Jo M. Holt

Confirmation of a unique intra-dimer cooperativity in the human hemoglobin ?1?1half-oxygenated intermediate supports the symmetry rule model of allosteric regulation

The contribution of the α1β1half‐oxygenated tetramer [αβ:αO2βO2] (species 21) to human hemoglobin cooperativity was evaluated using cryogenic isoelectric focusing. The cooperative free energy of binding, reflecting O2‐driven protein structure changes, was measured as 21ΔGc = 5.1 ± 0.3 kcal for the Zn/FeO2 analog. For the Fe/FeCN analog, 21ΔGc was estimated as 4.0 kcal after correction for a CN ligand rearrangement artifact, demonstrating that ligand rearrangement does not invalidate previous conclusions regarding this species. In the context of the entire Hb cooperativity cascade, which includes eight intermediate species, the 21 tetramer is highly abundant relative to the other doubly‐ligated species, providing strong support for the previously determined consensus partition function of O2 binding and for the Symmetry Rule model of hemoglobin cooperativity (Ackers et al., Science 1992;255:54–63). Cooperativity of normal human hemoglobin is shown to depend on site‐configuration, and not solely the number of O2 bound, nor the occupancy of α vs. β subunits. Verification of a unique contribution from the α1β1doubly‐oxygenated species to the equilibrium O2 binding curve strongly reinforces the Symmetry Rule interpretation that the α1β1dimer acts both as a structural and functional element in cooperative O2 binding. Proteins 2000;41:23–43.

Single residue modification of only one dimer within the hemoglobin tetramer reveals autonomous dimer function

The mechanism of cooperativity in the human hemoglobin tetramer (a dimer of αβ dimers) has historically been modeled as a simple two-state system in which a low-affinity structural form (T) switches, on ligation, to a high-affinity form (R), yielding a net loss of hydrogen bonds and salt bridges in the dimer–dimer interface. Modifications that weaken these cross-dimer contacts destabilize the quaternary T tetramer, leading to decreased cooperativity and enhanced ligand affinity, as demonstrated in many studies on symmetric double modifications, i.e., a residue site modified in both α- or both β-subunits. In this work, hybrid tetramers have been prepared with only one modified residue, yielding molecules composed of a wild-type dimer and a modified dimer. It is observed that the cooperative free energy of ligation to the modified dimer is perturbed to the same extent whether in the hybrid tetramer or in the doubly modified tetramer. The cooperative free energy of ligation to the wild-type dimer is unperturbed, even in the hybrid tetramer, and despite the overall destabilization of the T tetramer by the modification. This asymmetric response by the two dimers within the same tetramer shows that loss of dimer–dimer contacts is not communicated across the dimer–dimer interface, but is transmitted through the dimer that bears the modified residue. These observations are interpreted in terms of a previously proposed dimer-based model of cooperativity with an additional quaternary (T/R) component.

Effects of NaCl on the linkages between O2 binding and subunit assembly in human hemoglobin: titration of the quaternary enhancement effect

Oxygen binding by human hemoglobin (Hb) and the coupled reactions of dimer-tetramer assembly were studied over a range of NaCl concentrations (from 0.08 M to 1.4 M) at pH 7.4 and 21.5 degrees C. A strategy of multi-dimensional analysis was employed [G.K. Ackers and H.R. Halvorson, Proc. Natl. Acad. Sci. U.S.A., 91, (1974) 4312] to optimize the resolution of the contributions to cooperativity and their heterotropic salt linkages at each stoichiometric degree of O2 binding. A wide range of Hb concentration was utilized at each [NaCl] in which O2-linked subunit assembly reactions contributed significantly to the positions and shapes of the binding isotherms. Kinetic determinations yielded forward and reverse rate constants for assembly of the unligated species. Amplitudes for the assembly rate data had concentration dependences in agreement with the independently determined dimer-tetramer assembly constants of oxyhemoglobin. Concentration-dependent binding isotherms were analyzed, in combination with the kinetically determined equilibrium constants, to yield salt-linked components of cooperativity at the four stages of oxygenation. The principal results of this study were as follows. (i) Assembly of fully oxygenated Hb tetramers is opposed by NaCl: the dimer-to-tetramer equilibrium constant becomes two orders of magnitude less favorable over the [NaCl] range 0.08 M to 1.4 M. By contrast, for deoxy-Hb the assembly equilibrium constant is reduced only two-fold. (ii) Oxygen binding to dimers is non-cooperative over the entire salt range, whereas dimer affinity is slightly favored by increasing the NaCl concentration. (iii) Overall affinity of tetramers for O2 is opposed by NaCl, becoming an order of magnitude less favorable over the range employed. Most of this decrease occurs at the fourth binding step, which shows a large, salt-mediated quaternary enhancement effect; i.e., the assembly of dimers into tetramers at 0.08 M NaCl is accompanied by an eight-fold increase in O2 affinity. (iv) The quaternary enhancement effect at the last O2-binding step is titrated progressively by salt until it reaches a negligible value near the highest [NaCl] of this study. The lowest [NaCl] condition (0.08 M) elicits the greatest tetramer cooperativity with the largest maximal Hill coefficient and the greatest suppression of intermediates. Possible origins and mechanistic implications of these phenomena are considered.

The Hill coefficient: inadequate resolution of cooperativity in human hemoglobin.

