High pressure NMR studies of hemoproteins. The effect of pressure on the tertiary and quaternary structures of human adult ferrous hemoglobin

The effect of pressure on the tertiary and quaternary structures of human oxy, carbonmonoxy, and deoxyhemoglobin was examined by high pressure NMR spectroscopy at 300 MHz. The increased pressure displaced the ring current-shifted gamma 1-methyl resonance of beta E11 valine for oxy- and carbonmonoxyhemoglobin to the upfield side, whereas that of the alpha subunit was insensitive to pressure. Such a preferential pressure-induced upfield shift for the beta E11 valine gamma 1-methyl signal was also encountered for the isolated carbonmonoxy beta chain. For deoxyhemoglobin, hyperfine shifted resonances of the heme peripheral proton groups and the proximal histidyl NH proton for the beta subunit were pressure-dependent, in contrast to the pressure-insensitive responses for these resonances of the alpha subunit. These results indicate the structural nonequivalence of the pressure-induced structural changes in the alpha and beta subunits of hemoglobin. The exchangeable proton resonances due to the intra- and intersubunit hydrogen bonds which have been used as the oxy and deoxy quaternary structural probes were not changed upon pressurization. From all of above results, it was concluded that pressure induces the tertiary structural change preferentially at the beta heme pocket of the ferrous hemoglobin derivatives with the quaternary structure retained.     

A proton nuclear magnetic resonance study of the quaternary structure of human homoglobins in water.

Proton nuclear magnetic resonance spectra of human hemoglobins in water reveal several exchangeable protons which are indicators of the quaternary structures of both the liganded and unliganded molecules. A comparison of the spectra of normal human adult hemoglobin with those of mutant hemoglobins Chesapeake (FG4alpha92 Arg yields Leu), Titusville (G1alpha94 Asp yields Asn), M Milwaukee (E11beta67 Val yields Glu), Malmo (FG4beta97 His yields Gln), Kempsey (G1beta99 Asp yields Asn), Yakima (G1beta99 Asp yields His), and New York (G15beta113 Val yields Glu), as well as with those of chemically modified hemoglobins Des-Arg(alpha141), Des-His(beta146), NES (on Cys-beta93)-Des-Arg(alpha141), and spin-labeled hemoglobin [Cys-beta93 reacted with N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)iodoacetamide], suggests that the proton in the important hydrogen bond between the tyrosine at C7alpha42 and the aspartic acid at G1beta99, which anchors the alpha1beta2 subunits of deoxyhemoglobin (a characteristic feature of the deoxy quaternary structure), is responsible for the resonance at -9.4 ppm from water at 27 degrees. Another exchangeable proton resonance which occurs at -6.4 ppm from H2O is a spectroscopic indicator of the deoxy structure. A resonance at -5.8 ppm from H2O, which is an indicator of the oxy conformation, is believed to originate from the hydrogen bond between the aspartic acid at G1alpha94 and the asparagine at G4beta102 in the alpha1beta2 subunit interface (a characteristic feature of the oxy quaternary structure). In the spectrum of methemoglobin at pH 6.2 both the -6.4- and the -5.8ppm resonances are present but not the -9.4-ppm resonance. Upon the addition of inositol hexaphosphate to methemoglobin at pH 6.2, the usual resonance at -9.4 ppm is shifted to -10 ppm and the resonance at 6.4 ppm is not observed. In the spectrum of methemoglobin at pH greater than or equal to 7.6 with or without inositol hexaphosphate, the resonance at -5.8 ppm is present, but not those at -10 and -6.4 ppm, suggesting that methemoglobin at high pH has an oxy-like structure. Two resonances (at -8.2 and -7.3 ppm) which remain invariant in the two quaternary structures could come from exchangeable protons in the alpha1beta1 subunit interface and/or other exchangeable protons in the hemoglobin molecule which undergo no conformational changes during the oxygenation process. These exchangeable proton resonances serve as excellent spectroscopic probes of the quaternary structures of the subunit interfaces in studies of the molecular mechanism of cooperative ligand binding to hemoglobin.     

