Addition Of Inositol Hexaphosphate Research Articles

Antioxidative Pseudoenzymatic Mechanism of NAD(P)H Coexisting with Oxyhemoglobin for Suppressed Methemoglobin Formation.

Oxyhemoglobin (HbO2) coexisting with equimolar NADH retards autoxidation and oxidant-induced metHb formation based on the pseudocatalase (CAT) and pseudosuperoxide dismutase (SOD) activities. In this work, we compared the effects of NADH with those of NADPH and estimated the binding site of NAD(P)H to HbO2 to elucidate the antioxidative mechanisms. The results clarified that pseudo-CAT and pseudo-SOD activities of HbO2 coexisting with NADPH were similar to activities obtained with NADH. Prompt MetHb formation (<40 min) facilitated by oxidants (H2O2, NO, and NaNO2) was hindered by NADPH. These effects were similar to those of NADH. However, we found that NADPH is thermally unstable compared to NADH and that NADPH cannot sustain antioxidative effects for a long period of autoxidation to metHb such as 24 h. Lineweaver-Burk plots clarified that the Michaelis constants of these pseudoenzymatic activities are in the millimolar range. Addition of inositol hexaphosphate (IHP) and 2,3-diphosphoglycerate (DPG), which are known to bind not only with deoxyHb but also weakly with HbO2, showed competitive inhibition of pseudoenzymatic activities. These results suggest that the binding site of NADH and NADPH on HbO2 is the same as those of IHP and DPG. 31P nuclear magnetic resonance definitively showed 1:1 stoichiometric binding of NADH to HbO2. High-performance liquid chromatography analysis showed that NADH preferentially inhibited autoxidation of α-subunit heme. Docking simulations also predicted that the binding site of relaxed-state HbO2 with NAD(P)H is the same as those with IHP and DPG. Collectively, the pseudoenzymatic activities of HbO2 coexisting with NAD(P)H are induced by the 1:1 stoichiometric binding of NAD(P)H to HbO2.

Studies on nitrosyl hemes in Ni(II)–Fe(II) hybrid hemoglobins

Subunit heterogeneity within a particular subunit in hemoglobin A have been explored with electron paramagnetic resonance spectroscopy using the nitrosyl hemes in Ni-Fe hybrid Hb under various solution conditions. Our previous studies on the crystal structure of NiHb demonstrated the presence of subunit heterogeneity within alpha-subunit. To further cross check this hypothesis, we made a hybrid Hb in which either the alpha- or beta-subunit contains iron, which alone can bind to NO. By this way dynamic exchange between penta- and hexa-coordinated forms within a subunit was confirmed. Upon the addition of inositol hexa phosphate (IHP) to these hybrids, R to T state transition is observed for [alpha(2)(Fe-NO)beta(2)(Ni)] but such a direct transformation is less marked in [alpha(2)(Ni)beta(2)(Fe-NO)]. Hence the bond between N(epsilon) and Fe is fundamental to the structure-function relation in Hb, as the motion of this nitrogen triggers the vast transformation, which occurs in the whole molecule on attachment of NO.

Redox states of cerebral tissues of rats substituted by the liposome-encapsulated hemoglobin.

Many kinds of artificial blood that have the ability to transport oxygen into tissues have been proposed. Hemoglobin vesicles, in particular, have advantages over other ones because of their longer half-lives and lower viscosity in circulation.10,11,12 Neo Red Cells (NRC) is the liposome-encapsulated human hemoglobin, and the P50O2 of NRC is changeable in the range of 13–60 Torr in accord with the variation in the addition of inositolhexaphosphate (IHP) as an allosteric effector.8

The association reaction between hemoglobin and carbon monoxide as studied by the isolation of the intermediates. Implications on the mechanism of cooperativity.

The concentrations of the intermediates in the association reaction between human hemoglobin and CO at 20 degrees C, pH 7, under conditions of negligible dissociation of the ligand, were measured by cryogenic techniques. The monoligated species were predominant at all values of overall ligand bound studied. The analysis of the experimental data assuming a scheme of four consecutive reactions indicated that the binding rates increased in a continuous fashion. A significant acceleration after the binding of the second molecule of ligand occurred in the presence of 0.1 M KCl, but not with the addition of an excess of inositol hexaphosphate, indicating that major functional, and possibly structural, transitions occur at the diligated state. Differences in the concentrations of the intermediates in the same state of ligation were observed under all conditions. The analyses of the data on the basis of schemes of multiple pathways of reaction indicated that the beta subunits reacted about 1.5 times faster than the alpha subunits in the first ligation reaction. After the addition of inositol hexaphosphate, the alpha subunits reacted about 1.5 times faster than the beta subunits in the first ligation step, but the overall rate of the first CO binding step was unchanged.

