COMMUNICATION
ATP-induced Tetramerization and Cooperativity in Hemoglobin of Lower Vertebrates*

Carlos F. S. Bonafe, Adriana Y. Matsukuma, and Maria S. A. MatsuuraDagger

From the Departamento de Bioquímica, Instituto de Biologia, Universidade Estadual de Campinas, 13083-970, Campinas, São Paulo, Brazil

    ABSTRACT
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Abstract
Introduction
Procedures
Results & Discussion
References

The importance of intraerythrocytic organic phosphates in the allosteric control of oxygen binding to vertebrate hemoglobin (Hb) is well recognized and is correlated with conformational changes of the tetramer. ATP is a major allosteric effector of snake Hb, since the absence of this nucleotide abolishes the Hb cooperativity. This effect may be related to the molecular weight of about 32,000 for this Hb, which is compatible with the dimeric form. ATP induces a pH-dependent tetramerization of deoxyHb that leads to the recovery of cooperativity. This phenomenon may be partially explained by two amino acid replacements in the beta  chains (CD2 Glu-43 right-arrow Thr and G3 Glu-101 right-arrow Val), which result in the loss of two negative charges at the alpha 1beta 2 interface and favors the dissociation into dimers. The ATP-dependent dimer left-right-arrow  tetramer may be physiologically important among ancient animal groups that have similar mutations and display variations in blood pH that are governed by these animals' metabolic state. The enormous loss of free energy of association that accompanies Hb oxygenation, and which is also observed at a much lower intensity in higher vertebrate Hbs, must be taken into consideration in allosteric models. We propose that the transition from a myoglobin-like protein to an allosteric one may be of evolutionary significance.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results & Discussion
References

In vertebrates, hemoglobin (Hb) exists as a tetramer in its intraerythrocytic environment, and it is this form that is involved in the classic structural change from a low to a high O2 affinity molecule in the presence of increasing O2 concentrations. This phenomenon, known as cooperativity, is reflected in the sigmoidal shape of the O2 saturation curve.

Protons and organic phosphate are important in the physiological transport of O2 in most vertebrate groups, since they stabilize the low affinity form of Hb (1, 2).

Previous studies have demonstrated an oxygen-induced dissociation of snake Hb at physiological pH and Hb concentration, as well as in the presence of high levels of organic phosphate (3). The structural basis of this phenomenon is the replacement of amino acid residues beta -CD2-43 and beta -G3-101 at the alpha 1beta 2 interface which is responsible for tetramer stabilization. These key residues, normally both glutamic acid, are replaced by threonine and valine, respectively, in snake Hb (4). This loss of negative charges would disturb the interface contact, leading to a pronounced tendency of Hb to dissociate into dimers. Since these residues are also replaced in most hemoglobins from ectothermic animals (5-9), this suggests that a dissociation of Hb occurs during oxygenation. The physiological role of such Hb dissociation is considered in the present investigation.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results & Discussion
References

Hemoglobin Preparation-- Adult snakes of both sexes weighing 200-400 g were obtained from the Instituto Butantã (São Paulo) and were kept in the laboratory until bleeding. The hemolysate was prepared as described by Rossi-Fanelli and Antonini (10) and was freed of salts and small organic molecules by passage through a Sephadex G-25 column (2.0 × 90 cm) equilibrated with 1 mM Tris-HCl, pH 9.0 (11), to produce "stripped" Hb.

Measurement of Redox Potentials-- The redox titrations were carried out according to Antonini et al. (12). Five milliliters of Hb solution (140 µM as heme) were deoxygenated in a tonometer and then transferred anaerobically, with continuous flush of N2, to the titration half-cell, which contained 0.1 M Tris-HCl plus 0.1 M NaCl (pH range: 7.0-8.0). Thionin was added as a mediator in a molar ratio to protein of 2-4%. The oxidation of deoxyHb was performed by the stepwise addition of a degassed solution of 5 mM potassium ferricyanide. The measured electrode potentials were refereed to the normal hydrogen electrode (13). The oxidation-reduction potential at 50% of oxidation provided the midpoint potential (E1/2).

O2-Hb Equilibrium-- The experiments were performed at 20 °C in 0.1 M Tris-HCl buffer of different pH values containing 0.1 M NaCl, using a tonometric-spectrophotometric method (14). The protein concentration was 80 µM (as heme).

