Cooperative effect of inositol hexakisphosphate, bezafibrate, and clofibric acid on the spectroscopic properties of the nitric oxide derivative of ferrous human hemoglobin

The cooperative effect of inositol hexakisphosphate (IHP), bezafibrate (BZF), and clofibric acid (CFA) on the spectroscopic (EPR and absorbance) properties of the nitric oxide derivative of ferrous human hemoglobin (HbNO) has been investigated quantitatively. In the presence of IHP, BZF, and CFA, the X-band EPR spectra and the absorption spectra in the Soret region of HbNO display the same basic characteristics described in the presence of 2,3-diphosphoglycerate (2,3-DPG), which have been attributed to a low affinity conformation of the tetramer. Addition to HbNO of two allosteric effectors together (such as IHP and BZF, or IHP and CFA) further stabilizes the low affinity conformation of the ligated hemoprotein (i.e., HbNO). Moreover, in the presence of saturating amounts of IHP, the affinity of BZF and CFA for HbNO increases by about fifteenfold. Likewise, in the presence of both IHP and BZF, as well as in IHP and CFA, the oxygen affinity for ferrous human hemoglobin (Hb) is reduced with respect to that observed in the presence of IHP, BZF, or CFA alone, which in turn is lower than that reported in the absence of any allosteric effector. All the data were obtained at pH 7.0 (in 1.0 × 10⁻¹ M N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid]/NaOH buffer system plus 1.0 × 10⁻¹ M NaCl), as well as at 100 K and/or 20°C. The results here reported represent clearcut evidence for the cooperative and specific (i.e., functionally relevant) binding of IHP, BZF, and CFA to Hb.     

Influence of allosteric effectors on the heme conformation of dromedary ferrous nitrosylhemoglobin detected by XANES spectroscopy

The changes of the Fe heme-active site conformation of dromedary (Camelus dromedarius) nitrosylhemoglobin (HbNO) induced by inositol hexakisphosphate (IHP) and chlofibric acid (CFA) have been studied by using X-ray absorption near-edge structure (XANES) spectroscopy. Structural information has been determined by multiple scattering analysis of the Fe K-edge XANES spectra. The proximal histidine is found to move away from iron centers by about 0.4 Angstrom on the average over the four hemes upon binding of CFA or stoichiometric amount of IHP. In molar excess of polyanion or in the simultaneous presence of IHP, CFA and chloride, the proximal histidine moves back to a position very close to that observed in pure buffer; yet, the structure modulation induced by the allosteric effectors is not completely reversible. Such findings parallel with the functional properties and the spectroscopic (e.g., EPR and absorbance) characteristics of HbNO.     

Different structural effects of allosteric modulators on subunits of tetrameric ferrous nitrosylated human hemoglobin: an EPR spectroscopic study

 The X-band EPR spectroscopic features of the ferrous nitrosylated derivative of α(Fe)₂β(Co)₂ and of α(Co)₂β(Fe)₂ metal hybrids of human hemoglobin (Hb) have been investigated at pH 7.0 and analyzed in parallel with those of the native nitrosylated tetramer (HbNO). The effect of 2,3-biphosphoglycerate (BPG), inositol hexakisphosphate (IHP) and bezafibrate (BZF) has been investigated in order to understand the perturbations induced on α and β subunits in the tetramer by the binding of allosteric effectors. A large perturbation is observed in both subunits upon BZF binding, while in the case of IHP only α-chains are affected; on the other hand, BPG leaves both chains essentially unperturbed. Thus, different binding modes of allosteric effectors to HbNO may occur, and the simultaneous addition of two effector molecules, namely BPG and BZF or IHP and BZF to HbNO, brings about different alterations of the X-band EPR spectroscopic properties. This behavior indicates that the intramolecular communication pathway(s) between the heme and the binding pockets of the heterotropic ligands (i.e., IHP and BZF, or BPG and BZF) are different, leading to distinct structural perturbations. Received: 19 September 1997 / Accepted: 16 December 1997     

A biochemical--biophysical study of hemoglobins from woolly mammoth, Asian elephant, and humans