Isolation and characterization of the triply oxidized derivative of a cross-linked hemoglobin

Hemoglobin A, cross-linked between Lys 99 alpha 1 and Lys 99 alpha 2, was used to obtain a partially oxidized tetramer in which only one of the four hemes remains reduced. Because of the absence of dimerization, asymmetric, partially oxidized derivatives are stable. This is evidenced by the fact that eight of the ten possible oxidation states could be resolved by analytical isoelectric focusing. A triply oxidized hemoglobin population HbXL+3 was isolated whose predominant component was (alpha + alpha +, beta + beta 0). This triferric preparation was examined as a possible model for the triliganded state of ferrous HbA. The aquomet and cyanomet derivatives were characterized by their CD spectra and their kinetic reactions with carbon monoxide. CD spectra in the region of 287 nm showed no apparent change in quaternary structure upon binding ligand to the fourth, ferrous heme. The spectra of the oxy and deoxy forms of the cyanomet and aquomet derivatives of HbXL+3 differed insignificantly and were characteristic of the normal liganded state. Upon addition of inositol hexaphosphate (IHP), both the oxy and deoxy derivatives of the high-spin triaquomet species converted to the native deoxy conformation. In contrast, IHP had no such effect on the conformation of the low-spin cyanomet derivatives of HbXL+3. The kinetics of CO combination as measured by stopped-flow and flash photolysis techniques present a more complex picture. In the presence of IHP the triaquomet derivative does bind CO with rate constants indicative of the T state whether these are measured by the stopped-flow technique or by flash photolysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Spin equilibria in human methemoglobin: effects of bezafibrate and inositol hexaphosphate as measured by susceptometry and visible spectroscopy

The effects of inositol hexaphosphate (IHP) and a second allosteric effector, bezafibrate, on the spin-state equilibria of the mixed-spin derivatives of ferric human hemoglobin A are examined. Changes in spin-state equilibrium are monitored by measuring absorption spectra in the visible region (460-700 nm) as well as by direct measurements of magnetic susceptibility by means of a superconducting fluxmeter. The addition of IHP at pH 6.5 results in a measurable shift in the spin equilibria of these derivatives toward higher spin. However, the addition of bezafibrate in the presence of IHP results in still larger shifts toward the high-spin form. The changes in the free energies of the spin-state equilibria resulting from the combination of these two effectors are similar in magnitude to that which results from the R-state to T-state transition in carp hemoglobin.

Intrinsic fluorescence of carp hemoglobin: a study of the R → T transition

The intrinsic fluorescence of hemoglobins is known to respond to ligand-induced changes in the quaternary structure of the protein. Carp hemoglobin is an interesting model to study the quaternary transition since its R ↔ T equilibrium is pH-dependent and at low pH, in the presence of organic phosphate, it remains in the T or ‘deoxy’ quaternary structure, even when saturated with ligand. In this study, using front-face fluorometry, we show that the intrinsic fluorescence intensity exhibited by carp carboxyhemoglobin increases as the pH is lowered below 6.5 in the presence of inositol hexaphosphate. At low pH, carp methemoglobin is less affected by the addition of inositol hexaphosphate than is the CO derivative, while little or no change is observed in the met-azide derivative. We conclude: (1) the exact nature of the R to T state transition induced by inositol hexaphosphate differs for carp carboxy-, met- and met-azide hemoglobin derivatives; (2) the chromophores responsible for the changes observed with absorption spectroscopy may not be the same as those chromophores responsible for the fluorescence differences; and (3) α46-Trp is tentatively assigned as one source of fluorescence emission. Furthermore, fluorescence properties of carp hemoglobin are compared to those of human hemoglobin.

