Asymmetric distribution of cooperativity in the binding cascade of normal human hemoglobin. 1. Cooperative and noncooperative oxygen binding in Zn-substituted hemoglobin

The complete binding cascade of human hemoglobin consists of eight partially ligated intermediates and 16 binding constants. Each intermediate binding constant can be evaluated via dimer-tetramer assembly when ligand configurations within the tetramer are fixed through the use of hemesite analogs. The Zn/Fe analog, in which the nonbinding Zn2+ heme substitutes for deoxy Fe2+ heme, also permits direct measurement of O2 binding to the remaining Fe2+ hemesites within the symmetrically ligated Hb tetramers. Measurement of O2 binding over a range of Zn/Fe Hb concentrations to both alpha-subunits (species 23) or to both beta-subunits (species 24) shows noncooperative binding and incomplete saturation of the available Fe2+ hemesites. In contrast, the asymmetrically ligated Zn/FeO2 species 21, in which both oxygens are bound to one of the dimers within the tetramer, exhibits positive cooperativity and >90% ligation under atmospheric conditions. These properties are confirmed in the present study by measurement of the rate constant for tetramer dissociation to free dimer. The binding constants thus derived for these partially ligated intermediates are consistent with the stoichiometric constants measured for native hemoglobin by standard O2 binding techniques, providing additional evidence that Zn2+-heme substitution provides an excellent deoxy hemoglobin analog. There is no evidence that Zn-substitution stabilizes a low-affinity form of the tetramer, as previously suggested. These characterizations demonstrate distinct, nonadditive physical properties of the doubly ligated tetrameric species, yielding an asymmetric distribution of cooperativity within the cascade of O2 binding by human hemoglobin.     

A general formulation of the free energy of interaction in cooperative ligand binding: application to hemoglobin

A general method for calculating the change in free energy of cooperative interaction upon partial or complete liganding of a protein containing an arbitrary number of binding sites is presented. This method is used to estimate the change in free energy of interaction upon oxygenation of hemoglobin under physiological conditions, which is compared to the internal energy change upon binding oxygen to the non-interacting binding site.     

Resolvability of free energy changes for oxygen binding and subunit association by human hemoglobin.

Probability distributions of the free energy changes for oxygen binding, subunit association, and quaternary enhancement by human hemoglobin were obtained from Monte Carlo simulations performed on two independent sets of variable protein concentration equilibrium oxygen-binding data. Uncertainties in unliganded and fully liganded dimer to tetramer association free energy changes (0 delta G'2 and 4 delta G'2) were accounted for in the simulations. Distributions of the dimer to tetramer association free energy changes for forming singly and triply liganded tetramers (1 delta G'2 and 3 delta G'2) are well defined and quite symmetric, whereas that for forming doubly liganded tetramers (2 delta G'2) is poorly defined and highly asymmetric. The distribution of the dimer stepwise oxygen-binding free-energy change (delta g'2i) is well defined and quite symmetric as are those of the tetramer stepwise oxygen-binding free-energy changes for binding the first and last oxygens to tetramers (delta g'41 and delta g'44). Distributions of the intermediate tetramer stepwise oxygen-binding free-energy changes (delta g'42 and delta g'43) are poorly defined and highly asymmetric, but are compensatory in that their sum (delta g'4[2 + 3]) is again well defined and nearly symmetric. Distributions of the free energy changes corresponding to the tetramer product Adair oxygen binding constants (delta G'4i) are well defined and quite symmetric for i = 1, 3, 4 but not for i = 2. The distribution of delta g'44 - delta g'2i (the quaternary enhancement free energy change) is relatively narrow, nearly symmetric, and confined to the negative free-energy domain. This suggests that the quaternary enhancement free energy change (a) may be resolved with good confidence from this data and (b) is finite and negative under the conditions of these experiments. Our results also suggest two different four-state combinatorial switch models that provide accurate characterization of hemoglobin's functional behavior.     

Description of the molecular mechanism of cooperativity in human hemoglobin cannot be limited to a first-order free energy coupling concept

