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

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The monomeric insect (Chironomus thummi thummi) haemoglobins CTT III and CTT IV show an alkaline Bohr effect. The amplitude of the Bohr effect curve of CTT IV is about twice as large as that of CTT III. In particular, at low pH a time‐dependent ‘slow’ decrease in p50 upon cyclic oxygenation/deoxygenation is observed which is larger if dithionite, instead of ascorbate, is the reducing agent. The decrease of p50 (increase in affinity) correlates with the ratio of haem‐rotational components exhibiting an increase of the ‘myoglobin‐like’ haemrotational component with high O2 affinity and high stability of the globin‐haem complex. The replacement of protohaem IX by mesohaem IX and deuterohaem IX, respectively, causes an increase in O2 affinity following the order: proto < meso < deutero CTT Hbs. The Bohr effect, however, seems not to be affected by these porphyrin side‐group substitutions. The O2 affinity is modulated by steric effects due to the substituents in position 2 and 4 via variation of the protein‐haem interactions which influence the O2 release. The replacement of iron by cobalt in proto and meso CTT IV leads to an increase of the p50 by two to three orders of magnitude. Neither central metal nor vinyl replacement affect the Bohr effect. The natural CTT Hbs III and IV analyzed for mono‐componential kinetic systems exhibit pH‐dependent O2 off‐rate constants: 300 s−1 (at pH 5.6) and 125 s−1 (at pH 9.7) for CTT III, and 550 s−1 (at pH 5.4) and 100 s−1 (at pH 9.0) for CTT IV. Inflection points and amplitudes of the log koff/pH plots correspond to those obtained from the Bohr effect curves indicating again a larger Bohr effect for CTT IV than for CTT III. In contrast, the O2 on‐rate constants are pH‐independent (kon= 1.15—1.26x108 M−1 s−1). Thus, the Bohr effect is completely controlled by the off‐rate constants. Analysis for bi‐componential kinetic systems employing the eigenfunction expansion method clearly identifies two kinetic components for proto‐IX and deutero‐IX CTT Hbs which can be attributed to the two haem‐rotational components x and y (x and y differ due to an 180° rotation of the haem group about the α,γ‐meso axis; y is the myoglobin‐like haem‐rotational component). Meso‐IX CTT Hbs show only one kinetic component, although two haem‐rotational components have been manifested by NMR. After replacement of protohaem IX in these haemoglobins by the ‘symmetric’ protohaem III or protohaem XIII only one kinetic component appears. The orders of koff values [proto‐IX(x) ∼ proto‐XIII > proto‐III ≧ proto‐IX(y) CTT Hbs for low pH and proto‐IX(x) ≧ proto‐XIII > proto‐III ∼ proto‐IX(y) CTT Hbs for high pH] reflect the modulation of koff, O2 affinity, and Bohr effect exerted by specific contacts of the porphyrin substituents in position 1–4 with protein sites named A, B, C and D. The permutations of the porphyrin substituents in position 1–4 result in characteristic changes of the interactions at the protein sites A, B, C and D. The globin‐haem complex stability is largest and the koff value is smallest, if a vinyl, i.e. in proto‐IX(y) and proto‐III CTT Hbs, interacts with site C. The Bohr effect is largest if a vinyl, i.e. in proto‐IX(x) and proto‐III CTT Hbs, interacts with site B. Thus, the contact of vinyl with site B is an indicator of the t⇌r conformation transition. Copyright © 1986, Wiley Blackwell. All rights reserved

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European Journal of Biochemistry

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