Quaternary structures and low frequency molecular vibrations of haems of deoxy and oxyhaemoglobin studied by resonance Raman scattering

The influence of quaternary structure on the low frequency molecular vibrations of the haem within deoxyhaemoglobin (deoxy Hb) and Oxyhaemoglobin (oxy Hb) was studied by resonance Raman scattering. The FeO₂ stretching frequency was essentially identical between the high affinity (R) state (Hb A) and low affinity (T) state (Hb Kansas and Hb M Milwaukee with inositol hexaphosphate). However in deoxy Hb, only one of the polarized lines showed an appreciable frequency shift upon switch of quaternary structure, i.e. 215 to 218 cm^?1 for the T state (Hb A, des-His(146β) Hb, and des-Arg(141α) Hb (pH 6.5)) and 220 to 221 cm^?1 for the R state (des-Arg(141α) Hb (pH 9.0), des-His(146β)-Arg(141α) Hb and NES des-Arg(141α) Hb). Based on the observed ⁵⁴Fe isotopic frequency shift of the corresponding Raman lines of deoxy Hb A (214 → 217 cm^?1), of deoxy NES des-Arg Hb (220 → 223 cm^?1), of the protoporphyrinato-Fe(II)-(2-methylimidazole) complex in the ferrous high spin state (207 → 211 cm^?1) and of deoxymyoglobin (220 → 222 cm^?1) (Kitagawa et al., 1979), and on substitution of perdeuterated for protonated 2-methylimidazole in the deoxygenated picket fence complex (T_pivPP)Fe²⁺ (2-MeIm) (209 → 206 cm^?1), and on the results of normal co-ordinates calculation carried out previously, we proposed that the 216 cm^?1 line of deoxy Hb is associated primarily with the FeN_ε(HisF8) stretching mode and accordingly that the FeN_ε(HisF8) bond is stretched in the T state due to a strain exerted by globin.     

A proton nuclear magnetic resonance investigation of proximal histidyl residues in human normal and abnormal hemoglobins. A probe for the heme pocket.

Proton nuclear magnetic resonance spectroscopy at 250 MHz has been used to investigate the conformations of proximal histidyl residues of human normal adult hemoglobin, hemoglobin Kempsey [beta 99(G1) Asp leads to Asn], hemoglobin Osler [beta 145(HC2) Tyr leads to Asp], and hemoglobin McKees Rocks [beta 145(HC2) Tyr leads to Term] around neutral pH in H2O at 27 degrees C, all in the deoxy form. Two resonances that occur between 58 and 76 ppm downfield from the water proton signal have been assigned to the hyperfine shifted proximal histidyl NH-exchangeable protons of the alpha- and beta-chains of deoxyhemoglobin. These two resonances are sensitive to the quaternary state of hemoglobin, amino acid substitutions in the alpha 1 beta 2-subunit interface and in the carboxy-terminal region of the beta-chain, and the addition of organic phosphates. The experimental results show that there are differences in the heme pockets among these four hemoglobins studied. The structural and dynamic information derived from the hyperfine shifted proximal histidyl NH-exchangeable proton resonances complement that obtained from the ferrous hyperfine shifted and exchangeable proton resonances of deoxyhemoglobin over the spectral region from 5 to 20 ppm downfield from H2O. The relationship between these findings and Perutz's stereochemical mechanism for the cooperative oxygenation of hemoglobin is discussed.     

Study of the conversion of GDP-mannose into GDP-fucose in Nereids: a biochemical marker of oocyte maturation

The high-resolution proton nuclear magnetic resonance spectra of carp hemoglobin have been compared to those of human normal adult hemoglobin. Carp deoxy and carbonmonoxy hemoglobins in the deoxy-type quaternary state exhibit two downfield exchangeable proton resonances as compared to four seen in human normal adult deoxyhemoglobin. This suggests that two of the hydrogen bonds present in human normal adult deoxyhemoglobin are absent or occur in very different environments in carp hemoglobin. One of the exchangeable proton resonances of carp hemoglobin, while present in the deoxy-type quaternary state of the carbonmonoxy and deoxy derivatives, is absent in the oxy-type quaternary state of both, in agreement with the assignments of these quaternary structures by other methods. The ring-current-shifted proton resonances (sensitive tertiary structural markers) of carp carbonmonoxyhemoglobin are substantially different from those of human normal adult hemoglobin. The aromatic proton resonance region of carp hemoglobin has fewer resonances than that of human normal adult hemoglobin, consistent with its much reduced histidine content. The hyperfine-shifted proximal histidyl NH-exchangeable proton resonances of carp hemoglobin suggest that during the transition from the oxy to the deoxy quaternary structure, there is a greater alteration in the heme pocket of one type of subunits (presumably the beta chain) than that in the other subunit. The present results suggest that there are differences in both tertiary and quaternary structures between carp and human normal adult hemoglobins which could contribute to the great differences in the functional properties between these two proteins.     