Studies of the linkage between ligand binding and subunit assembly of oligomeric proteins have extensively used the concept of free energy coupling. The "order" of these free energy couplings was introduced [Weber, G. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 7098-7102] as the number of subunits that must be liganded to alter specific intersubunit interactions. This concept dictates that the ligation of fewer subunits has no effect, but once the order number of subunits becomes ligated, the specific intersubunit interaction energy between those particular subunits is completely eliminated. Weber's report claims that the free energy coupling between oxygen binding and the dimer-tetramer subunit assembly in stripped human hemoglobin A is "first order". This conclusion is based on the analysis of a set of previously published equilibrium constants [Mills, F. C., Johnson, M. L., & Ackers, G. K. (1976) Biochemistry 15, 5350-5362]. I subsequently reported that the original experimental data, from which the equilibrium constants were derived, are consistent with both the first-order and "second-order" free energy coupling concepts [Johnson, M. L. (1986) Biochemistry 25, 791-797]. I also demonstrated that more precise recent experimental data [Chu, A. H., Turner, B. W., & Ackers, G. K. (1984) Biochemistry, 23, 604-617] are consistent with both the first-order and second-order free energy coupling concepts. A recent article [Weber, G. (1987) Biochemistry 26, 331-332] disagrees that the oxygen-binding data for human hemoglobin A are consistent with a second-order model.(ABSTRACT TRUNCATED AT 250 WORDS)     

Some aspects of cooperativity in human hemoglobin

The cooperativity in hemoglobin can be described by the Hill parameter n, the free energy of interaction ΔF₁ and the allosteric free energy ΔF_A. By this latter is meant here the free energy change associated with the transition from the deoxy to the oxy conformation in hemoglobin. In this paper some general relations between n, ΔF₁ and ΔF_A are given. A method is presented by which ΔF_A can be calculated from oxygenation data.     

The binding of CO2 to human hemoglobin.

CO2-dissociation curves of concentrated human deoxy- and carbonmonoxyhemoglobin at 37 degrees, pH 7.6 to 7.0, PCO2 equal to 10 to 160 mm Hg, have been obtained by a rapid mixing and ion exchange technique. The CO2-dissociation curves for deoxyhemogloblin can only be fitted by assuming two classes of binding sites for carbon dioxide. The simplest way to account for the experimental data is to assume that the alpha-amino groups of the alpha and beta chains react with carbon dioxide with affinities that differ by at least a factor of 3. No difference in reactivity with CO2 was found among the four terminal alpha-amino groups of carbonmonoxyhemoglobin.     

Thyrotropin receptors in normal human thyroid. Nonclassical binding kinetics not explained by the negative cooperativity model

Saturation analysis of equilibrium binding of iodinated thyrotropin (125I-TSH) to normal human thyroid preparations yielded linear Scatchard plots under non-physiological conditions of pH 6.0 or 20 mM Tris/acetate buffer, pH 7.4. The apparent equilibrium dissociation constant of this binding was approximately 10(-8) M. By contrast, nonlinear plots were obtained under standard conditions of pH 7.4 and 40 mM Tris/acetate buffer. Resolution of the components of these curves by computer analysis revealed the presence of at least two classes of binding sites, one of which is of a low capacity and high affinity (approximately 10(-10) M) consistent with receptor binding. The other component is of a high capacity and lower affinity. Binding to non-target tissues of muscle, parathyroid, mammary carcinoma, and placenta was only demonstrable at pH 6.0 or in 20 mM Tris/acetate buffer, pH 7.4, yielding linear Scatchard plots with similar binding affinity (approximately 10(-8)M) to normal thyroid but much reduced capacity. Preincubation of thyroid tissue at 50 degrees C resulted in an apparent selective loss of the high affinity component of binding measured under standard conditions. Kinetic experiments on the dissociation of bound 125I-TSH were undertaken to determine whether the non-linearity of Scatchard plots was due to two or more classes of binding sites or negative cooperativity. It was found that the experimental determinant that is presently ascribed to a negative cooperativity phenomenon regulating receptor affinity (i.e. an enhanced dilution-induced dissociation rate in the presence of excess native hormone), although apparently hormone-specific, was demonstrated under nonphysiological binding conditions and in non-target tissue. Significantly, the phenomenon was found under conditions of pH 6.0 or 20 mM Tris where a linear Scatchard plot was obtained. The evidence thus suggests that 125I-TSH binds to heterogeneous binding sites (of which the high affinity is probably the receptor for TSH) and that the enhanced dilution-induced dissociation of bound hormone by native hormone for this system, is only a characteristic of the low affinity binding site (maybe gangliosides).     

Calculation of the free energy and cooperativity of protein folding

Calculation of the free energy of protein folding and delineation of its pre-organization are of foremost importance for understanding, predicting and designing biological macromolecules. Here, we introduce an energy smoothing variant of parallel tempering replica exchange Monte Carlo (REMS) that allows for efficient configurational sampling of flexible solutes under the conditions of molecular hydration. Its usage to calculate the thermal stability of a model globular protein, Trp cage TC5b, achieves excellent agreement with experimental measurements. We find that the stability of TC5b is attained through the coupled formation of local and non-local interactions. Remarkably, many of these structures persist at high temperature, concomitant with the origin of native-like configurations and mesostates in an otherwise macroscopically disordered unfolded state. Graph manifold learning reveals that the conversion of these mesostates to the native state is structurally heterogeneous, and that the cooperativity of their formation is encoded largely by the unfolded state ensemble. In all, these studies establish the extent of thermodynamic and structural pre-organization of folding of this model globular protein, and achieve the calculation of macromolecular stability ab initio, as required for ab initio structure prediction, genome annotation, and drug design.     

