Isomeric incorporation of the haem into monomeric haemoglobins of Chironomus thummi thummi 3. Comparative study of components, I, III and IV.

13C-NMR studies on 13CO complexes of the components III and IV of the monomeric haemoglobins from the subspecies Chironomus thummi thummi have been carried out in order to confirm the existence of two conformational isomers differing by the isomeric incorporation of the haem group and the extent of the Bohr effect. In addition, the allosteric linkage between the ligand binding site and the Bohr proton binding site in the component IV is described from investigations of various pH-dependent proton resonances including the C-2 proton resonances of the titratable histidines. In comparison to the data obtained for the component III it is assumed that in both conformational isomers of the component IV the allosteric linkage between the distal site of the haem and the main Bohr proton donating group is present. From corresponding NMR investigations of the component I an isomeric incorporation of the haem group into this monomeric haemoglobin seems unlikely. Also, any correlation between the ligand binding site and a titratable group of the protein had not been found in this haemoglobin, in agreement with previous results of other laboratories that a Bohr effect in the component I cannot be detected.

Isomeric incorporation of the haem into monomeric haemoglobins of Chironomus thummi thummi. 1. Isolation of chemically homogeneous haemoglobins. Evidence for the isomerism of the haem in the component III.

With high-resolution NMR spectroscopy it has been possible to detect various types of isomerisms in the monomeric component III of the haemoglobins of Chironomus thummi. In the component III prepared from commercially available larvae of Ch. thummi a chemical heterogeneity has been found to occur at the position E6 (Ile or Thr). The imidazole C-2 proton resonance of the adjacent His-E7 is split because of this alternative occupancy. From larvae of an inbreeding of the single subspecies Chironomus thummi thummi a component III material has been isolated in which the position E6 is occupied by one single amino acid. An additional isomerism of this haemoglobin III is produced by an isomeric incorporation of the haem group into the haem pocket. This isomerism has been established from the complex signal pattern of the mesoproton resonances of the diamagnetic ligated haemoglobin III. Other evidences of this isomerism have been obtained from an analysis of th high-field proton resonances.

Isomeric incorporation of the Haem into monomeric haemoglobins of Chironomus thummi thummi. 2. The Bohr effect of the component III explained on a molecular basis and functional differences between the two isomeric structures.

The Bohr effect in the monomeric haemoglobin component III of Chironomus thummi thummi is described on a molecular basis using various pH-dependent proton resonances. One of the three titratable histidines changes its pK from 7.9 in the unligated state to 7.7 upon ligation with CO and up to 7.0 upon ligation with O2. It can be concluded from an analysis of the C-2 proton resonance intensities that these shifts in pK occur in only one of the two conformational isomers of the component III, whereas in the second isomer these pK shifts are not observed. The differences between these functional properties are also derived from pH-dependent changes of the mesoproton resonances as well as of the methyl resonances of Ile-E11. The conformational change of the component III, which is connected with the Bohr effect, influences a variety of other resonances which could not be assigned. The pK values derived from the pH dependence of these changes agree exactly with the pK value of the Bohr proton donating group, which is supposed to be His-G2. Also the corresponding signal intensities indicate the presence of two conformational isomers in the haemoglobin component III. Although the change in chemical shift of the His-G2 C-2 proton resonance is only small upon deprotonation of the imidazole ring, it has been shown with potentiometric and calorimetric methods that this histidine is deprotonated in this pH range. Considering the tertiary structure obtained from an X-ray study of Steigemann and Weber we can readily explain the conformational changes in the Bohr active isomer of the component III. With binding of ligands dislocations of side chains at the proximal site produce a change in the interactions of His-G2 with the carboxyl group of the C-terminal methionine-H22.