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

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 degrees 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 degrees C are: inflection point of the Bohr-effect curve at pH 7.1, number of Bohr protons -- deltalog p1/2 (O2)/deltapH = 0.36 mol H+/mol O2 and amplitude of the Bohr-effect curve deltalogp1/2 (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 degrees C which follows the order: proto less than meso less than 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 gparallel = 2.035 and by the low-field shift of gparallel. The ESR spectra of oxy cobalt proto- and oxy cobalt meso-CTT IV are dependent of pH. The cobalt hyperfine constants coinciding with gparallel - 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 gparallel. 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.

Bohr effect in monomeric insect haemoglobins controlled by O2 off-rate and modulated by haem-rotational disorder

“…Both CTT I11 and CTT IV show only an alkaline Bohr effect with an inflection point in the range of pH 7.2-7.4. The pK shifts induced by ligand binding and monitored at the Bohr proton site correspond with the amplitudes of the Bohr effect curves [5,7] (see also Table 2 in [12]). The lack of the distal histidine in these haemoglobins is a further advantage for model studies, as polar (i.e.…”

Proton-magnetic-resonance investigation of the dynamics of the conformational transition in allosteric monomeric insect hemoglobins

“…dvl, is the chemical shift difference between t and r states at 25°C; its value is converted to Hz for use in Eqn (3) by multiplying by 200 for deoxy CTTs and by 360 for all cyano-met CTTs. k,, was computed using Eqn (7) Hemoglobin Heme Solvent The peak from the minor component, 2-H: marked by an asterisk, was not included in the simulation first approximation we are observing reversible first-order processes for all of our systems. Under these circumstances, it is most convenient to compare the rates of t e r interconversion among the various CTT hemoglobins at the pK, for which Eqn (3) reduces V; = f r = 0.5, 7, = z, = z) to:…”

“…Nevertheless, the rate of cyano-met CTT IV is slightly faster by a factor of < 2 than that of cyano-met CTT 111. Since CTT IV exhibits a larger Bohr effect than CTT I11 [4,5,7,81 and for each hemoglobin component the heme orientation A possesses the larger Bohr effect [7], there appears to be no simple correlation between the amplitude of the Bohr effect curve and the rate of the t$r transition.…”

“…Comparison of the relative magnitudes of the 'H-NMR spectral changes [lo-141 with the amplitudes of the Bohr effect curve as well as the variation of the pKvalue of the observed spectral transition allows the association of such processes with the Bohr proton. Thus, the transition described in Eqn Changes in 'H-NMR [6, 9 -141 and electron spin resonance (ESR) [7] spectral parameters for the various complexes upon raising the pH give clear evidence for the structural transition with variable pK.…”

“…The Bohr effect is controlled by a single proton (Bohr proton). The binding of the Bohr proton induces a tertiary conformational transition from r (high-pH form, high-affinity state) to t (low-pH form, low-affinity state) [7, 81. This allosteric transition can be simply formalized as shown in Eqn (1) t e r + H + where the two conformation states are designated as t (tense) and r (relaxed).…”