Proton nuclear magnetic resonance study of the ferrous derivatives of the dimeric and tetrameric hemoglobin from the mollusc Scapharca inaequivalvis

Proton NMR spectra have been measured for the two hemoglobins from the mollusc Scapharca inaequivalvis: HbI, a homodimer, and HbII, a heterotetramer. These hemoglobins are endowed with a unique subunit assembly, since the heme carrying E and F helices are involved in the major intersubunit contact. In the far-downfield region of hyperfine-shifted resonances the spectra of HbI and HbII in the deoxy state show respectively one (66.7 ppm) and two (67.8 and 63.6 ppm) exchangeable signals of the proximal histidine N delta H groups, the resonance position being indicative of a significant strain in the iron-imidazole interaction. In the hydrogen-bonded proton region, inter- and intrasubunit hydrogen-bonded proton signals have been detected for both hemoglobins. Deoxy-HbI shows two unique downfield resonances at 11.83 and 11.51 ppm which disappear in the oxygenated state, suggesting that the corresponding hydrogen bonds are involved in the stabilization of the tertiary and/or quaternary structure of the deoxy form. HbII shows even smaller changes in this region upon changes in ligation state. These results therefore provide further proof that, at variance with the vertebrate hemoglobin tetramer, the unique subunit assembly of these proteins is stabilized mainly by hydrophobic interactions.     

Proton nuclear magnetic resonance studies of hemoglobin Malm?: implications of mutations at homologous positions of the alpha and beta chains.

The abnormal human hemoglobin Malm? (beta97FG4 His leads to Gln) has been studied and its properties are compared with those of normal adult hemoglobin A. The data presented here show that the ring-current shifted proton resonances of both HbCO and HbO2 Malm? are very different from the corresponding forms of Hb A. The hyperfine shifted proton resonances of deoxy-Hb Malm? do not differ drastically from those of deoxy-Hb A. This result, together with the finding that the exchangeable proton resonances of the deoxy form of the two hemoglobins are similar, suggests that unliganded Hb Malm? can assume a deoxy-like quaternary structure both in the absence and presence of organic phosphates We have also compared the properties of Hb Malm? with those of Hb Chesapeake (alpha92FG4 Arg leads to Leu). This allows us to study the properties of two abnormal human hemoglobins with mutations at homologous positions of the alpha and beta chains in the three-dimenstional structure of the hemoglobin molecule. Our present results suggest that the mutaion at betaFG4 has its greatest effect on the teritiary structure of the heme pocket of the liganded forms of the hemoglobin while the mutation at alphaFG4 alters the deoxy structure of the hemoglogin molecule but does not alter the teriary structure of the heme pockets of the liganded form of the hemoglobin molecule. Both hemoglobins undergo a transition from the deoxy (T) to the oxy (R) quaternary structure upon ligation. The abnormally high oxygen affinities and low cooperativities of these two hemoglobins must therefore be due to either the structural differences which we have observed and/or to an altered transition between the T and R structures.     

Gelation of sickle hemoglobin. III. Nitrosyl hemoglobin.

Sickle cell nitrosyl hemoglobin was examined for gelation by an ultracentrifugal method previously described (Briehl &; Ewert, 1973) and by birefringence. In the presence of inositol hexaphosphate gelation which exhibited the endothermic temperature dependence seen in gels of deoxyhemoglobin S was observed by both techniques. In the absence of inositol hexaphosphate no gelation was observed, nor did nitrosyl hemoglobin A exhibit gelation. On the assumption that gelation is dependent on the deoxy or T (low ligand affinity) as opposed to the oxy or R (high ligand affinity) quaternary structure this supports the conclusion that nitrosyl hemoglobin S in inositol hexaphosphate assumes the T structure, in contrast to the other liganded ferrohemoglobin derivatives oxy and carbon monoxide hemoglobin. Assuming further that the quaternary structures and isomerizations are the same in hemoglobins A and S it can also be concluded that nitrosyl hemoglobin A in inositol hexaphosphate assumes the T state. Since no gelation was seen in stripped nitrosyl hemoglobin S, inositol hexaphosphate serves to effect an R to T switch in this derivative. Thus R-T isomerization in nitrosyl hemoglobin occurs without change in ligand binding at the sixth position of the heme group confirming the conclusion of Salhany (1974) and Salhany et al. (1974).Lowering of the pH toward 6 favors gelation of NO hemoglobin S as it does of deoxy and aquomethemoglobin S (Briehl &; Ewert, 1973,1974), consistent with a favoring of the T structure due to strengthening of the interchain salt bridges and the binding of inositol hexaphosphate and/or changes in site-to-site interactions on which gelation depends.     