This study is aimed at investigating the molecular basis of environmental adaptation of woolly mammoth hemoglobin (Hb) to the harsh thermal conditions of the Pleistocene ice ages. To this end, we have carried out a comparative biochemical-biophysical characterization of the structural and functional properties of recombinant hemoglobins (rHb) from woolly mammoth (rHb WM) and Asian elephant (rHb AE) in relation to human hemoglobins Hb A and Hb A(2) (a minor component of human blood). We have obtained oxygen equilibrium curves and calculated O(2) affinities, Bohr effects, and the apparent heat of oxygenation (ΔH) in the presence and absence of allosteric effectors [inorganic phosphate and inositol hexaphosphate (IHP)]. Here, we show that the four Hbs exhibit distinct structural properties and respond differently to allosteric effectors. In addition, the apparent heat of oxygenation (ΔH) for rHb WM is less negative than that of rHb AE, especially in phosphate buffer and the presence of IHP, suggesting that the oxygen affinity of mammoth blood was also less sensitive to temperature change. Finally, (1)H NMR spectroscopy data indicates that both α(1)(β/δ)(1) and α(1)(β/δ)(2) interfaces in rHb WM and rHb AE are perturbed, whereas only the α(1)δ(1) interface in Hb A(2) is perturbed compared to that in Hb A. The distinct structural and functional features of rHb WM presumably facilitated woolly mammoth survival in the Arctic environment.     

Effect of inositol hexakisphosphate on the spectroscopic properties of the nitric oxide derivative of ferrous horse and bovine hemoglobin

The effect of inositol hexakisphosphate (IHP) on the spectroscopic (EPR and absorbance) properties of the nitric oxide derivative of ferrous horse and bovine hemoglobin (Hb) has been investigated. In the absence of IHP, the nitric oxide derivative of ferrous horse Hb shows spectroscopic properties similar to those of the corresponding derivative of ferrous human Hb that are generally taken as typical of the high affinity state of tetrametric hemoproteins. Similar to human Hb, the addition of IHP to the nitric oxide derivative of ferrous horse Hb induces a transition toward a species characterized by spectral properties typical of the low affinity state of hemoglobins. Nevertheless, the equilibrium constant for IHP binding to the nitric oxide derivative of ferrous horse Hb (= 1.5 x 10(2) M-1) is much lower than that reported for the association of the polyphosphate to the same derivative of ferrous human Hb (greater than 3 x 10(5) M-1). Conversely, the spectroscopic properties of the nitric oxide derivative of ferrous bovine Hb are characteristic of the low affinity state of tetrameric hemoproteins, both in the absence and in the presence of IHP. These results, taken together with the behavior of the nitric oxide derivative of ferrous human Hb, provide further evidence for the peculiar oxygen binding properties of horse and bovine Hb.     

Effect of bezafibrate and clofibric acid on the spectroscopic properties of the nitric oxide derivative of ferrous human hemoglobin.

The effect of bezafibrate (BZF) and clofibric acid (CFA) on the spectroscopic (EPR and absorbance) properties of the nitric oxide derivative of ferrous human hemoglobin (HbNO) has been investigated quantitatively. In the presence of BZF and CFA, the X-band EPR spectra and the absorption spectra in the Soret region of HbNO display the same basic characteristics described in the presence of inositol hexakisphosphate (IHP) and 2, 3-diphosphoglycerate (2,3-DPG). Next, in the presence of these allosteric effectors, the oxygen affinity for ferrous human hemoglobin (Hb) is reduced. These findings indicate that BZF and CFA, as already reported for IHP and 2, 3-DPG, induce the stabilization of a low affinity conformation of the ligated hemoprotein (i.e., HbNO). Values of the apparent equilibrium constant for BZF and CFA binding to HbNO (K) are 1.5(+/- 0.2) x 10(-2) M and 2.8(+/- 0.3) x 10(-2) M, respectively, at pH 7.0 (in 0.1 M N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]/NaOH buffer system plus 0.1 M NaCl) and 20 degrees C. The results reported here represent clearcut evidence for BZF and CFA specific (i.e., functionally relevant) binding to a ligated derivative of Hb (i.e., HbNO).     

