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Bohr-effect and pH-dependence of electron spin resonance spectra of a cobalt-substituted monomeric insect haemoglobin

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Abstract

The monomeric haemoglobin IV from Chironomus thummi thummi (CTT IV) exhibits an alkaline Bohr-effect and therefore it is an allosteric protein. By substitution of the haem iron for cobalt the O2 half-saturation pressure, measured at 25‡ C, increases 250-fold. The Bohr-effect is not affected by the replacement of the central atom. The parameters of the Bohr-effect of cobalt CTT IV for 25‡ C are: inflection point of the Bohr-effect curve at pH 7.1, number of Bohr protons -δlog p1/2 (O2)/gDpH=0.36 mol H+/mol O2 and amplitude of the Bohr-effect curve δlog p1/2 (O2)=0.84. The substitution of protoporphyrin for mesoporphyrin causes a 10 nm blue-shift of the visible absorption maxima in both, the native and the cobalt-substituted forms of CTT IV. Furthermore, the replacement of vinyl groups by ethyl groups at position 2 and 4 of the porphyrin system leads to an increase of O2 affinities at 25‡ C which follows the order: proto < meso < deutero for iron and cobalt CTT IV, respectively. Again, the Bohr-effect is not affected by the replacement of protoporphyrin for mesoporphyrin or deuteroporphyrin. The electron spin resonance (ESR) spectra of both, deoxy cobalt proto- and deoxy cobalt meso-CTT IV, are independent of pH. The stronger electron-withdrawing effect by protoporphyrin is reflected by the decrease of the cobalt hyperfine constants coinciding with g∥=2.035 and by the low-field shift of g∥. The ESR spectra of oxy cobalt proto- and oxy cobalt meso-CTT IV are dependent of pH. The cobalt hyperfine constants coinciding with g∥=2.078 increase during transition from low to high pH. The pH-induced ESR spectral changes correlate with the alkaline Bohr-effect. Therefore, the two O2 affinity states can be assigned to the low-pH and high-pH ESR spectral species. The low-pH form (low-affinity state) is characterized by a smaller, the high-pH form (high-affinity state) by a larger cobalt hyperfine constant in g∥. The correlation of the cobalt hyperfine constants of the oxy forms with the O2 affinities is discussed for several monomeric haemoglobins. The Co-O-O bond angle in cobalt oxy CTT IV is characterized by an ozonoid type of binding geometry and varies little during the pH-induced conformation transition. Due to the lack of the distal histidine in CTT IV no additional interaction via hydrogen-bonding with dioxygen is possible; this is reflected by the cobalt hyperfine constants.

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  1. Abteilung Physiologische Chemie, Rheinisch-WestfÄlische Technische Hochschule Aachen, Schneebergweg 213, D-5100, Aachen, Federal Republic of Germany

    K. Gersonde, H. Twilfer & M. Overkamp

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  1. K. Gersonde
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  2. H. Twilfer
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  3. M. Overkamp
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Gersonde, K., Twilfer, H. & Overkamp, M. Bohr-effect and pH-dependence of electron spin resonance spectra of a cobalt-substituted monomeric insect haemoglobin. Biophys. Struct. Mechanism 8, 189–211 (1982). https://doi.org/10.1007/BF00535459

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