Two monomeric haemoglobins of the insect Chironomus thummi thummi are described which, though their 0,-binding curves are exactly hyperbolic (Hill coefficient n = 1.00), do exhibit a Bohr effect (haemoglobin 111, 0.28 at 25 "C; haemoglobin IV, 0.42 at 25 "C). Thus, these haemoglobins can exist in two conformational states differing in 0,-affinity. The states are characterized also by different A S values ; the acid conformation with low affinity possessing a low, the alkaline conformation with high affinity a higher A X value.I n this paper the Bohr effect is regarded from generalizing aspects, i. e. as an interaction between the proton-binding site and the 6th coordination point of the iron which can therefore be studied as well with oxidised haemoglobin. I n the case of the oxidised form of haemoglobin the allosteric interaction between the proton-binding site and the coordinated H,O was the basis of our experiments.Absorbance and ellipticity of the oxidised form of haemoglobin measured at different wavelengths as a function of pH result in titration curves differing in the position of their inflection points. On the basis of the circuit process described previously [21], the percentage distribution of the constituent conformation isomers according to pH and the allosteric pK values (pKa:! = 7.30; pKal = 7.00) were calculated.In electron spin resonance spectroscopy the heterotropic allosterism is revealed by a change in the symmetry of the intramolecular electric field of the H,O-derivative. The acid conformation with rhombic symmetry ( g i l # 912) is transformed into the alkaline conformation with axial symmetry. The acid conformation is further characterized by five hyperfine structure lines in the gill signal, showing the d-electrons to interact with two N-nuclei in the z-direction. The hyperfine structure lacking in the alkaline conformation indicates a larger distance of the proximal imidazole from the iron. The pH-dependent variation of this distance controlls the affinity of the sixth ligand.A possible molecular mechanism of the Bohr effect of haemoglobin I11 is described, considering the interactions, revealed by the atomic model [8].It is shown that the Bohr effect mechanism in Chironomus haemoglobin is based on the same conditions of tertiary structure as in the case of vertebrate haemoglobins [ 7 ] . These conditions of tertiary structure, however, are realized with completely differing primary structures.The oxygen-binding properties of isolated haemoglobins of Chironomus has recently been described by several authors [l -31. Chromatographically isolated haemoglobins of Chironomus thummi thummi were shown by Sick and Gersonde [l] to possess hyperbolic 0,-binding curves and more or less strong Bohr effects. Formanek and Amit [2], however, failed to find a Bohr effect for haemoglobin 111, the individual haemoglobin characterized by sequence [4] and X-ray structure analysis [ 5 ] . Lampe and coworkers [3] have found Hill coefficients up to n = 2 a t pH 9 for haemoglobins of Chironomus plumosus, und...