Heterotropic Allosterism of Monomeric Haemoglobins of Chironomus thummi thummi

Abstract

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...

Resonance Raman evidence for the mechanism of the allosteric control of O2-binding in a cobalt-substituted monomeric insect hemoglobin

“…Monomeric CTT III from the lymph of insect larvae of Chironomus thummi thummi was purified and identified as described elsewhere (7,21,30). The hemoglobin was desalted, lyophilized, and stored at -300C in the met form.…”

“…A single proton (Bohr proton) controls the binding of dioxygen (21). The changes in ligand affinity occuring over a range from pH 5 to 10 can be exactly described by the Bohr-effect curve (log P50-vs. -pH plot) (7). Inflection points of the Bohr-effect curves at pH 7.1-7.4 indicate an alkaline Bohr effect.…”

“…Extremely high affinities have been observed for liver fluke hemoglobin (Dicrocoelium dibranchiata hemoglobin) (1,2) and for leghemoglobin (3)(4)(5), which are both monomeric. The monomeric (6,7) and the dimeric (6,8,9) Chironomus hemoglobins also exhibit considerably high dioxygen affinities, whereas low dioxygen affinities have been found for the tetrameric vertebrate hemoglobins ( 10).…”

“…The Bohr effect of CTT IV is larger than that of CTT III (7). A large body of structural (17,18), thermodynamic (6,7,19,20), and spectroscopic (21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35) data already exists for these monomeric insect hemoglobins. The influence of heme metal exchange and of porphyrin side group substitutions on the Bohr effect of these hemoglobins has also been investigated (25,36).…”