Metastable CO binding sites in the photoproduct of a novel cooperative dimeric hemoglobin.

The infrared absorption spectrum of the CO-photoproduct from Scapharca inaequivalvis hemoglobin (Hbl) at 10 K yields only a single line in the "B" state region at 2132 cm-1. This is the same frequency as the B1 line observed in photodissociated vertebrate HbCO and MbCO. No evidence was found for the B2 line detected in vertebrate hemoglobins and myoglobin in the 2118-2120 cm-1 region. These data demonstrate that the protein does not have the same conformationally accessible ligand-binding sites as do vertebrate hemoglobins and myoglobins. The absence of the B2 line indicates that only a single weak site is accessible to the photolyzed CO molecule. These results are in accord with geminate rebinding experiments and ligand escape pathway calculations which have shown that the distal properties of Hbl are distinct from those of tetrameric hemoglobins and vertebrate myoglobins.     

Studies of oxygen binding energy to hemoglobin molecule.

The fractional oxygen saturation of hemoglobin and of its isolated α-chains have been determined by the application of the technique of Mossbauer effect while the oxygen equilibrium pressure was measured directly. By comparing the experimental data so obtained with theoretically derived oxygen saturation curves for hemoglobin and isolated α-chains the values of the oxygen pairing energy and oxygen binding energy to hemoglobin molecule have been found to be 0.043 ± 0.004 and 0.60 ± 0.06 eV respectively.     

Two-step ligand binding and cooperativity. A model to describe the cooperative binding of myosin subfragment 1 to regulated actin.

The binding of actin to myosin subfragment 1 (S1) has been shown to occur as a two-step reaction. In the first step actin is weakly bound and then the complex isomerizes to the "rigor type" acto-S1 complex (Coates, J. H., A. H. Criddle, and M. A. Geeves, 1985 Biochem. J., 232:351-356). We propose here a model in which troponin/tropomyosin (Tn/Tm) controls the actin-S1 interaction by inhibiting the isomerization step. In this model the (actin)7 Tn/Tm unit is assumed to exist in two states: open and closed. S1 can bind to either of the two states but only the open form allows the isomerization reaction to take place. We demonstrate that this model can account for the cooperative binding of S1 and S1 nucleotide complexes to actin. The model provides a way of integrating both the effects of calcium and nucleotide on actin-S1 interactions.     

What the intermediate compounds in ligand binding to hemoglobin tell about the mechanism of cooperativity

The populations of the intermediates in concentrated solutions of hemoglobin A0 equilibrated at various PCO values, pH 7.0, 0.1 M KCl, and 20 degrees C, have been determined using cryogenic methods. Data on CO saturations and distributions of intermediates were analysed in terms of the free energies of dimer-tetramer assembly of the intermediates (G.K. Ackers and F.R. Smith, Annu. Rev. Biophys. Chem. 16 (1987) 583). The cooperative free energy value of the singly ligated species was approximately one-half the total cooperative energy. The cooperative free energy value of the doubly ligated species was not significantly different from that of carboxyhemoglobin. Because of experimental error, the observed difference in concentrations among the populations of the doubly ligated species cannot be taken as indicative of their functional heterogeneity. Additional studies on some NO intermediates have emphasized that (alpha 1 beta 1)(alpha 2 beta 2)X, a key intermediate in the formulation of the 'third-state' hypothesis in the deoxy/cyanomethemoglobin system, has a free energy value for dimer-tetramer assembly which is critically dependent on the nature of the ligand X as suggested by Ackers and Smith (reference as cited above).     

Oxygen-linked CO2 binding to isolated beta subunits of human hemoglobin.

