The Bohr Proton‐Binding Site in a Monomeric Haemoglobin

Abstract

Individual haemoglobin III from Chironomus thummi thummi is a monomeric protein (Mr= 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‐G19 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 pK 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 at 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 at 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.In 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.