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

Abstract

The monomeric insect (Chironomus thummi thummi) haemoglobins CTT 111 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 111. In particular, at low pH a time-dependent 'slow' decrease in p s o upon cyclic oxygenation/deoxygenation is observed which is larger if dithionite, instead of ascorbate, is the reducing agent. The decrease of p s o (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 p.30 by two to three orders of magnitude. Neither central metal nor vinyl replacement affect the Bohr effect.The natural CTT Hbs 111 and IV analyzed for mono-componential kinetic systems exhibit pH-dependent O2 off-rate constants: 300 s-' (at pH 5.6) and 125 s-l (at pH 9.7) for CTT 111, and 550 s-l (at pH 5.4) and 100 s-' (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 111. In contrast, the O2 on-rate constants are pH-independent (Icon = 1.15 -1.26 x lo8 M-' s-l). 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 qy-meso axis; y is the The monomeric insect haemoglobins CTT I11 and CTT IV exhibit a Bohr effect [l -31 and therefore serve as simple allosteric model systems [4, 51. Changes in ligation or in pH have no effect on the molecular mass of these haemoglobins which are therefore monomeric under all conditions [2, 61. The pH-dependent ligand afinity is controlled by a single proton (Bohr proton) [2,3,7 -91. NMR titration experiments [7] and histidine assignments based on X-ray structure analysis [lo] provided evidence that the allosteric site in CTT 111 (Bohr proton binding site) is a salt bridge formed by the imidazole group of His-G2 and the C-terminal carboxyhc group of Met-H22. At low pH, this salt bridge stabilizes the tense tertiary structure (t state) characterized by low O2 affinity. At high pH, by dissociation of the Bohr proton, the salt bridge is opened leading to a transition of the terti...

Allosteric Regulation and Temperature Dependence of Oxygen Binding in Human Neuroglobin and Cytoglobin

“…This observation is consistent with the invariance of resonance Raman and visible spectra of the CO derivative within the same pH range (9,34,39). Also, in the monomeric globin of the insect Chironomus thummi thummi, the Bohr effect is larger when O 2 rather than CO is the heme ligand (40). These observations support the conclusion that in NGB the heme-bound O 2 , which in globins, including NGB (9), has a Fe(III)O 2 .…”

Structural Dynamics in the Active Site of Murine Neuroglobin and Its Effects on Ligand Binding

“…Both globins also have a positively charged amino acid (arginine instead of lysine) at position 67. Remarkably, as in murine Ngb, the heme binds to the globin in two orientations in Chironomus Hb (42,43).…”

Insulin & Related Proteins - Structure to Function and Pharmacology

“…Differences of possible physiological relevance have been reported for two other heme proteins, however. A difference of 27 mV in the reduction potential of the two isomers of cytochrome has been found (Walker et al, 1988), and the Bohr effect of Chironomus thummi thummi hemoglobin has been shown to be different for the two isomers (Gersonde et al, 1986).…”