T-quaternary structure of oxy human adult hemoglobin in the presence of two allosteric effectors, L35 and IHP

The cooperative O(2)-binding of hemoglobin (Hb) have been assumed to correlate to change in the quaternary structures of Hb: T(deoxy)- and R(oxy)-quaternary structures, having low and high O(2)-affinities, respectively. Heterotropic allosteric effectors have been shown to interact not only with deoxy- but also oxy-Hbs causing significant reduction in their O(2)-affinities and the modulation of cooperativity. In the presence of two potent effectors, L35 and inositol hexaphosphate (IHP) at pH 6.6, Hb exhibits extremely low O(2)-affinities (K(T)=0.0085mmHg(-1) and K(R)=0.011mmHg(-1)) and thus a very low cooperativity (K(R)/K(T)=1.3 and L(0)=2.4). (1)H-NMR spectra of human adult Hb with these two effectors were examined in order to determine the quaternary state of Hb in solution and to clarify the correlation between the O(2)-affinities and the structural change of Hb caused by the heterotropic effectors. At pH 6.9, (1)H-NMR spectrum of deoxy-Hb in the presence of L35 and IHP showed a marker of the T-quaternary structure (the T-marker) at 14ppm, originated from inter- dimeric α(1)β(2)- (or α(2)β(1)-) hydrogen-bonds, and hyperfine-shifted (hfs) signals around 15-25ppm, caused by high-spin heme-Fe(II)s. Upon addition of O(2), the hfs signals disappeared, reflecting that the heme-Fe(II)s are ligated with O(2), but the T-marker signals still remained, although slightly shifted and broadened, under the partial pressure of O(2) (P(O2)) of 760mmHg. These NMR results accompanying with visible absorption spectroscopy and visible resonance Raman spectroscopy reveal that oxy-Hb in the presence of L35 and IHP below pH 7 takes the ligated T-quaternary structure under the P(O2) of 760mmHg. The L35-concentration dependence of the T-marker in the presence of IHP indicates that there are more than one kind of L35-binding sites in the ligated T-quaternary structure. The stronger binding sites are probably intra-dimeric binding sites between α(1)G- and β(1)G-helices, and the other weaker binding site causes the R→T transition without release of O(2). The fluctuation of the tertiary structure of Hb seems to be caused by both the structural perturbation of α(1)β(1) (or α(2)β(2)) intra-dimeric interface, where the stronger L35-binding sites exist, and by the IHP-binding to the α(1)α(2)- (or β(1)β(2)-) cavity. The tertiary structural fluctuation induced by the allosteric effectors may contribute to the significant reduction of the O(2)-affinity of oxy-Hb, which little depends on the quaternary structures. Therefore, the widely held assumptions of the structure-function correlation of Hb - [the deoxy-state]=[the T-quaternary structure]=[the low O(2)-affinity state] and [the oxy-state]=[the R-quaternary structure]=[the high O(2)-affinity state] and the O(2)-affiny of Hb being regulated by the T/R-quaternary structural transition - are no longer sustainable. This article is part of a Special Issue entitled: Allosteric cooperativity in respiratory proteins.     

Antagonistic interaction between oxygenation-linked lactate and CO2 binding to human hemoglobin

Oxygen binding to hemoglobin (Hb) depends on allosteric effectors (CO(2), lactate and protons) that may increase drastically in concentration during exercise. The effectors share common binding sites on the Hb molecules, predicting mutual interaction in their effects on Hb (de)oxygenation. We analysed the effects of lactate and CO(2), separately and in combination, on O(2) binding of purified human Hb at 37 degrees C and physiological pH and chloride values. We demonstrate pH-dependent, inhibitory interactions between lactate binding and CO(2) binding (carbamate formation); at pH 7.4, physiological CO(2) tension ( approximately 43 mm Hg) reduced lactate binding more markedly ( approximately 75%), than lactate (50 mM) inhibited carbamate formation ( approximately 25%). In contrast to previous studies on blood and Hb solutions, we moreover find that added lactate neither 'reverses' oxylabile carbamate formation (resulting in lower carbamate levels in deoxyHb than in oxyHb) nor exerts greater allosteric effects on Hb-O(2) affinity than equal increases in chloride ion concentrations.     

Molecular and physicochemical characterization of hemoglobin from the high-altitude Taiwanese brown-toothed shrew (Episoriculus fumidus)