Preparing a quality control material for blood gases, pH and hemoglobinometry using stroma-free hemoglobin solution

Properties of a quality control material for blood gases and pH should be similar to normal human whole blood with respect to oxygen buffering and acid-base behaviour. A hemoglobin solution may potentially fulfill this. However, the drawbacks of such a solution are the high oxygen affinity (lowp50), especially when it is prepared from human blood, and the improper concentration of bicarbonate. Bicarbonate is added to human stroma-free hemoglobin solution (SFHS), prepared as described previously, to obtain the desired pH and pCO2 combinations. Tonometry was used to determine the appropriate concentration of bicarbonate, which is 22.5 mmol/L to obtain an acidotic, and 29 mmol/L for both an alkalotic, and normal pH and pCO2 combination. Inositolhexaphosphate (IHP) is added to SFHS containing bicarbonate to obtain a normal p50 (around 3.55 kPa). Tonometry was used to determine the molar ratio of IHP/Hb4 (mol/mol) at which this is achieved. The molar ratios of IHP/Hb4 are 1.52, 1.74 and 3.40 for preparations with an acidotic, normal and alkalotic pH, respectively. In human SFHS nHill is 2.55 in the absence of IHP,nHill is at minimum 1.71 at a molar ratio IHP/Hb4 of 1.86 and increases to 2.53 at a molar ratio IHP/Hb4 of 5.04 and higher. Because the p50 will decrease with chi Hi this was studied at molar ratios of IHP/Hb4 of 0, 2 and 4, which covers the range of ratios as used. At these molar ratios of 0, 2 and 4, the decrease in p50 is 0.017 kPa/%Hi, 0.023 kPa/%Hi and 0.028 kPa/%Hi, respectively. Because bovine Hb was p50 near that of normal human blood, it is also used. The oxygen affinity shows a small decrease (p50 increases from 3.05 to 5.27 kPa) on addition of IHP. In the absence of IHP, nHill is 2.51 and nHill is at maximum 3.35 at molar ratios IHP/Hb4 between 3.00 to 4.56. At higher molar ratios nHill decreases to 2.90.

Quaternary structure has little influence on spin states in mixed-spin human methemoglobins.

A key feature of the Perutz stereochemical model for cooperativity in hemoglobin is a strong coupling between quaternary structure and the spin state of the heme iron [Perutz, M. F. (1979) Annu. Rev. Biochem. 48, 327-386]. While this coupling appears to be present for carp azide methemoglobin, it should also be present for all liganded forms of human methemoglobin that exhibit a thermal high-spin in equilibrium low-spin equilibrium. To test this hypothesis, we have measured the changes in spin equilibria upon conversion of six mixed-spin forms of human methemoglobin from the R (high-affinity) to the T (low-affinity) quaternary structure by addition of inositol hexaphosphate. These experiments were done with a sensitive superconducting magnetic susceptibility instrument on solutions at 20 degrees C in 20 mM maleate buffer, pH 6. The data show zero or small increases in high-spin content upon switching from R to T, changes that are equivalent to a relative stabilization of the high-spin form by only 0-300 cal mol-1 heme-1. These changes in energy are far less than the 1200 cal mol-1 heme-1 predicted from the Perutz stereochemical model [Cho, K. C., & Hopfield, J. J. (1979) Biochemistry 18, 5826-5833]. That is, these data do not support a view that the low affinity of the T state is due to restraints acting through the iron-proximal histidine linkage. The mechanistic implications of these results and the differences between species and ferric ligands are discussed.

Proton nuclear magnetic resonance investigation of the allosteric transition in ligated and unligated carp hemoglobin: Evidence for structural heterogeneity in the heme pocket

The proton nuclear magnetic resonance spectra of carp hemoglobin (Hb) in the unligated deoxy and ligated met-cyano and met-azido forms have been recorded as a function of pH and upon addition of inositol hexaphosphate. All protein derivatives yield spectra that are consistent with appreciable molecular heterogeneity in the heme cavity. The pattern of heme methyl hyperfine shifts in carp met-cyano Hb indicates that this heterogeneity arises from the presence of heme rotational disorder, as found in native myoglobin. In carp deoxy Hb, the T → R transition manifests itself in nuclear magnetic resonance spectral changes similar to those found in modified human Hb species; namely, a decrease in heme methyl and an increase in proximal histidyl imidazole ring NH hyperfine shifts indicative of a strengthening of the iron-histidine bond. The met-cyano complex exhibits heme methyl hyperfine shifts similar to the analogous R state complex of Hb A; addition of inositol hexaphosphate did not give evidence for a quaternary structural change. Carp met-azido Hb in the R state also closely resembles the electronic structure of the Hb A complex. Addition of inositol hexaphosphate appeared to effect at least a partial conversion to a T state with larger high-spin content than that observed for T state human metHbN 3.

Mechanism of autoxidation of hemoglobin Kempsey (beta 99 Asp----Asn).

