Entropy-Driven Intermediate Steps of Oxygenation May Regulate the Allosteric Behavior of Hemoglobin (original) (raw)
Related papers
Biochemistry, 1993
We have reported [Bucci, E., Fronticelli, C., t Grycznski, Z. (1991) Biochemistry 30,3195-31991 that in human and bovine hemoglobins the release of heat at the subsequent steps of oxygenation is not constant. This is especially evident in the binding of the third 0 2 molecule, which is an endothermic event. This phenomenon was attributed to peculiar conformations of the intermediates of oxygenation, not included in the fundamental R / T transition of the system. To test this hypothesis, we have explored the effect of conformational constraints on the thermodynamics of the intermediates of oxygenation. The assumption was that intramolecular constraints would stabilize the intermediates into conformations similar to the R and T forms reducing the variability of their enthalpies. We have analyzed the temperature dependence of the oxygen binding isotherms of human hemoglobin cross-linked either between the ,882 or between the a99 lysines by bis(3,5-dibromosalicyl)fumarate. The measurements were performed at pH 9.0 in 0.1 M borate buffer in order to avoid thermal effects due to oxygen-linked binding of anions and protons. The data were analyzed singularly by local procedures and simultaneously using global procedures. The two cross-links had opposite effects. The cross-link between the @-subunits decreased while that between the a-subunits increased the endothermic behavior of the third step of oxygenation. Also, the cross-link between the &subunits increased the fractional amount of the triligated species at intermediate stages of oxygenation, while that between the a-subunits decreased this quantity to hardly detectable values. These data are consistent with the hypothesis that the R / T transition in hemoglobin involves novel conformations not included in the R / T system. It is also speculated that the novel conformations of the intermediates of oxygenation is similar to the Y structure of hemoglobin Ypsilanti. The cooperativity of ligand binding to hemoglobin tetramers is produced by a scarcity of the intermediate species of ligation between 0 and 4 ligands per molecule, which defies the statistics This work was supported, in part, by Grants PHS NIH HLBI-33629 (E.B. and C.F.) and HLBI-13164 (E.B. and C.F.1, by funds from the Medical Biotechnology Center (J.H.C.), and by the Polish Scientific Council KBN (A.R.). Computer time and facilities were supported in part by the computer networkof the University of Maryland at Baltimore, MD, and at College Park, MD. Z.G. is the recipient of a fellowship of the American Heart Association, Maryland affiliate.
Enthalpy Change of Allosteric Transition in Human Haemoglobin A
Journal of The Chinese Chemical Society, 2004
Oxygen equilibria of haemoglobin were analysed according to a binding isotherm proposed by Amire (Bull. Chem. Soc. Jpn. 1994, 67, 7) 1 to obtain the intrinsic oxygen association constants to the molecule. Two sets of binding sites in haemoglobin were identified, which were ascribed to R2 and T forms of the molecule. The average intrinsic association constants determined as a function of temperature gave a heat of oxygenation of-76 ± 4 kJ mol-1 (tetramer). A microcalorimetrically determined heat of deoxygenation of oxyhaemoglobin by dithionite gave-267 ± 10 kJ mol-1 (tetramer). From these results, the heat of allostery of-234 ± 24 kJ mol-1 for haemoglobin tetramer was obtained, yielding allosteric energy per salt bridge of-29 ± 3 kJ. This result suggests that salt-bridge may, in fact, be thermochemically equivalent to hydrogen bonds.
Cooperative ligand binding of crosslinked hemoglobins at very high temperatures
Journal of Molecular Biology, 1990
Human hemoglobin was reacted with the bifunctional reagent bis(3,5-dibromosalicyl) fumarate to yield a derivative (Hb ~) crosslinked between the two a-chains; when the reaction was carried out with HbA already crosslinked between the twofl-chains by 2-nor-2formylpyridoxal 5'-phosphate, a doubly crosslinked derivative (Hb a~) was obtained. We have observed that both modified hemoglobins are extremely stable up to temperatures of at least 85°C.
Allosteric Effectors Influence the Tetramer Stability of Both R- and T-states of Hemoglobin A
Journal of Biological Chemistry, 2006
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.