    RESULTS AND DISCUSSION
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Abstract
Introduction
Procedures
Results & Discussion
References

To gain insight into the possible physiological role of pH and ATP in the subunit assembly of snake (Helicops modestus) Hb, we investigated the Hb-O2 equilibrium as a function of proton concentration in the presence or absence of ATP (Fig. 1). Stripped snake Hb showed a high affinity for O2 and no allosterism, in accordance with a molecular mass compatible with the dimeric form (3, 10, 15). In the presence of organic phosphates, the molecule became cooperative (nH = 2) with a low O2 affinity at a pH up to 7.4. With increasing pH, the Hb gradually lost cooperativity, suggesting a weakening of the electrostatic interaction between ATP and Hb. As a result, the latter tended to assume the properties of stripped Hb. The pH sensitivity cannot be attributed exclusively to a classic Bohr effect in view of the dimerization process that is also present.


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Fig. 1.   Effect of ATP on the O2 equilibrium of H. modestus Hb at different pH values. The buffer used was 0.1 M Tris-HCl containing 0.1 M NaCl, at 20 °C, and the Hb concentration was 80 µM as heme. open circle , stripped Hb; bullet , Hb in the presence of 1.0 mM ATP. Inset, nH values derived from a Hill plot.

Based on these unusual findings, we investigated the redox potential of snake Hb under the same conditions as those used for the O2 equilibrium curves in order to better understand the Hb properties in the presence of ATP at different pH values. This approach, applied to either tetrameric Hbs or myoglobins, has been employed to show the conversion of the deoxy to the met form and its close correlation with oxygenation equilibrium curves, since the potentiometric curves share similarities with the equilibrium ligand binding curves for Hb (12, 16-20). Fig. 2A illustrates that snake Hb had a peculiar behavior in this experiment. The redox potential of stripped Hb did not change with a pH of up to 7.6, but decreased at higher pH values. The resulting curve was similar to that of myoglobin and corroborated our expectation that stripped Hb is dissociated even in the deoxygenated form. The progressive decrease in Eh observed with increasing pH in both stripped and ATP-Hb is correlated to the extend of water ionization on the sixth coordinate of heme iron (18). ATP dramatically changed the redox equilibrium profile. In the presence of ATP, the Eh value at pH up to 7.22 was constant and higher than in the absence of the nucleotide. However, the Eh value decreased sharply in the pH range of 7.22-7.38. This observation is consistent for a tetrameric Hb in which the classic allosteric model is found. The redox equilibrium curve at pH > 7.80 superposed the stripped Hb curve, indicating the complete release of ATP from its binding site. In the pH range of 7.38-7.80, the redox potential presented a curve compatible with equilibrium between dimers and ATP-bound tetramers. From Fig. 2A we estimated the quantitative contribution of the different molecular forms of Hb (Fig. 2B). The dissociation of tetrameric Hb into dimers was observed primarily between pH 7.38 and 7.55 and varied from 0 to 80%.


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Fig. 2.   A, effect of ATP on the oxidation-reduction equilibrium of H. modestus Hb at different pH values. open circle , stripped Hb; bullet , Hb in the presence of 1.0 mM ATP. The buffer used was 0.1 M Tris-HCl containing 0.1 M NaCl at 20 °C, and the Hb concentration was 140 µM as heme. The dashed line indicates the theoretical tetramer behavior. A, inset: Hill plots of the oxidation-reduction equilibrium curves for H. modestus stripped Hb (open symbols) and in the presence of 1.0 mM ATP (closed symbols). B, estimation of the proportion of dimeric and tetrameric Hb in the presence of ATP as a function of pH, based on the above figure. For each pH value, the fraction corresponding to the tetramer, Falpha 2beta 2, and dimer, Falpha beta , was calculated based on the theoretical data for the tetramer (dashed line of Eh curve in the presence of ATP, which corresponds to the theoretical behavior if ATP were bound) and on experimental data for the dimer (Eh curve in the absence of ATP): Falpha 2beta 2 = (Ehalpha beta  - Eh)/(Ehalpha beta  - Ehalpha 2beta 2); Falpha beta  = 1 - Falpha 2beta 2.

The inset in Fig. 2A shows the corresponding Hill plots of the redox equilibrium. At pH 7.0, the oxidated Hb retained its tetrameric form, indicating that ATP remains bound independently of the degree of oxidation. At pH 7.80, the Hb dissociated and had the same nH values as stripped Hb. However, at pH 7.38, the biphasic behavior indicated that above 50% of oxidation, R-met Hb became very unstable and immediately dissociated into dimers.