The mechanism of autoxidation of oxyhemoglobin Kempsey (beta 99 asp----asn) was studied at pH 7.0, 37 degrees C, using isoelectric focusing electrophoresis. During autoxidation, two intermediate hemoglobins, (alpha 2+ beta 3+)2 and (alpha 3+ beta 2+)2 were observed, and these were consecutively changed to methemoglobin. The autoxidation rates of oxyhemoglobin Kempsey were reduced about 43% by the addition of catalase and superoxide dismutase, but were not significantly changed by the addition of inositol hexaphosphate. This abnormal hemoglobin autoxidized more slowly than normal hemoglobin A. The differences in the autoxidation rates between these hemoglobins were explained by the changes in quaternary structure of these proteins. The reaction rate constant, k+1, k+2, k+3 and k+4 of each step including the reactions: (formula; see text) were determined by the analysis of fractional changes in these hemoglobin derivatives during the autoxidation. The difference in the reaction rate constants between k+1 and k+3 was explained by the nonequivalence of alpha and beta chains in tetrameric oxyhemoglobin Kempsey. The reaction mechanism of autoxidation of hemoglobin Kempsey was discussed on the basis of these kinetic results.

Electron paramagnetic resonance studies on cobalt hemoglobin, iron-cobalt hybrid hemoglobins, and their related model complexes. Characterization of proximal histidine binding to porphyrin cobalt(II) ion and its transition associated with subunit interaction.

Electron paramagnetic resonance studies have been conducted on iron-cobalt hybrid deoxyhemoglobins and cobalt deoxyhemoglobin. Examination of alpha (Co)2 beta (Fe)2 hybrid hemoglobin at two different microwave frequencies revealed two sets of axial-symmetric electron paramagnetic resonance (EPR) signals rather than the anisotropic splitting of the g signal as reported previously [Ikeda-Saito, M., Yamamoto, H., & Yonetani, T. (1977) J. Biol. Chem. 252, 8639-8644]. One of these two sets is a characteristically broad spectrum with g = 2.38 and g parallel = 2.03; the other, a sharp spectrum with g = 2.33 and g parallel = 2.03. The alpha-subunits of deoxy-CoHb also exhibited a complex spectrum with a mixture of these two different types of signals. The relative compositions of these two signals were calculated for deoxy-alpha (Co)2 beta (Fe)2 and the alpha-subunits of CoHb, which were profoundly sensitive to the pH of the medium and also to the presence of allosteric effectors. A positive correlation was noticed between the spectral characteristics and the equilibrium extent of oxygenation, showing that the broad component may be associated with the electronic state of the porphyrin metal ion in T-state hemoglobin and the sharp component with that in the R state. In contrast, the complementary hybrid alpha (Fe)2 beta (Co)2 and the beta-subunits in CoHb showed relatively narrow spectra under similar conditions, and only small changes were noted with variation of pH and addition of inositol hexaphosphate. These spectra were close to the sharp component of the alpha-subunits associated with the R state. Comparison of the hemoglobin EPR spectra with those of model cobaltous porphyrin complexes revealed that the restraint that is operative at the fifth ligand position of the prosthetic group in the T state is released in the R state. These ligand binding properties were discussed in relation to the regulation of oxygen binding to hemoglobin.

The kinetic mechanism of heme binding to human apohemoglobin.

The formation of hemoglobin from free CO-heme and apoprotein is, at minimum, a two-step process. The first reaction involves very rapid equilibration of the heme groups with hydrophobic regions of the globin molecule. The spectrum of the resultant heme-globin complex is analogous to that of CO-heme incorporated into phospholipid membranes. The Soret absorbance maximum of this species is at about 413 nm, which suggests strongly that the iron-histidine bond has not been formed. The equilibrium dissociation constant for the formation of the heme-globin complex is 6.2 +/- 2 microM at pH 7.2 and 10 degrees C. This constant increases with increasing pH, with the addition of inositol hexaphosphate, with modification of the protoporphyrin vinyl groups, and with increasing glycerol concentration. The second step in hemoglobin formation is unimolecular and appears to involve a large conformational transition which results in iron-histidine bond formation and the appearance of a native CO-hemoglobin spectrum. The rate of this process is 500 +/- 150 s-1 at pH 7.2 and 10 degrees C and is little affected by changes in pH, the addition of organic phosphates, or heme modification. Increasing the solvent viscosity to about 10 c.p. by the addition of 50% glycerol causes a 10-fold decrease in the rate of this conformational change. This suggests that large protein movements, presumably folding around the heme, are involved in the final stages of hemoglobin formation. Iron-histidine bond formation does not appear to be a rate-limiting step.

Librational modes in liganded and unliganded hemoglobin as seen by fluorescence spectroscopy.