A proton nuclear Overhauser effect investigation of the subunit interfaces in human normal adult hemoglobin

High-resolution proton nuclear magnetic resonance spectroscopy and nuclear Overhauser effects for the low-field exchangeable proton resonances of human normal adult hemoglobin in aqueous solvents are being used to confirm and extend the assignments of these resonances to specific protons at the intersubunit interfaces of the molecule. Most of these exchangeable proton resonances of human normal adult hemoglobin have been found to be absent in the spectra of isolated alpha or beta subunits. This finding indicates that they are specific spectral markers for the quaternary structure of the hemoglobin tetramer. Based on the nuclear Overhauser effect results, we have assigned the exchangeable proton resonance at +7.4 ppm downfield from H2O to the hydrogen-bonded proton between alpha 103(G10)His and beta 108(G10)Asn at the alpha 1 beta 1 interface. The nuclear Overhauser effect results have also confirmed the assignments of the exchangeable proton resonances at +9.4 and +8.2 ppm downfield from H2O previously proposed by workers in this laboratory based on a comparison of human normal adult hemoglobin and appropriate mutant hemoglobins. This independent confirmation of previously proposed assignments is necessary in view of the possible long-range conformational effects of single amino-acid substitutions in mutant hemoglobin molecules.     

Assessment of the alpha 1 beta 2 contact structure of valency hybrid hemoglobins by ultraviolet difference spectra

We have developed a rapid and useful method for purification of valency hybrid hemoglobins (alpha 2+ beta 2 and alpha 2 beta 2+: + denotes ferric heme) from a hemoglobin solution oxidized partially with ferricyanide by preparative high-performance liquid chromatography. This method does not involve the separation of hemoglobin subunits and the reconstitution of ferric and partner ferrous subunits. Using the valency hybrid hemoglobins thus prepared, the effect of the ferric spin state on the alpha 1 beta 2 subunit boundary structure was investigated by measuring the ultraviolet difference absorption spectra between the deoxy and the oxy valency hybrids associated with various ferric ligands (fluoride, aquo, azide and cyanide). All derivatives of both alpha 2+ beta 2 and alpha 2 beta 2+ showed the difference spectra characteristic of R-T quaternary structural transition. However, the magnitude of the difference spectral peak observed near 288 nm was larger for high-spin derivatives than for low-spin ones. The magnitude of the peak for the valency hybrid hemoglobin was closely correlated with the difference in the free energy of oxygen binding between the R and T states. Since the R state of high-spin hybrids is considered to be identical to that of low-spin hybrids, we concluded from these results that the alpha 1 beta 2 subunit boundary structure plays an important role in regulating the oxygen affinity of deoxy T state.     

Effect of hydrostatic pressure on ligand binding to hemoglobin.

Increase in hydrostatic pressure to 1000 atm increased the affinity of human and menhaden (Brevoortia tyrannus) hemoglobins for oxygen. With necessary assumptions about the form of the equilibrium curve, and after correction for changes in pH and volume due to pressure, the increase in affinity is about 2-fold for both hemoglobins. At pH 6.5, Hill's n for menhaden hemoglobin is near 1, and it is believed to remain in the T state, whereas human hemoglobin undergoes a T to R transition. This suggests that the R-T equilibrium is not disturbed by pressure. In direct experiments the binding of a fluorescent effector (8 hydroxy-1,3,6-pyrene (trisulfonic acid) to deoxyhemoglobin was not changed by pressure. The binding of n-butylisocyanide to hemoglobin and to myoglobin is also greater at high pressures, similarly suggesting that the R-T transition is not involved in the pressure effect.     

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.     