The Hill coefficient nH is a dimensionless parameter that has long been used as a measure of the extent of cooperativity. Originally derived from the oxygen-binding curve of human hemoglobin (Hb) by A. V. Hill in 1910, and reinvented by J. Wyman several decades later, nH is indexed to the stoichiometry of ligation and is indirectly related to the overall cooperative free energy for binding all four oxygen ligands. However, the overall cooperative free energy of Hb ligation can be measured directly by experimental methods. The microscopic cooperative free energies that relate to energetic coupling between specific subunit pairs can also be experimentally determined, while the Hill coefficient is, by its nature, a macroscopic parameter that cannot detect differences among specific subunit-subunit couplings. Its continued use in studies of the mechanism of cooperativity in Hb is therefore of increasingly limited value.

Analyzing intermediate state cooperativity in hemoglobin.

Publisher Summary The phenomenon of cooperative ligand binding is dramatically demonstrated in the distinctive sigmoidal O 2 binding curve of human hemoglobin (Hb), which facilitates efficient delivery of O 2 from the lungs to tissues undergoing oxidative metabolism. Although a great deal of physical and chemical characterization has been amassed in its study, the mechanism of cooperativity in Hb is currently undergoing major revision, based on the results of more modern experimental strategies implemented in the 1990s. The renovation is occurring at the most fundamental levels of understanding of how these molecules carries out its functions, including the number of quaternary structures the tetramer may assume and which of them dominates in solution, as well as which heme sites communicate with each other in the various stoichiometric and site-combinatorial forms of ligation. This chapter outlines the methodologies employed in the site-specific combinatorial investigation of the partially ligated Hb intermediates. The methods for analyzing physical properties of distinct partially ligated intermediates have been used to develop a high-resolution understanding of cooperative ligand binding not available from the methods that yield average parameters and a low-resolution glimpse of cooperativity. The cornerstone of this approach has been the characterization of each of the ligation intermediates of Hb by linkage thermodynamics, independent of the structural and energetic models of cooperativity that dominated Hb research in previous decades. This chapter outlines the model-independent experimental approaches used to evaluate the contribution of each partially ligated Hb intermediate to the well-known sigmoidal O 2 binding curve.

[1] Pathway of allosteric control as revealed by intermediate states of hemoglobin

Publisher Summary The new level of understanding of the energetics of ligand binding cooperativity in hemoglobin (Hb) has resulted in a new mechanistic interpretation of the allosteric switch, incorporating results of an extensive experimental dissection of the multistep ligand-binding process into its component intermediate steps. This chapter reviews the most important elements of the mechanism, along with the experimental strategies by which it has been deduced. The fundamental energetic and structural properties of the Hb molecule are outlined. The Hb α 2 β 2 tetramer binds four 0 2 molecules with successively more favorable equilibrium constants: the fourth 0 2 binds approximately 200-fold as strongly as the first. The most prominent structural feature of the Hb tetramer in the symmetry rule mechanism is the dimer-dimer interface. The formation and release of tertiary constraint are fundamental driving forces of cooperative ligand binding in Hb. The work described is an outgrowth of the realization obtained from analysis of Hb models that experimental information on the intermediate ligation states is critically required for adequately deciphering the Hb allosteric mechanism, that is, for solving the partition function for oxygen binding. This partition function is a quantitative mathematical representation of relationships between the equilibrium constants for dominant ligation events and major structural transitions.

Kinetic Trapping of a Key Hemoglobin Intermediate

The complete binding cascade of human hemoglobin consists of a series of partially ligated intermediates. The individual intermediate binding constants cannot be distinguished in O(2) binding curves, however, each constant can be determined from the O(2)-induced change in assembly constant for the α(2)β(2) tetramer from its constituent αβ dimers. The characterization of these O(2) binding constants has shown the Hb cascade to be asymmetric in nature, with binding dependent upon the specific distribution of O(2) among the four hemesites. A stopped-flow approach to measuring the dissociation constant of a key doubly ligated intermediate, that in which one dimer is oxygenated and the other is not, is described. The intermediate is transiently formed in the absence of O(2) and then allowed to dissociate in the presence of O(2). The free dimers thus released are trapped by the plasma protein haptoglobin, the rate limiting step being that of tetramer dissociation. The kinetic constant observed for the dissociation of this intermediate confirms the value for its equilibrium O(2) binding constant, previously determined under equilibrium conditions by subzero isoelectric focusing.

Biothermodynamics, Part A. Preface.

This volume is the continuation in a series of Methods in Enzymology volumes which promotes thermodynamics as an important tool for the study of biological systems. One of the many examples of biological thermodynamics is the cooperative binding of oxygen by hemoglobin. Hemoglobin is the quintessential example of a ligand-binding protein. Most biochemistry textbooks explain that the hemoglobin tetramer exists in two structural states, a low-affinity structure without oxygen bound and a high-affinity structure with oxygen bound. This is the classic two-state allosteric model as presented by Monod, Wyman, and Changeux (1965, J. Mol. Biol., 12, 88–118) and extended by Ackers and Johnson (1981, J. Mol. Biol. 147, 559–582). Unfortunately, this model tells us nothing about the specific molecular interactions that are altered by the binding of oxygen, which force the hemoglobin to shift to the alternative structural state. Investigating hemoglobin at this level is analogous to viewing only the first and last act of a tragedy by William Shakespeare (e.g., King Lear or Macbeth). The first act being a celebration and in the last act, the stage is full of dead bodies. While only the first and last acts are provocative, the truly wonderful part of a Shakespearean play is the character interactions in the intervening steps between the initial and final states, which force the last act to follow from the first act. Thermodynamics provides a conceptual and mathematical framework, that is, a “logic tool,” which allows the investigation of the specific molecular interactions and the concomitant energetics such as those that are altered by the binding of oxygen, which force the hemoglobin to shift to the alternative structural and/or association states.