Hemoglobin labeled with N-iodoacetylaminoethyl-5-naphthalene-1-sulfonate at the beta-93 cysteine shows normal allosteric properties including the Bohr effect and heme-heme-interaction, and the oxygen affinity is somewhat increased. Viscosity-resolved correlation times obtained from direct measurement of the decay of fluorescence anisotropy show values of 4.15 +/- 0.23 and 9.13 +/- 0.46 ns for the liganded and unliganded derivatives, respectively, in 0.05 M tris buffer, pH 7.5 at 15 degrees C. Addition of 1 mM inositol hexaphosphate to the liganded and unliganded derivatives results in correlation times of 9.31 +/- 0.87 and 43.69 +/- 16.04 ns, respectively. Similar values of correlation times are obtained from Perrin plots. Considering that the correlation times expected for the single subunits, the dimers, and the tetramers of the system are approximately 10-15, 20-30, and above 40 ns, respectively, it can be concluded that the molecule of hemoglobin becomes increasingly rigid upon removal of ligands and addition of inositol hexaphosphate. These observations support the hypothesis of a functional relevance of the internal flexibility of hemoglobin.

Resonance Raman investigation of nitric oxide bonding in nitrosylhemoglobin A and -myoglobin: detection of bound N-O stretching and Fe-NO stretching vibrations from the hexacoordinated NO-heme complex.

With excitation at 406.7 nm, we have observed the resonance Raman enhancement of the bound v(N-O) stretch at approximately 1623 cm-1 in nitrosylhemoglobin A and nitrosylmyoglobin, indicating the existence of a charge-transfer transition underlying the strong Soret band. The v(Fe-NO) stretch at 551 cm-1 has also been detected in the Soret as well as in the Q-band region, a phenomenon similar to the v(Fe-O2) and v(Fe-CO) stretches in oxy and carbon monoxy hemoproteins. It appears that these iron-ligand vibrations ay be resonance enhanced via porphyrin pi leads to pi transitions. Upon addition of inositol hexaphosphate at pH 6.0, the v(Fe-NO) stretch at 551 cm-1 and a low-frequency mode at 301 cm-1 exhibit an intensity decrease by approximately one-half. Contrary to the work of Stong et al. [Stong, J. D., Burke, J. M., Daly, P., Wright. P., & Spiro, T. G. (1980) J. Am. Chem. Soc. 102, 5815], who employed an excitation wavelength at 454.5 nm, we observed no intensity increase at 592 cm-1 attributable to the v(Fe-NO) stretch from the pentacoordinated NO-heme complex in the alpha subunits.

Interaction of methemoglobin with inositol hexaphosphate. Presence of the T state in human adult methemoglobin in the low spin state.

The interaction of human methemoglobin with inositol hexaphosphate (IHP) was examined by proton NMR spectroscopy. Upon addition of IHP, new proton peaks which are distinguishable from those observed in the absence of IHP appeared irrespective of the spin state of the ferric heme iron. the IHP-induced NMR peaks of low spin azide methemoglobin were not observed when azide was titrated to stripped aquomethemoglobin. Therefore, it was indicated that the IHP-induced peaks are not due to the slight dissociation of azide. Azide titration to aquomethemoglobin in the presence of IHP further revealed that the IHP-induced peaks are not due to a localized structural change but rather due to the presence of an IHP-induced new conformer. The NMR spectral intensity calibration for the 5-methylimidazole complex of methemoglobin showed that the intensity of the spectrum of stripped methemoglobin decreased by about 80% upon addition of IHP, suggesting the structural alterations in both of the alpha and beta subunits. The intensity of the IHP-induced peak at 31.1 ppm in azide methemoglobin spectrum decreased at both higher and lower pD values with its optimal around pD 6.7. The pD dependence of this peak was closely similar to that of the exchangeable proton NMR peak which has been used as a measure of the T quaternary conformation for human aquomethemoglobin (Huang, T.-H. (1979) J. Biol. Chem. 254, 11467-11474). From all of the above results, it was concluded that human low spin methemoglobin can be switched from the R to T quaternary structures by the binding of IHP. The allosteric constant L4 = [T4]/[R4] for the fully ligated low spin methemoglobin increased with decreasing temperature and was estimated to be 1 to 5 at room temperature.

The effect of complex-formation with polyanions on the redox properties of cytochrome c.