Red-toothed shrews (subfamily Soricinae) exhibit the highest mass-specific rates of O? consumption recorded among eutherian mammals, though surprisingly no data appears to be available on the functional characteristics of their hemoglobin (Hb). As a first step in addressing this shortcoming, we investigated the O? binding characteristics of Taiwanese brown-toothed shrew (Episoriculus fumidus) Hb and its temperature and pH dependence in the absence and presence of anionic red blood cell effectors. Although comparative data regarding the intrinsic O? affinity of other shrew species are currently unavailable, our data suggest that the sensitivity of this high-elevation endemic species' Hb to allosteric effector molecules is similar to that of the two lowland species of white-toothed (crocidurine) shrews examined to date. The efficient exploitation of blood O? reserves by E. fumidus appears to be achieved via synergistic modulation of O? affinity by Cl? and organic phosphates that moreover dramatically lowers the overall enthalpy of oxygenation of their Hb. Oxygen unloading is presumably further enhanced by a relatively high Bohr effect (ΔLog P??/ΔpH = -0.69) and marked reduction in the titratable histidine content (predicted low proton buffering value) of the component globin chains relative to human HbA. Notably, however, the limited data available suggest these latter attributes may be widespread among shrews and hence likely are not adaptations to chronic altitudinal hypoxia per se.     

R-state haemoglobin with low oxygen affinity: crystal structures of deoxy human and carbonmonoxy horse haemoglobin bound to the effector molecule L35

Although detailed crystal structures of haemoglobin (Hb) provide a clear understanding of the basic allosteric mechanism of the protein, and how this in turn controls oxygen affinity, recent experiments with artificial effector molecules have shown a far greater control of oxygen binding than with natural heterotropic effectors. Contrary to the established text-book view, these non-physiological compounds are able to reduce oxygen affinity very strongly without switching the protein to the T (tense) state. In an earlier paper we showed that bezafibrate (BZF) binds to a surface pocket on the alpha subunits of R state Hb, strongly reducing the oxygen affinity of this protein conformation. Here we report the crystallisation of Hb with L35, a related compound, and show that this binds to the central cavity of both R and T state Hb. The mechanism by which L35 reduces oxygen affinity is discussed, in relation to spectroscopic studies of effector binding.     

Spectroscopic properties of the nitric oxide derivative of ferrous man, horse, and ruminant hemoglobins: a comparative study.

The spectroscopic (EPR and absorbance) properties of the nitric oxide derivative of ferrous man, horse, buffalo, deer, mouflon, musk ox, ox, and reindeer hemoglobin (HbNO) have been investigated in the absence of any allosteric effector at pH 6.5 (in 0.1 M 2-[N-morpholino]ethanesulphonic acid/NaOH chloride-free buffer system), as well as at 100 K and/or 20 degrees C. Man and horse HbNO show spectroscopic properties that are generally taken as typical of the high affinity state of ferrous tetrameric Hb's; on the other hand, the spectroscopic properties of ruminant (i.e., buffalo, deer, mouflon, musk ox, ox, and reindeer) HbNO are characteristic of the low affinity conformation. These results are in keeping with the functional properties of the mammalian Hb's considered and have been related to the peculiar low oxygen affinity of ruminant Hb's.     

Anion- and pH-linked effects on the heme-iron geometry in ferrous nitrosylated monomeric myoglobins

 The cooperative effect of anions and proton concentration on the EPR spectroscopic properties of the ferrous nitrosylated derivative of monomeric Mb from loggerhead sea turtle (Caretta caretta), sperm whale (Physeter catodon), and horse (Caballus caballus) has been investigated between pH 4.5 and 9.0, at 100 K. In the absence of anions, an EPR spectrum characteristic of the hexa-coordinated species of ferrous nitrosylated Mb with an axial geometry is observed, which is unaffected by pH. On the other hand, a transition toward a species characterized by an EPR spectrum corresponding to a hexa-coordinated rhombic geometry takes place in the presence of phosphate, acetate, citrate, sulfate, and chloride. Only the hexa-coordinated form characterized by the rhombic EPR spectrum appears then to undergo a pH-dependent transition toward the penta-coordinated species. Present results show clear-cut evidence for the spectroscopic coupling of proton and anion binding sites with the Mb reactive center, indicating that an allosteric mechanism might modulate the proximal HisF8-heme-NO geometry in monomeric hemoproteins. Received: 15 December 1997 / Accepted: 15 June 1998     

Spectrophotometric, electron paramagnetic resonance and oxygen binding studies on the hemoglobin from the marine polychaete Perinereis aibuhitensis (Grübe): comparative physiology of hemoglobin

