The Bohr Proton‐Binding Site in a Monomeric Haemoglobin

Abstract

Individual haemoglobin I11 from Chironomus thummi thummi is a monomeric protein ( M , = 15500) which has recently been shown by us to be characterized by a simple Bohr effect. Three of the four histidines, the position of which in the polypeptide chain is known from sequence analysis, are distinguished by their titration characteristics, namely His-G2, His-GI9 and His-E5. The chemical shift of the C-2 proton resonances of the imidazole groups, which varies upon protonation of the histidine, has been used for the determination of the intrinsic p K values. His-F8, the proximally bound histidine, is not observable because its C-2 proton resonance is broadened.One of the three titratable histidines reveals a pK value which is varied in the range from 7.5 to 7.1, depending on the 6th ligand a t the haem iron. The relatively high pK value of this histidine and its abnormal value of the chemical shift of the C-2 proton resonance a t low pH indicate an interaction with a carboxylate group. This shift of the pK value caused by the binding of CO to reduced haemoglobin is in agreement with the interaction energy, calculated from the amplitude of the Bohr effect curve, and corresponds to 500 cal.I n this simple case the assignment of the C-2 proton resonances to the known histidines is possible considering the atomic model, described by Huber et al. [4]. The results indicate that His-G2 is the allosteric binding site; the remaining two detectable histidines are not involved in the allosteric interaction. The molecular mechanism of the Bohr effect in this haemoglobin is discussed.Contrary to the mammalian haemoglobins, the larval haemoglobins of the insect Chironomus thummi thummi are characterized by a simple, alkaline Bohr effect [I]. It is of special interest that two of these haemoglobins, namely individual haemoglobins I11 and IV, which are monomeric in the range of pH 5 to 10, also show this Bohr effect Recently we reported results of proton magnetic resonance studies of histidine residues of horse myoglobin [5]. I n the case of myoglobin nine of the eleven histidine C-2 proton magnetic resonances have been resolved and from the titration curves the intrinsic p K values of these histidines have been determined. I n this communication the results of a titration of the histidine residues of haemoglobin I11 are described. This haemoglobin has a molecular weight of 15800 [3] and a tertiary structure quite similar Abbreviations. NMR, nuclear magnetic resonance; Me,Si, tetramethylailane.to that of myoglobin [4]; however, only four histidines appear in the sequence : His-F8, His-G2, His-E5, and His-G19. This small number of histidines allows a more exact determination and assignment of the intrinsic pK values and of their possible variation in the case of conformational changes induced by ligand binding. Such a conformational change is re%ected by the Bohr effect.The Bohr effect describes a negative interaction between the 0,-binding site and proton-binding sites in the mammalian haemoglobins 16-81. The identification of the ...

Proton-magnetic-resonance investigation of the dynamics of the conformational transition in allosteric monomeric insect hemoglobins

“…Thus, the transition we are monitoring must represent the t e r conformational transition whose rate is independent of the rate of proton attack on the histidine G2, which is the Bohr proton site. This should not be unexpected, as the direct titration of the Bohr proton binding, His-G2, has been detected by N M R in the diamagnetic carbonmonoxy CTTs [6,[9][10][11]261. Since the chemical shift change with protonation for the His C-2 proton is almost 1 ppm, the absence of detectable line-broadening for this proton resonance dictates proton exchange at a rate much faster than the observed t+r transition.…”

“…This allosteric transition can be simply formalized as shown in Eqn (1) t e r + H + (1) where the two conformation states are designated as t (tense) and r (relaxed). The binding site of the Bohr proton which controls the t e r interconversion has been proposed to be the imidazole of His-G2 in CTT I11 [9]. Proton nuclear magnetic resonance is a particularly convenient probe for detecting pHmodulated structural changes in the heme pocket.…”

Isomeric incorporation of the haem group into two monomeric haemoglobins of Chironomus thummi thummi

“…Larvae of one particular subspecies Chironomus thummi thummi were grown in our laboratory. The components III and IV were isolated according to the procedure of Sick et al [7] . A final step of purification by isoelectrofocusing has been added.…”

Reduced Bohr Effect in NO‐Ligated Chironomus Haemoglobin

“…binding site was identified by proton-magneticresonance (PMR) studies of the titratable histidine residues of Chironomus haemoglobin 111. CO-ligation produces a shift in p K of one of these histidines [7].As NO-haemoglobin is considered as a n analogue of oxygenated haemoglobin [8,9,10] and is accessible to ESR spectroscopy whereas oxygenated haemoglobin is not, the present investigation was intended to demonstrate the Bohr effect in case of NO-haemoglobin by a comparative study of the pHdependent ESR spectra and the C-2 PMR spectra of His-G2 of Chironomus haemoglobin 111. However, although the three individual haemoglobins investigated are known to differ markedly between themselves with regard to the pH-dependence of their 02-binding properties, only very small differences and no pH-dependent changes of the ESR-spectra of the NO-derivatives were observed.…”

“…binding site was identified by proton-magneticresonance (PMR) studies of the titratable histidine residues of Chironomus haemoglobin 111. CO-ligation produces a shift in p K of one of these histidines [7].…”