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...

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.

Two hybrid species of haemoglobin M Iwate exist: apmet/?metgdeoxy and a p m e t j 3 p .These species differ in their ligand and effector binding properties.The apmet@metadeoxy hybrid is characterized by a Bohr effect, while the Hill coefficient is n = 1.00. The energy of the interaction between the Bohr protons and the ligand-binding site corresponds to the interaction energy found in monomeric haemoglobins and amounts to 0.55 kcal/mol. One molecule of 2,3-bisphosphoglycerate per mole is bound a t neutral pH, while a t alkaline pH two co-operative molecules are bound. Binding of 2,3-bisphasphoglycerate reduces the ligand affinity and shifts the pK value of the Bohr proton binding site to the alkaline region.hybrid shows a pH-dependent co-operativity of the ligand-binding sites ( n = 1.8 a t pH 9). The co-operativity is weakened by binding of protons or 2,3-bisphosphoglycerate. The latter lowers the ligand affinity, but in the alkaline region no influence is observed. Furthermore the Bohr effect curve is shifted to the alkaline region by this compound but the amplitude remains unchanged.The results of the present paper are interpreted in terms of intra-chain and /?-b inter-chain interactions, the latter being responsible for a consecutive change of the ligand affinity in a "frozen" T-state molecule. has shown that in the met form as well as in the deoxy form of Hb M Iwate helix E of the a-chains is shifted by approximately 0.2 nm in direction of the haem group. His-E7(58) occupies the 6th coordination site, while Tyr-FS is bound a t the 5th position of the iron. The latter can be followed from the low affinity of the a-chains towards external ligands [i, 5,6]. As the iron of the mutant a-chains remains ferric in vivo, 0, and CO are bound only to the haem groups of the ,&chains. The 0,-affinity, however, is much lower than that of normal haemoglobin; the Abbreviations. H b A, haemoglobin A: Hb M Iwate, haemoglobin M Iwate; P,-glycerate, 2,3-bisphosphoglycerate; ESR, electron spin resonance; NMR, nuclear magnetic resonance. 0,-dissociation curve was found to be hyperbolic, and no Bohr effect was observed [5].It is generally accepted that the heterotropic and homotropic allosteric interactions in haemoglobins are coupled; that means, a loss of the Bohr effect is accompanied by a loss of the haem-haem interaction and vice versa [7,8]. However, the Bohr effect of the mammalian haemoglobin is complicated, because the C-termini are involved in inter-chain salt bridges. Intra-and interchain interactions are coupled, therefore, in this tetrameric haemoglobin.

A monomeric allosteric haemoglobin from Chironomus thummi thummi was reconstituted with 57Fe-haem. This reconstituted haemoglobin was found to be identical to the non-reconstituted material with regard to the O2-binding properties and the visible spectra. The 270 MHz proton magnetic resonance of the bis(cyano)-57Fe-haemin shows that the reconstituted haem is identical with the non-reconstituted haem. Furthermore it has been proved by proton magnetic resonance that in Chironomus haemoglobins the prosthetic group is proto-haem IX. The ESR spectrum of the native nitrosyl haemoglobin demonstrates rhombic symmetry of the haem iron (gxx = 2.086, gyy=1.981, gzz=2.005) and hyperfine structures at gyy (aNε = 1.35 mT) and at gzz (a15NO = 3.05 mT, a14NO = 2.19 mT, aNε=0.715mT, a57Fe = 0.38 mT). The spectrum is independent of pH and can be classified as a type II spectrum following the classification of ref. 2. NO-binding obviously stabilizes the tertiary structure of this haemoglobin in a “tense” conformation with a relatively strong o bond of the 5th ligand (Nε of imidazole) and a relatively weak o bond of the 6th ligand (NO). Reaction of this haemoglobin with anionic, cationic and non-ionic detergents, respectively, leads to a transformation of the NO-ligated form into a “relaxed” conformation with a stretched or broken a bond of the 5th ligand (Nε of imidazole) and a strong σ bond of the 6th ligand (NO). The ESR spectrum of this modified NO-haemoglobin shows again a rhombic symmetry of the haem iron (gxx = 2.10, gyy = 2.06, gzz=2.010), but dramatically changes in the g tensors (low field shift), hyperfine structures and hyperfine splitting constants (a15NO=2.32 mT, a14 NO = 1.66 mT, a57Fe = 0.48 mT). The hyperfine splitting is isotropic. Transition from the “tense” conformation to the “relaxed” conformation corresponds with an increase of the spin density at the iron atom by 26% and a decrease of the spin density at the NO ligand by 25%. The spin density at the Nε of imidazole strongly decreases in the “relaxed” conformation, so that a hyperfine splitting of this ligand is not any more resolved. These results demonstrate the trans-effect of the proximal imidazole which in haemoglobins controls the binding properties of the external ligand in trans-position.