Biochemistry, 2004
A novel model linking the thermodynamics and kinetics of hemoglobin's allosteric (R f T) and ligand binding reactions is applied to photolysis data for human HbCO. To describe hemoglobin's kinetics at the microscopic level of structural transitions and ligand-binding events for individual [ij]ligation microstates ( ij R f ij T, ij R + CO f (i+1)k R, and ij T + CO f (i+1)k T), the model calculates activation energies, ij ∆G ‡ , from previously measured cooperative free energies of the equilibrium microstates (Huang, Y., and Ackers, G. K. (1996) Biochemistry 35, 704-718) by using linear free energy relations
Biophysical Journal, 2004
Mouse and human neuroglobins, as well as the hemoglobins from Drosophila melanogaster and Arabidopsis thaliana, were recombinantly expressed in Escherichia coli, and their ligand-binding properties were studied versus temperature. These globins have a common feature of being hexacoordinated (via the distal histidine) under deoxy conditions, as evidenced by a large amplitude for the alpha absorption band at 560 nm and the Soret band at 426 nm. The transition from the hexacoordinated form to the CO bound species is slow, as expected for a replacement reaction Fe-His / Fe / FeCO. The intrinsic binding rates would indicate a high oxygen affinity for the pentacoordinated form, due to rapid association and slow (100 ms-1 s) dissociation. However, the competing protein ligand results in a much lower affinity, on the order of magnitude of 1 torr. In addition to decreasing the affinity for external ligand, the competitive internal ligand leads to a weaker observed temperature dependence of the ligand affinity, since the difference in equilibrium energy for the two ligands is much lower than that of ligand binding to pentacoordinated hemoglobin. This effect could be of biological relevance for certain organisms, since it could provide a globin with an oxygen affinity that is nearly independent of temperature.
Conformational kinetics of triligated hemoglobin
Biophysical Journal, 1985
ABSTRACr We have used the method of modulated excitation (Ferrone, F. A., and J. J. Hopfield, 1976, Proc. Natl. Acad. Sci. USA. 73:4497-4501), with an improved apparatus and a revised analytical procedure, to measure the rate of conformational change between the oxy (R) and deoxy (T) conformations of triligated carboxy-hemoglobin A at pH 6.5 and 7.0. We have found the rates to be kRT = 1.2 x I03 s'-and kTR = 3.5 x 103 s' for pH 6.5, while for pH 7.0, kRT = 1.0 X 103 S-l, and kTR 3.0 x 103 s-'. The value for L3, the equilibrium constant between conformations, was virtually unchanged between pH 6.5 and 7.0. While the rates measured here differ from those obtained in the original use of this method, these new rates are fully consistent with the original data when analyzed by the revised procedures presented here. When taken with other kinetic and equilibrium data, our measurements suggest that the transition state between structures is dominated by the behavior of the T quaternary structure. Finally, a spectral feature near the HbCO Soret peak has been observed that we ascribe to an allosteric perturbation of the spectra of the liganded hemes.
Molecular basis of thermal stability in truncated (2/2) hemoglobins
Biochimica et Biophysica Acta (BBA) - General Subjects, 2014
Background: Understanding the molecular mechanism through which proteins are functional at extreme high and low temperatures is one of the key issues in structural biology. To investigate this phenomenon, we have focused on two instructive truncated hemoglobins from Thermobifida fusca (Tf-trHbO) and Mycobacterium tuberculosis (Mt-trHbO); although the two proteins are structurally nearly identical, only the former is stable at high temperatures. Methods: We used molecular dynamics simulations at different temperatures as well as thermal melting profile measurements of both wild type proteins and two mutants designed to interchange the amino acid residue, either Pro or Gly, at E3 position. Results: The results show that the presence of a Pro at the E3 position is able to increase (by 8°) or decrease (by 4°) the melting temperature of Mt-trHbO and Tf-trHbO, respectively. We observed that the ProE3 alters the structure of the CD loop, making it more flexible.
An allosteric model of hemoglobin
Archives of Biochemistry and Biophysics, 1972
One assumption of the Monod, Wyman, and Changeux (MWC) allosteric model of hemoglobin is that the ligand binding energies depend only upon the conformation R or T, and are independent of the degree of ligation. Canonical values of the equilibrium and kinetic constant,s for the R and T forms are obtained from the measurements, mainly by Gibson and Roughton, of the last and first ligands binding to hemoglobin. These values, differing by a factor of ~240 in equilibrium constants, are compared wit.h the values measured for mutant and chemically modified hemoglobins which are frozen in one quaternary structure or another. From these comparisons we conclude that nearly all of the affinity differences between the R and T forms are contributed by t,he quaternary structures and that the parameters for 02 binding are to a first order independent of the degree of ligation. Kinetic and equilibrium constants of t,he mutants, Hemoglobin J Capetown and Chesapeake which appear to be intermediate between the canonical values for the R and T forms are shown to be consistent with those values by postulating an earlier switch from T to R than is observed in HbA, as was originally discussed by Edelstein.