Coupling of tertiary and quaternary changes in human hemoglobin: a 1D and 2D NMR study of hemoglobin Saint Mandé (beta N102Y)

Hemoglobin Saint Mandé (beta N102Y) is a low-affinity mutant with the substitution site situated in the quaternary-sensitive alpha 1 beta 2 interface. In adult hemoglobin the Asn102 beta contributes to the stability of the liganded (R) state, forming a hydrogen bond with Asp94 alpha. The quaternary and tertiary perturbations subsequent to the Tyr for Asn substitution in monocarboxylated hemoglobin Saint Mandé have been investigated by one- and two-dimensional nuclear magnetic resonance (NMR) spectroscopy. Analysis of the one-dimensional NMR spectra of the liganded and unliganded samples in 1H2O provides evidence that both R and T quaternary structures of Hb Saint Mandé are different from the corresponding ones in HbA. In the monocarboxylated form of the mutant hemoglobin, at acid pH, we have observed the disappearance of an R-type hydrogen bond and the appearance of a new one whose proton resonates like a deoxy T marker. Using two-dimensional NMR methods and on the basis of previous results on the monocarboxylated HbA, we have obtained a significant number of resonance assignments in the spectra of monocarboxylated Hb Saint Mandé at pH 5.6 in the presence or absence of a strong allosteric effector, inositol hexaphosphate. This enabled us to characterize the tertiary conformational changes (relative to the liganded normal hemoglobin) triggered by the quaternary-state modification. The observed structural variations are confined within the heme pocket regions but concern both the alpha and beta subunits. Most of them, localized in the C, F, G, and FG segments, could result directly from the side-chain substitution, while others, such as Leu141 beta, could be explained only by long-range interactions.     

NMR study of hybrid hemoglobins containing unnatural heme: effect of heme modification on their tertiary and quaternary structures

The effect of heme modification on the tertiary and quaternary structures of hemoglobins was examined by utilizing the NMR spectra of the reconstituted [mesohemoglobin (mesoHb), deuterohemoglobin (deuteroHb)] and hybrid heme (meso-proto, deutero-proto) hemoglobins (Hbs). The heme peripheral modification resulted in the preferential downfield shift of the proximal histidine N1H signal for the beta subunit, indicating nonequivalence of the structural change induced by the heme modification in the alpha and beta subunits of Hb. In the reconstituted and hybrid heme Hbs, the exchangeable proton resonances due to the intra- and intersubunit hydrogen bonds, which have been used as the oxy and deoxy quaternary structural probes, were shifted by 0.2-0.3 ppm from that of native Hb upon the beta-heme substitution. This suggests that, in the fully deoxygenated form, the quaternary structure of the reconstituted Hbs is in an "imperfect" T state in which the hydrogen bonds located at the subunit interface are slightly distorted by the conformational change of the beta subunit. Moreover, the two heme orientations are found in the alpha subunit of deuteroHb, but not in the beta subunit of deuteroHb, and in both the alpha and beta subunits of mesoHb. The tertiary and quaternary structural changes in the Hb molecule induced by the heme peripheral modification were also discussed in relation to their functional properties.     

Proton nuclear magnetic resonance and biochemical studies of oxygenation of human adult hemoglobin in deuterium oxide.

The proton nuclear magnetic resonance spectrum of human adult deoxyhemoglobin in D2O in the region from 6 to 20 ppm downfield from the proton resonance of residual water shows a number of hyperfine shifted proton resonances that are due to groups on or near the alpha and beta hemes. The sensitivity of these resonances to the ligation of the heme groups and the assignment of these resonances to the alpha and beta chains provide an opportunity to investigate the cooperative oxygenation of an intact hemoglobin molecule in solution. By use of the nuclear magnetic resonance correlation spectroscopy technique, at least two resonances, one at approximately 18 ppm downfield from HDO due to the beta chain and the other at approximately 12 ppm due to the alpha chain, can be used to study the binding of oxygen to the alpha and beta chains of hemoglobin. The present results using approximately 12% hemoglobin concentration in 0.1 M Bistris buffer at pD 7 and 27 degrees C with and without organic phosphate show that there is no significant line broadening on oxygenation (from 0 to 50% saturation) to affect the determination of the intensities or areas of these resonances. It is found that the ratio of the intensity of the alpha-heme resonance at 12 ppm to that of the beta-heme resonance at 18 ppm is constant on oxygenation in the absence of organic phosphate but decreases in the presence of 2,3-diphosphoglycerate or inositol hexaphosphate, with the effect of the latter being the stronger. On oxygenation, the intensities of the alpha-heme resonance at 12 ppm and of the beta-heme resonance at 18 ppm decreases more than the total number of deoxy chains available as measured by the degree of O2 saturation of hemoglobin. This shows the sensitivity of these resonances to structural changes which are believed to occur in the unligated subunits upon the ligation of their neighbors in an intact tetrameric hemoglobin molecule. A comparison of the nuclear magnetic resonance data with the populations of the partially saturated hemoglobin tetramers (i.e., hemoglobin with one, two, or three oxygen molecules bound) leads to the conclusion that in the presence of organic phosphate the hemoglobin molecule with one oxygen bound maintains the beta-heme resonance at 18 ppm but not the alpha-heme resonance at 12 ppm. These resluts suggest that some cooperativity must exist in the deoxy quaternary structure of the hemoglobin molecule during the oxygenation process. Hence, these results are not consistent with the requirements of two-state concerted models for the oxygenation of hemoglobin. In addition, we have investigated the effect of D2O on the oxygenation of hemoglobin by measuring the oxygen dissociation curves of normal adult hemoglobin as a function of pH in D2O andH2O media. We have found that (1) the pH dependence of the oxygen equilibrium of hemoglobin (the Bohr effect) in higher pH in comparison to that in H2O medium and (2) the Hill coefficients are essentially the same in D2O and H2O media over the pH range from 6.0 to 8.2...     

