Conformation-induced circular dichroism in hemoglobin of Chironomus th. thummi

Abstract

The conformational isomers of Chironomus haemoglobin first postulated on the strength of the O2‐binding properties [24] and demonstrated by electron spin resonance spectroscopy [23] could now be confirmed by measurement of the circular dichroism.All the circular dichroism bands of the oxidized haemoglobin from Chironomus in the visible and Soret region are induced or influenced conformationally. The pH‐dependency of the extinction and the ellipticity of the individual bands follows a circuit process with four reaction steps, two of which describe the allosteric transformation and two the protolytic reaction of water at the 6th coordination point of the haem iron. With the aid of fit calculations we get the protolytic constants pK′2= 8.1 and pK”2= 8.3 and the allosteric constants pKa1= 7.3 and pKa2= 7.1.The existence of conformationally induced circular dichroism bands becomes obvious, if one compares the separated haemoglobins showing different degrees of allosterism. Thus for instance the individual haemoglobin IV, showing the smallest amplitude of the Bohr effect, is characterized by a minimal conformationally‐induced change of ellipticity. The individual haemoglobin VIII with the highest amplitude of Bohr effect shows the strongest conformationally‐induced circular dichroism bands.The pH‐dependency of the absorption spectra in the visible range displays further characteristics of conformational isomerism. The 4‐component system to be expected from the circuit process reveals at sufficient distance between the allosteric and protolytic pK values a system of two 2‐component mixtures, which can be transformed into each other depending on pH and thus produce “isosbestic triangles”.The helix content of the Chironomus haemoglobins is not influenced by the described change of conformation and amounts to 60 and 70% according to the individual haemoglobins.The results are discussed under the aspect of isomerism of conformations and allosterism controlled by the trans‐effect of the 5th ligand of the haem iron.