The physicochemical properties of giant hemoglobin (Hb) of the marine polychaete Perinereis aibuhitensis were extensively studied and the following results were obtained. (1) Light absorption spectra of the oxy, deoxy, CO, met, and cyanomet derivatives were similar to those for human Hb, except for a somewhat peculiar shape and pH-dependence of the met derivative, and high absorbance values around 277 nm for all these derivatives of Perinereis Hb. Abnormal pH dependence for the met derivative was confirmed by powder electron parmagnetic resonance (EPR) spectroscopy, which revealed that a water molecule does not coordinate to the heme iron as a sixth ligand. The high absorption around 277 nm is indicative of the existence of some non-heme polypeptide chains and/or a high content of aromatic residues in the molecule. (2) UV difference and derivative spectra revealed oxygenation-induced conformational changes in the protein moiety that are related to the degree of cooperativity. (3) The EPR spectrum for the nitrosyl derivative showed well-resolved triplet-triplet splittings due to 14N, indicating that the proximal residue is probably a histidine. (4) The oxygen affinity and cooperativity of this Hb were pH-dependent. Mg2+ markedly increased the oxygen affinity, the Bohr effect, and the cooperativity, which was maximal at physiological pH. CO2 and anions such as 2,3-diphosphoglycerate and inositol hexaphosphate had no effect on the oxygenation properties. Thus, different from vertebrate Hb, the oxygen-binding properties of this Hb are regulated by divalent cations which bind preferentially to the oxy form. The low temperature-dependence of oxygen affinity observed for this Hb is a sign of adaptation to the environment by this poikilothermic organism. (5) By using a graphic method, the minimal functional unit that preserves the full cooperativity (allosteric unit) was inferred to be the one containing 6 heme groups and its significance is discussed in connection with the structural hierarchy of the molecule.     

Hemoglobin-ligand binding: understanding Hb function and allostery on atomic level

The major physiological function of hemoglobin (Hb) is to bind oxygen in the lungs and deliver it to the tissues. This function is regulated and/or made efficient by endogenous heterotropic effectors. A number of synthetic molecules also bind to Hb to alter its allosteric activity. Our purpose is to review the current state of Hb structure and function that involves ensemble of tense and relaxed hemoglobin states and the dynamic equilibrium of the multistate due to the binding of endogenous heterotropic or synthetic allosteric effectors. The review also discusses the atomic interactions of synthetic ligands with the function or altered allosteric function of Hb that could be potentially harnessed for the treatment of diseases. This article is part of a Special Issue entitled: Protein Structure and Function in the Crystalline State.     

Effect of zeta-globin substitution on the O2-transport properties of Hb S in vitro and in vivo

Hemoglobin zeta(2)beta(2)(S) is generated by substituting embryonic zeta-globin subunits for the normal alpha-globin components of Hb S (alpha(2)beta(2)(S)). This novel hemoglobin has recently been shown to inhibit polymerization of Hb S in vitro and to normalize the pathological phenotype of mouse models of sickle cell disease in vivo. Despite its promise as a therapeutic tool in human disease, however, the basic O(2)-transport properties of Hb zeta(2)beta(2)(S) have not yet been described. Using human hemoglobins purified from complex transgenic-knockout mice, we show that Hb zeta(2)beta(2)(S) exhibits an O(2) affinity as well as a Hill coefficient, Bohr response, and allosteric properties in vitro that are suboptimally suited for physiological O(2) transport in vivo. These data are substantiated by in situ analyses demonstrating an increase in the O(2) affinity of intact erythrocytes from mice that express Hb zeta(2)beta(2)(S). Surprisingly, though, co-expression of Hb zeta(2)beta(2)(S) leads to a substantial improvement in the tissue oxygenation of mice that model sickle cell disease. These analyses suggest that, in the context of sickle cell disease, the beneficial antisickling effects of Hb zeta(2)beta(2)(S) outweigh its O(2)-transport liabilities. The potential structural bases for the antisickling properties of Hb zeta(2)beta(2)(S) are discussed in the context of these new observations.     

Heterotropic effectors control the hemoglobin function by interacting with its T and R states--a new view on the principle of allostery

Careful analyses of precise oxygenation curves of hemoglobin (Hb) clearly indicate that, contrary to the common belief, allosteric effectors exert a dramatic control of the oxygenation characteristics of the protein by binding not only to the T (unligated), but also to the R (ligated) state, in a process that is proton-driven and involves proton uptake. The most striking functional changes were obtained when the allosteric effectors were bound to the fully ligated Hb: the oxygen affinity decreased dramatically, Bohr effect was enhanced, and cooperativity of oxygen ligation was almost absent, emulating a Root effect-like behavior. However, structural analysis, such as Cys beta 93 sulfhydryl reactivity and ultraviolet circular dichroism, confirmed that the ligated Hb was in fact in the R state, despite its extremely low affinity state features. These findings provide a new global view for allosteric interactions and invoke for a modern interpretation of the role of allosteric effectors and a reformulation of the Monod-Wyman-Changeaux model for control of allosteric systems, and other complementary models as well.     