The substantial differences in the Hb properties described above assume a great significance when the physiological state of ectothermic vertebrates are considered. Several studies have reported large blood pH changes when ectothermic animals are subjected to different temperature or stress conditions (21-23). In such situations, the proton concentration would be particularly important in influencing the binding of ATP to Hb, thereby altering the protein's O2 affinity. These functional properties may be present in a large array of animals from related groups, since the replacement of amino acid residues at key positions of alpha 1beta 2 contact is present (5-9).

Fig. 3 proposes a general model for O2 transport by snake and other related vertebrate Hbs in which the ATP plays a central role. In the dormancy state, when low O2 transport is required and the blood pH is increased, the Hb exists in a dimeric form that acts as a reserve supply of O2 in a manner similar to myoglobin. In stress or high activity, the decrease in pH promotes ATP-induced tetramerization and allosterism, thereby resulting in a significant O2 release. Thus in this dynamic interchange, ATP and pH changes serve to integrate the physiology of O2 supply. This novel model provides new insight into O2 transport when compared with higher vertebrates in which the cooperative ligand binding of Hb is based on a switching between quaternary states of the Hb tetramer with different O2 affinities (24).


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Fig. 3.   In vivo model of O2 transport by snake Hb.

From a thermodynamic aspect, the classic T-R model of Monod-Changeux-Wyman (MCW model) does not take into account the free energy of association between alpha beta dimers (25). This situation was considered by Weber (26), who demonstrated that O2 binding to human Hb is a first order reaction that is inconsistent with the two-state model. It is noteworthy that the dissociation of snake Hb is an extreme example of decreasing the Gibbs energy of association between dimers, since progressive oxygenation is linked with dissociation into more reactive dimeric species. Thus, the presence of the classic R state is theoretical and difficult to detect experimentally (Fig. 1), except in metHb obtained by redox potential experiments (Fig. 2A). In Fig. 4, we propose a diagram of the Gibbs free energy of O2 binding with snake ATP-Hb in comparison with stripped human Hb. The most striking feature is the inversion of the free energy of association between oxygenated dimers despite the presence of ATP. Moreover, the binding of the first/second O2 molecule results in a much higher affinity of tetrameric snake Hb to further O2 binding than is the case with human Hb.


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Fig. 4.   Gibbs free energy levels of Hb subunit association and oxygen binding by human (A) and snake (B) Hb. D = dimer; T = tetramer; X = O2. Delta G(n) = free energy of subunit association with "n" molecules of O2. Delta G(2,n) and Delta G(4,n) = free energy of O2 binding of dimer and tetramer, respectively, with "n - 1" molecules of O2.

The evolutionary adaptation, study of Hb structure and function, has been extensively discussed (27, 28). The Agnatha, lampreys, and hagfish, the most primitive group of vertebrates, have monomeric Hb in which the O2 transport mechanism is accomplished by a dimer left-right-arrow  monomer transition. This disaggregation leads to a higher affinity state and allows cooperative behavior (29-31). The singular properties of snake Hb reported here may point to the origin of a stable dimeric molecule that could have important evolutionary implications for heme-heme interactions. The alpha beta dimeric form may represent an intermediary evolutionary stage of the classic allosterism of vertebrate Hbs, where ATP serves as a central allosteric mediator. The molecular properties of such Hbs may reflect the physiological functions of ectothermic Hb, particularly the adaptations to exogenous and endogenous factors such as ambient hypoxia, temperature, activity, and dormancy. Finally, the mechanism of dimer-tetramer transitions during O2 transport may represent an intermediate stage of evolution to the stable tetrameric Hb found in higher vertebrates.

    ACKNOWLEDGEMENT

We are grateful to Prof. Stephen Hyslop for helpful discussions.

    FOOTNOTES

* This work was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Proc. 95-1245/7; Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq); and Fundação de Amparo ao Ensino e à Pesquisa (FAEP-UNICAMP) e Serviço de Apoio ao Estudante (SAE-UNICAMP).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. de Bioquímica, Instituto de Biologia, Universidade Estadual de Campinas, 13083-970, Campinas, SP, Brazil. Tel.: 55-19-788-7953; Fax: 55-19-289-3124; E-mail: bonafe{at}obelix.unicamp.br.

    REFERENCES
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Abstract
Introduction
Procedures
Results & Discussion
References

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