ATP-induced Tetramerization and Cooperativity in Hemoglobin of Lower Vertebrates

Abstract

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 ␤ chains (CD2 Glu-43 3 Thr and G3 Glu-101 3 Val), which result in the loss of two negative charges at the ␣ 1 ␤ 2 interface and favors the dissociation into dimers. The ATP-dependent dimer 7 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.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 O 2 affinity molecule in the presence of increasing O 2 concentrations. This phenomenon, known as cooperativity, is reflected in the sigmoidal shape of the O 2 saturation curve.Protons and organic phosphate are important in the physiological transport of O 2 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 ␤-CD2-43 and ␤-G3-101 at the ␣ 1 ␤ 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 PROCEDURESHemoglobin Preparation-Adult snakes of both sexes weighing 200 -400 g were obtained from the Instituto Butantã (Sã o 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 thro...

Rattlesnake Hemoglobins: Functional Properties and Tetrameric Stability

“…Our results concerning the Bohr effect for Hb-II (figure 2a) contrast with the behavior described above for Helicops modestus hemoglobin [6]. The stripped form of Hb-II showed between pH 7 to 7.5 an alkaline Bohr effect of -0.6 H + /heme, and in the presence of ATP -0.44 H + /heme.…”

“…Therefore, the idea proposed [6] that most of ectothermic animals have dimeric hemoglobins in the oxygenated form, as proposed for Liophis miliaris and Helicops modestus is not valid, and should be supported by experimental evidences.…”

“…In 1999 it was reported [6] that the stripped hemolysate of the semi-aquatic snake Helicops modestus displayed high affinity for O2-binding, and absence of both Bohr effect and cooperativity, what was interpreted as agreeing with the expected behavior for dimeric Hb. In the presence of organic polyphosphates, the hemoglobin showed low oxygen affinity and O2-binding became cooperative (at pH below 7.5), functional features compatible with the tetrameric form.…”

“…Bonafé et al [6] studied the hemolysate from Helicops modestus, reporting that the stripped hemoglobin has high oxygen affinity, absence of cooperativity and no oxygenlinked proton binding. These functional properties would agree with the existence of a dimeric form of that hemoglobin, as proposed for the semi-aquatic snake Liophis miliaris.…”

“…Subsequently, this mechanism was extended to other snake species, such as, Bothrops alternatus [5], Boa constrictor [3] and Helicops modestus [6]. For Mastigodryas bifossatus hemoglobins [7] the authors concluded that there were no clear evidences supporting the dissociation hypothesis, at least for that species.…”