Ligand‐Specific Bohr Effect in Haemoglobins

Abstract

The GO binding properties of monomeric (Hb I11 and IV), dimeric, and tetrameric Chironomus haemoglobins as well as of the monomer-analogue valence hybrid (aFmet /Imet /Ideox') of Hb M Iwate were studied and compared with the respective 0, binding properties. All these haemoglobins show hyperbolic 0, binding curves and a Bohr effect. The amplitudes of the 0, Bohr effect curves range from dlog pO,(,,r, = 0.3 (Hb 111) to 2.4 (tetrameric Chironomus haemoglobin).The ligands CO and 0, differ not only in affinity but also with regard to the Bohr effect. The GO affinities of the various Chironomus haemoglobins are 60 to 490 times larger than the 0, affinities and the amplitudes of the CO Bohr effect curves are 1.25 to 8 times smaller than those of the respective 0, Bohr effect curves. I n the case of Hb M Iwate, however, the CO Bohr effect is larger than the 0, Bohr effect by the factor 1.25 (2,3-bisphosphoglycerate-reacted H b M) and 1.4 (stripped Hb M).The ligand-specific differences in the magnitude of the Bohr effect cannot be explained solely by the allosteric interaction connected with the trans-effect of the complex. The differences are discussed in terms of steric influences on the binding geometry of the 6th ligand by protein side chains a t the distal side of the haem group. The titration behaviour of the three titratable histidine residues of H b I11 was studied by proton magnetic resonance (PMR) spectroscopy [3]. Ligation with CO produces a shift of the pK value of the histidine G2. This residue was identified, therefore, as the Bohr proton-binding site. As the PMR measurements required periods of several hours at 30 "C, it was not possible to use 0, as 6th ligand because of autoxidation of the haemoglobin. However, the kind of ligand bound to the 6th position seems to play an important role with respect to the extent of the pH-dependent change of the affinity for, in contrast to the experiments with CO, the PMR studies on ferric and ferrous H b I I I , ligated with Dedicated to Prof. Dr Hans Netter (Institut fiir Physiologische Chemie und Physikochemie der Christian-AlbrechtsUniversitat zu Kiel) on the occasion of his 75th birthday.Abbreviations. Hb I, Hb I11 and Hb IV, monomeric haemoglobins I, I11 and IV from Chironomus thummi thummi; Hb M Iwate, human haemoglobin M Iwate; PMR, proton magnetic resonance. The CO equilibrium studies were extended also to other monomeric, dimeric and tetrameric haemoglobins of Chironomus and to a mutant tetrameric haemoglobin (Hb M Iwate) which is characterized by one or two GO binding sites, respectively [6,7]. CN-MATERIALS AND METHODS HaemoglobinsMonomeric haemoglobin I, 111, and IV fromChironornus thummi thummi were isolated as described in [3].Eur. J. Biochem. 45 (1974)

A Calorimetric Study of the CO Bohr Effect of Monomeric Haemoglobins

“…The observed shift in pK value for the Bohr proton binding group, identified as histidine-G2, has been fully correlated to the pH dependence of the half-saturation pressure [4,5]. A suggested molecular mechanism for the long-range interaction between the ligand reaction and Bohr proton site depends upon the movement of the proximal histidine in Van der Waals contact with the C-terminal methionine residue [5].The structural and spectroscopic information obtained with these monomeric haemoglobins offer a unique opportunity to study the thermodynamic aspects of intra-chain allosterism. We have therefore carried out a series of direct calorimetric measurements on the CO binding reaction at different values of pH.…”

“…Under these conditions the ligand affinity is found to be smaller than that at high pH values. The observed shift in pK value for the Bohr proton binding group, identified as histidine-G2, has been fully correlated to the pH dependence of the half-saturation pressure [4,5]. A suggested molecular mechanism for the long-range interaction between the ligand reaction and Bohr proton site depends upon the movement of the proximal histidine in Van der Waals contact with the C-terminal methionine residue [5].…”

“…The pH dependence of the measured equilibrium half-saturation pressure was also found to depend upon the particular ligand (0, or CO) involved in the reaction to the haem [4]. The largest effect was noted for oxygen.…”

“…The monomeric haemoglobins 111 and IV from Chironomus thummi thummi were prepared as described previously [5]. The lyophilized salt-frce material was dissolved in 0.2 M maleate, bis-Tris, or Tris buffers.…”

“…In this protein the Bohr proton ionization site has been identified by nuclear magnetic resonance as a single histidine [4,5]. The X-ray structural studies by Huber et al [6,7] and the results of chemical shifts observed by nuclear magnetic resonance [5] indicate that this histidine forms a salt bridge with the C-terminal carboxyl group of methionine-H22. The distance between the Bohr proton site and the iron of the haem group is about 1.5 nm.…”