Solution 1H‐NMR studies of the heme cavity were performed for the cyanomet complexes of monomeric hemoglobins III and IV from the insect Chironomus thummi thummi, each of which exhibit marked Bohr effects. The low pH 5, paramagnetic (S= 1/2) derivatives were selected for study because the large dipolar shifts provide improved resolution over diamagnetic forms and allow distinction between the two isomeric heme orientations [Peyton, D. H., La Mar, G. N. & Gersonde, K. (1988) Biochim. Biophys. Acta 954, 82–94]. The crystal structure for the low‐pH form of the hemoglobin III derivative, moreover, has been reported and showed that the functionally implicated distal His58 side chain adopts alternative orientations, either in or out of the pocket [Steigemann, W. & Weber, E. (1979) J. Mol. Biol. 127, 309–338]. All heme pocket residues for the low‐pH forms of the two hemoglobins were located, at least in part, and positioned in the heme cavity on the basis of nuclear Overhauser effects to the heme and each other, dipolar shifts, and paramagnetic‐induced relaxation. The resulting structure yielded the orientation of the major axis of the paramagnetic susceptibility tensor. The heme pocket structure of the cyanomet hemoglobins III and IV were found to be indistinguishable, with both exhibiting a distal His58 oriented solely into the heme cavity and in contact with the ligand, and with two residues, Phe100 and Phe38, exhibiting small but significant displacements in solution relative to hemoglobin III in the crystal.

Free radicals have been postulated to play an important role as mediators in the pathogenesis of shock syndrome and multiple-organ failure. We attempted to directly detect the increased formation of radicals by Electron Spin Resonance (ESR) in animal models of shock, namely the endotoxin (ETX) shock or the hemorrhagic shock of the rat. In freeze-clamped lung tissue, a small but significant increase of a free radical signal was detected after ETX application. In the blood of rats under ETX shock, a significant ESR signal with a triplet hyperfine structure was observed. The latter ESR signal evolved within several hours after the application of ETX and was localized in the red blood cells. This signal was assigned to a nitric oxide (NO) adduct of hemoglobin with the tentative structure [alpha 2+ NO)beta 3+)2. The amount of hemoglobin-NO formed, up to 0.8% of total hemoglobin, indicated that under ETX shock a considerable amount of NO was produced in the vascular system. This NO production was strongly inhibited by the arginine analog NG-monomethyl-arginine (NMMA). The ESR signal of Hb-NO was also observed after severe hemorrhagic shock. There are three questions, namely (i) the type of vascular cells and the regulation of the process forming such a large amount of NO during ETX shock, (ii) the pathophysiological implications of the formed NO, effects which have been described as cytotoxic mediator, endothelium-derived relaxing factor (EDRF) or inhibitor of platelet aggregation, and (iii) the possible use of Hb-NO for monitoring phases of shock syndrome.

the porphyrin system and the hindering distal amino acid side group to change their positions so that the whole ligandheme-protein system again reaches an energy minimum. In contrast to a ligand-heme model system, the structure of liganded hemoglobin is extremely sensitive to temperature and electrostatic interaction (variations in salt concentration and pH). Bond length and binding geometry may therefore be sensitive indicators of the actual state of the ligandheme-protein interactions in hemoglobins. Since the numerous conformations of this system may be removed in a crystal, one needs more information about these subtle interactions in solution and under physiological conditions. At present, there is evidence that the fine-structure of CTT III in solution is not the same as it is in crystals. By proton NMR in solution (8,19), CTT III has been shown to exist in two forms, which differ in heme orientation, related by an t180°r otation about the a,-y-meso axis at least in the metHbCN

The GO binding properties of monomeric (Hb I11 and IV), dimeric, and tetrameric Chironomus haemoglobins as well as of the monomer-analogue valence hybrid (aFmet /Imet /Ideox') of Hb M Iwate were studied and compared with the respective 0, binding properties. All these haemoglobins show hyperbolic 0, binding curves and a Bohr effect. The amplitudes of the 0, Bohr effect curves range from dlog pO,(,,r, = 0.3 (Hb 111) to 2.4 (tetrameric Chironomus haemoglobin).The ligands CO and 0, differ not only in affinity but also with regard to the Bohr effect. The GO affinities of the various Chironomus haemoglobins are 60 to 490 times larger than the 0, affinities and the amplitudes of the CO Bohr effect curves are 1.25 to 8 times smaller than those of the respective 0, Bohr effect curves. I n the case of Hb M Iwate, however, the CO Bohr effect is larger than the 0, Bohr effect by the factor 1.25 (2,3-bisphosphoglycerate-reacted H b M) and 1.4 (stripped Hb M).The ligand-specific differences in the magnitude of the Bohr effect cannot be explained solely by the allosteric interaction connected with the trans-effect of the complex. The differences are discussed in terms of steric influences on the binding geometry of the 6th ligand by protein side chains a t the distal side of the haem group. The titration behaviour of the three titratable histidine residues of H b I11 was studied by proton magnetic resonance (PMR) spectroscopy [3]. Ligation with CO produces a shift of the pK value of the histidine G2. This residue was identified, therefore, as the Bohr proton-binding site. As the PMR measurements required periods of several hours at 30 "C, it was not possible to use 0, as 6th ligand because of autoxidation of the haemoglobin. However, the kind of ligand bound to the 6th position seems to play an important role with respect to the extent of the pH-dependent change of the affinity for, in contrast to the experiments with CO, the PMR studies on ferric and ferrous H b I I I , ligated with Dedicated to Prof. Dr Hans Netter (Institut fiir Physiologische Chemie und Physikochemie der Christian-AlbrechtsUniversitat zu Kiel) on the occasion of his 75th birthday.Abbreviations. Hb I, Hb I11 and Hb IV, monomeric haemoglobins I, I11 and IV from Chironomus thummi thummi; Hb M Iwate, human haemoglobin M Iwate; PMR, proton magnetic resonance. The CO equilibrium studies were extended also to other monomeric, dimeric and tetrameric haemoglobins of Chironomus and to a mutant tetrameric haemoglobin (Hb M Iwate) which is characterized by one or two GO binding sites, respectively [6,7]. CN-MATERIALS AND METHODS HaemoglobinsMonomeric haemoglobin I, 111, and IV fromChironornus thummi thummi were isolated as described in [3].Eur. J. Biochem. 45 (1974)

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