and T-states of Hemoglobin A (original) (raw)

2006, Journal of Biological Chemistry

The contribution of heterotropic effectors to hemoglobin allostery is still not completely understood. With the recently proposed global allostery model, this question acquires crucial significance, because it relates tertiary conformational changes to effector binding in both the R-and T-states. In this context, an important question is how far the induced conformational changes propagate from the binding site(s) of the allosteric effectors. We present a study in which we monitored the interdimeric interface when the effectors such as Cl ؊ , 2,3-diphosphoglycerate, inositol hexaphosphate, and bezafibrate were bound. We studied oxy-Hb and a hybrid form (␣FeO 2) 2-(␤Zn) 2 as the T-state analogue by monitoring heme absorption and Trp intrinsic fluorescence under hydrostatic pressure. We observed a pressure-dependent change in the intrinsic fluorescence, which we attribute to a pressure-induced tetramer to dimer transition with characteristic pressures in the 70-200-megapascal range. The transition is sensitive to the binding of allosteric effectors. We fitted the data with a simple model for the tetramer-dimer transition and determined the dissociation constants at atmospheric pressure. In the R-state, we observed a stabilizing effect by the allosteric effectors, although in the T-analogue a stronger destabilizing effect was seen. The order of efficiency was the same in both states, but with the opposite trend as inositol hexaphosphate > 2,3-diphosphoglycerate > Cl ؊. We detected intrinsic fluorescence from bound bezafibrate that introduced uncertainty in the comparison with other effectors. The results support the global allostery model by showing that conformational changes propagate from the effector binding site to the interdimeric interfaces in both quaternary states. Hemoglobin (1) is a tetrameric protein, which plays a vital role in the transport of oxygen. It consists of two dimers of ␣ and ␤ subunits that reversibly bind and release oxygen (1). The description of this cooperative phenomenon has been most frequently derived from the Monod-Wyman-Changeux (MWC) 2 two-state allosteric model (2) that attributes cooperativity to a rapid equilibrium between two conformations of distinct oxygen affinity of the whole tetramer. These distinct states are the fully unliganded T-state and the fully ligated R-state. Szabo and Karplus (3) modified the two-state model incorporating the stereochemical mechanism suggested by Perutz (4) for the T to R switch, and introduced ligation-induced tertiary changes within the T-state. In this extended model (MWC-SK), it was proposed that cooperativity still works through a ligation-induced shift in the equilibrium of states T and R, but the model attributed importance in the conformational switch to certain changes at the inter-and intrasubunit interfaces. Upon ligation in the T-state, the network of intersubunit interactions become perturbed, some (e.g. salt bridges) become broken up to release the characteristic strain of the T-state. The mechanism involves a rotation of one dimer with respect to the other, thus reaching the more relaxed R-state (5). It has been widely reported that some molecules, referred to as heterotropic allosteric effectors, considerably lower the oxygen affinity of the T-state upon binding to HbA but not to the heme (6-9). Structural studies in the T-state showed that these allosteric effectors primarily bind to the central cavity of HbA (10-12). The modulation of the oxygen dissociation curves by allosteric effectors is addressed in the extended MWC model by the assumption that allosteric effectors bind specifically to the somewhat larger central cavity of the T-state and stabilize this conformation. This shifts the R/T equilibrium in favor of the T-state and consequently lowers the overall affinity to oxygen (8, 13). The well known Bohr effect and results reported for Cl Ϫ , also influencing the oxygen affinity of the T-state (14, 15), show that, in a broader sense, H ϩ and Cl Ϫ can also be considered as being members of the family of allosteric effectors. Extended studies on the effect of allosteric effectors, however, indicated that they not only bind to the T-state but also to the R-state (16, 17). The modulation of the oxygen association constants was shown to occur at a much broader scale (65-fold change in K T and 2000-fold change in K R ; see Ref. 18 for details) * This work was supported by a collaborative grant from the Fogarty International Center, Award TW005924 (to T. Y. and J. F.), National Science Foundation of Hungary Grants OTKA T049213 (to L. S.