Isomeric Incorporation of the Haem into Monomeric Haemoglobins of Chironomus thummi thummi

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Abstract
The Bohr effect in the monomeric hemoglobin component III of C. t. thummi is described on a molecular basis using various pH-dependent proton resonances. One of the 3 titratable histidines changes its pK from 7.9 in unligated state to 7.7 upon ligation with CO and up to 7.0 upon ligation with O2. From an analysis of the C-2 proton resonance intensities these shifts in pK occur in only 1 of the 2 conformational isomers of the component III, whereas in the 2nd 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. The corresponding signal intensities indicate the presence of 2 conformational isomers in the hemoglobin 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 is 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 the conformational changes in the Bohr active isomer of the component III is explained. 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.