It is known that most of the oxygen-linked carbamate which is formed in normal adult human hemoglobin (Hb A) is confined to the beta subunits rather than to the alpha subunits. In order to find out if similar differences exist in the isolated protomers of Hb A we have measured the effect of various pressures of carbon dioxide (pCO2) on the oxygen affinity in the following heme pigments: isolated alpha and beta subunits with free --SH groups (alphaSH, betaSH), mercurated beta subunits (betaPMB), myoglobin (Mb), and betaSH/PLP in which the terminal alpha-amino group of betaSH was irreversibly blocked with pyridoxal phosphate (PLP). Similar measurements were done on Hb A and the fraction of oxygen-linked carbamate calculated from the effect of pCO2 (at constant pH) on the oxygen half-saturation pressure (p50). A distinct influence of CO2 on p50 was observed in betaSH which was absent in betaSH/PLP and thus indicates that the terminal alpha-amino group mediates the oxygen-linked binding of CO2 in betaSH as it does in the beta subunits of Hb A. However, the fraction of oxygen-linked carbamate was much less dependent on pH and pCO2 in betaSH than in Hb A. Neither alphaSH, betaPMB, or Mb, all of which are known to exist largely or wholly as monomers but have free terminal alpha-amino groups, showed a shift of p50 upon addition of CO2. As both betaSH and betaSH/PLP were shown to be tetrameric molecules, we conclude from this study that homotetramers composed of isolated beta subunits do exhibit a reciprocal interaction between the binding of O2 and CO2.     

Effect of reversed heme orientation on circular dichroism and cooperative oxygen binding of human adult hemoglobin

We found that recombinant human adult hemoglobin (rHb A) expressed in Escherichia coli showed heterogeneity of components with the intensity of a positive CD band at 260 nm and that it could be resolved into three components (SP-1, SP-2, and SP-3) by SP-Sepharose column chromatography. 1H NMR revealed that SP-1 is identical with native Hb A, while SP-2 and SP-3 largely contain the reversed heme isomer in both the alpha and beta subunits, with contents of approximately 50 and >80% in SP-2 and SP-3, respectively. Rotation of the heme 180 degrees about the 5,15-meso axis (reversed heme) causes an interexchange of the methyl groups at positions 2 and 7 with the vinyl groups at positions 8 and 3, respectively. To examine the effect of the modification of the heme-protein contact on the structure and function of Hb A, we compared the 1H NMR, CD, and oxygen binding properties of the three components with those of native Hb A. Native Hb A exhibits a distinct positive CD band in both the near-UV and Soret regions, but rHb A with reversed heme exhibits a very weak positive CD band at 260 nm and a prominent negative CD band in the Soret region. Cooperativity, as measured by Hill's n value, decreased from 3.18 (SP-1) to 2.94 (SP-2) to 2.63 (SP-3) with an increase in the reversed heme orientation. The effect of an allosteric effector, inositol hexaphosphate (IHP), on the oxygen binding properties was also reduced in rHb A with reversed heme. These results indicate that changes in the heme-globin contact exert a discernible influence on CD spectra and cooperative oxygen binding.     

The contribution of the asymmetric alpha 1beta 1 half-oxygenated intermediate to human hemoglobin cooperativity.

Considerable controversy remains as to the functional and structural properties of the asymmetric alpha1beta1 half-oxygenated intermediate of human hemoglobin, consisting of a deoxygenated and an oxygenated dimer. A recent dimer-tetramer equilibrium study using [Zn(II)/Fe(II)-O(2)] hybrid hemoglobins, in which Zn-protoporphyrin IX mimics a deoxyheme, showed that the key intermediate, [alpha(Fe-O(2))beta(Fe-O(2))][alpha(Zn)beta(Zn)], exhibited an enhanced tetramer stability relative to the other doubly oxygenated species. This is one of the strongest findings in support of distinctly favorable intra-dimer cooperativity within the tetramer. However, we present here a different conclusion drawn from direct O(2) binding experiments for the same asymmetric hybrid, [alpha(Fe)beta(Fe)][alpha(Zn)beta(Zn)], and those for [alpha(Fe)beta(Zn)](2) and [alpha(Zn)beta(Fe)](2). In this study, the O(2) equilibrium curves for [alpha(Fe)beta(Fe)][alpha(Zn)beta(Zn)] were determined by an O(2)-jump stopped-flow technique to circumvent the problem of dimer rearrangement, and those for [alpha(Fe)beta(Zn)]( 2) and [alpha(Zn)beta(Fe)]( 2) were measured by using an Imai apparatus. It was shown that the first and second O(2) equilibrium constants for [alpha(Fe)beta(Fe)][alpha(Zn)beta(Zn)] are 0.0209 mmHg(-1) and 0.0276 mmHg(-1), respectively, that are almost identical to those for [alpha(Fe)beta(Zn)](2) or [alpha(Zn)beta(Fe)](2). Therefore, we did not observe large difference among the asymmetric and symmetric hybrids. The discrepancy between the present and previous studies is mainly due to previously observed negative cooperativity for [alpha(Fe)beta(Zn)](2) and [alpha(Zn)beta(Fe)](2), which is not the case in our direct O(2) binding study.