1. The stable complex formed between mammalian cytochrome c and phosvitin at low ionic strength was studied by partition in an aqueous two-phase system. Oxidized cytochrome c binds to phosvitin with a higher affinity than reduced cytochrome c. The difference was equivalent to a decrease of the redox potential by 22 mV on binding. 2. Complex-formation with phosvitin strongly inhibited the reaction of cytochrome c with reagents that react as negatively charged species, such as ascorbate, dithionite, ferricyanide and tetrachlorobenzoquinol. Reaction with uncharged reagents such as NNN'N'-tetramethylphenylenediamine and the reduced form of the N-methylphenazonium ion (present as the methylsulphate) was little affected by complex-formation, whereas oxidation of the reduced cytochrome by the positively charged tris-(phenanthroline)cobalt(III) ion was greatly stimulated. 3. A similar pattern of inhibition and stimulation of reaction rates was observed when phosvitin was replaced by other macromolecular polyanions such as dextran sulphate and heparin, indicating that the results were a general property of complex-formation with polyanions. A weaker but qualitatively similar effect was observed on addition of inositol hexaphosphate and ATP. 4. It is suggested that the effects of complex-formation with polyanions on the reactivity of cytochrome c with redox reagents are mainly the result of replacing the positive charge on the free cytochrome by a net negative charge. Any steric effects on polyanion binding are small in comparison with such electrostatic effects.

Raman difference spectroscopy of tertiary and quaternary structure changes in methaemoglobins.

There have been several resonance Raman scattering investigations of the effect of inositol hexaphosphate (IHP) on methaemoglobins. In those studies the sensitivity for detecting frequency differences was limited to 1-2 cm-1, and consequently frequency differences were not detected although spectral intensity differences due to changes in spin equilibria were. Shelnutt et al. recently reported on the observation of frequency differences induced by changes in the quaternary structure of chemically modified deoxyhaemoglobins. An improved Raman difference spectroscopic technique with 0.1 cm-1 sensitivity allowed the detection of these differences. We report here the application of this technique to a series of methaemoglobins with and without the addition of IHP. In addition to the intensity changes resulting from changes in the spin equilibria, we have observed frequency differences. In all liganded methaemo-globins that we examined a decrease in frequency of the mode in the 1,370 cm-1 region was observed on addition of IHP. In those in which a quaternary structure change is known to occur the frequency difference is greater than 0.5 cm-1. In those in which no quaternary structure change occurs [metHbA(CN-) and methHbA(N-3)] the frequency difference is smaller (approximately 0.15 cm-1).

Interaction of human adult methemoglobin in low-spin state with inositol hexaphosphate. A proton magnetic resonance study

The hyperfine-shifted proton nuclear magnetic resonance (NMR) spectra of the low-spin complexes of human adult methemoglobin were found to be much altered by the addition of inositol hexaphosphate (IHP). The stoichiometry and pH-dependence of IHP binding, and the spin equilibrium of azide methemoglobin are parallel to those of high-spin human methemoglobin and of carp methemoglobin, both of which are proposed to be switched from the R to T states with IHP. The present NMR results show that IHP affects the structure of human methemoglobin regardless of the spin state of the heme iron, suggesting that there is no correspondence between quaternary structure and the spin state of ferric heme iron.

Effect of Removal of a Salt-Bridge on the Oxygen Binding Properties and the Electronic Structure of Heme in Cobalt-Iron Hybrid Hemoglobin

In order to clarify the role of salt-bridges in hemoglobin, the oxygen equilibrium curves and electron paramagnetic resonance (EPR) spectra of cobalt-iron hybrid hemoglobins were determined. The EPR spectra of deoxy alpha(Co)2 beta(Fe)2 could be interpreted as a mixture of two distinct paramagnetic species: one showed a maximum of the first derivative spectrum at g = 2.39 and the other at g = 2.33. The oxygen equilibrium curves of the hybrid indicated that the former is assignable to the T structure and the latter to the R structure. The cooperativity of oxygen binding of alpha(Co)2 beta(Fe)2 exhibited a maximum at g = 2.33, which is characteristic of the R structure, regardless of the pH. Addition of inositol hexaphosphate (IHP) to des-Arg alpha(Co)2 beta(Fe)2 restored the cooperativity of oxygen binding, which implies that the deoxygenated form of des-Arg alpha(Co)2 beta(Fe)2 is converted to the T structure upon addition of IHP. However, the EPR signal at g = 2.39 was not restored upon conversion to the T structure by addition of IHP. It is therefore concluded that the EPR spectrum of the deoxy alpha(Co) subunit depends both on the quaternary structure and on the localized strain at the heme.