Domain-specific effector interactions within the central cavity of human adult hemoglobin in solution and in porous sol-gel matrices: evidence for long-range communication pathways

The water-filled central cavity of human adult hemoglobin (Hb A) is the binding or interaction site for many different allosteric effectors. Oxygen binding titrations reveal that pyrenetetrasulfonate (PyTS), a fluorescent analogue of 2,3-diphosphoglycerate, behaves like an allosteric effector. The ligation state, pH, and concentrations of other effectors (IHP, L35, and chloride) alter PyTS fluorescence for both solution-phase and sol-gel-encapsulated Hb samples. These conditions also alter the resonance Raman spectra and rates of geminate recombination of CO-ligated Hb. Together, these results demonstrate that there are conformational and functional consequences resulting from interactions between specific domains of the central cavity and individual effectors as well as from long-range synergistic effects that are mediated through the central cavity.     

The nicotinamide adenine dinucleotides as allosteric effectors of human hemoglobin

The oxygen binding properties of human hemoglobin are appreciably altered by the nicotinamide dinucleotides NADH, NADP+, and NADPH. These cofactors are important in the control of many metabolic pathways and in providing reductive potential for a number of enzymatic reactions, including in vivo reduction of methemoglobin. Specific binding of these cofactors to hemoglobin and their potential for acting as allosteric modifiers of hemoglobin function have not been previously recognized. Detailed oxygen binding studies utilizing a thin-layer method suggest that the nicotinamide dinucleotides bind with high affinity to the deoxyhemoglobin tetramer at the beta chain anion-binding site and stabilize the low affinity "T-state" conformation. Stripped Hb A in 0.05 M N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer, pH 6.5, at 20 degrees C is half-saturated at a pO2 of 1.6 mm Hg. In the presence of 0.5 mM NADH, NADP+, or NADPH, the P50 is raised to 3.8, 7.1, and 12.5 mm Hg, respectively. The Bohr factor for stripped Hb A in 0.05 M HEPES buffer is sensitive to these effectors and is raised from 0.25 to about 0.65 by the addition of NADPH. The data suggest that routine use of these effectors in studies of human hemoglobin variants or the allosteric mechanism of Hb A be considered carefully. The relatively low intraerythrocytic levels of the nicotinamide dinucleotides in relation to hemoglobin dictate that these cofactors cannot significantly affect in vivo oxygen delivery. However, the converse is theoretically possible. The binding of the cofactors to hemoglobin and the preferential binding of their reduced forms may affect cofactor-dependent metabolic processes in red blood cells.     

The acid-alkaline transition of a sea turtle myoglobin: coexistence at high pH of high- and low-spin forms

The microenvironment of the iron in a sea turtle Dermochelys coriacea myoglobin is studied using the spectroscopic techniques EPR and optical absorption. Optical absorption spectra in the visible region suggest a great homology between turtle Mb and other myoglobins, such as those from whale, human and elephant. The pK of the acid-alkaline transition is 8.4 slightly lower than the pK of whale and equal to that of elephant myoglobin. The EPR spectrum at pH 7.0 is characteristic of a high-spin configuration with axial symmetry (gx = gy = 5.95). At higher pH, this signal changes in a way different from that observed for whale myoglobin. We observe for turtle Mb both the formation of a low-spin configuration with rhombic symmetry (gx = 2.56, gy = 2.20, gz = 1.90) and of a high-spin species with rhombic distortion (gx = 6.79, gy = 5.18, gz = 2.12). This suggests a lowering of symmetry at the haem, so that now the x and y directions are no more equivalent. This can be explained by amino acid substitution at the distal positions of haem or to off-axial positioning of distal residues. The coexistence at high pH (pH 11.0) of these two spin forms could be explained by the existence of two protein conformations, in which the crystal field splitting factor, delta, and the electron exchange energy are of the same order, allowing the presence of different configurations simultaneously. The presence of different kinds of haem is ruled out by the experiments with nitrosyl turtle Mb and turtle Mb-F showing spectra very similar to those of whale myoglobin. The pk of the acid-alkaline transition, 8.5, obtained from EPR spectra, agrees very well with results from optical absorption.