NMR studies of the quaternary structure and heterogeneity of nitrosyl- and methemoglobin.

NMR was used to study the quaternary structure of nitrosyl- and methemoglobin, the kinetics and equilibrium behavior of nitric oxide binding, and the oxidation of hemoglobin. The -9.6 ppm (from H2O) resonance was used as a measure of nitrosylhemoglobin molecules in the T quaternary structure. We found that stripped nitrosylhemoglobin is 70% in the T state below pH 6.4, and is in the R state above. Inositol hexaphosphate (IHP) raises this transition point to pH 7.5. For stripped aquomethemoglobin, the T marker at -10 ppm is absent. In IHP, at pH 6.5 all of the molecules are in the T state. At both higher and lower pH they shift to the R state. The intensity decreases to half of its maximum at pH 5.5 and 7.4. The relative affinity of nitric oxide binding to the alpha and beta subunits was inferred from the intensities of the resonances at -12 and -18 ppm. Under conditions in which nitrosylhemoglobin exists in the T state, NO binds to the alpha subunit 10 times more strongly than it does to the beta subunit. The kinetic experiments reveal that it binds to the two subunits at the same rate and that it dissociates at 5 x 10(-3) s-1 from the beta subunit and at 5 x 10(-4) s-1 from alpha subunit. At high pH, the two subunits are ligated at the same rate. Potassium ferricyanide oxidation, at pH 6.0 in the absence of IHP, is about 3 times more favorable for the alpha than the beta subunit. Addition of IHP raises this preferential oxidation slightly. At pH 8.44, both alpha and beta subunits were oxidized at the same rate.     

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 HbA 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 metHbN3.     

The dissociation of human deoxyhemoglobin and mixed state hemoglobin into monomer.

The experimental hybridizations between fully deoxygenated human and canine hemoglobins and between half-ligated human hemoglobin and canine cyanomethemoglobin show that new two hybrids in addition to the parent hemoglobins were clearly formed in the mixtures at the high concentration of KI. Thus, human deoxyhemoglobin under the present conditions is in an equilibrium with three species, tetramer in equilibrium dimer in equilibrium monomer. This means that the deoxyhemoglobin is in R-T equilibrium, and shifts considerably toward the R state under the present conditions. On the other hand, the half-ligated hemoglobin in 1.5 M KI becomes much more dissociable than the deoxy T state and appears to be completely transformed into the R state. Nevertheless, the co-operativity, n, is still high (n = 2.0).     

Coupling of the oxygen-linked interaction energy for inositol hexakisphosphate and bezafibrate binding to human HbA0

The energetics of signal propagation between different functional domains (i.e. the binding sites for O2, inositol hexakisphospate (IHP), and bezafibrate (BZF)) of human HbA0 was analyzed at different heme ligation states and through the use of a stable, partially heme ligated intermediate. Present data allow three main conclusions to be drawn, and namely: (i) IHP and BZF enhance each others binding as the oxygenation proceeds, the coupling free energy going from close to zero in the deoxy state to -3.4 kJ/mol in the oxygenated form; (ii) the simultaneous presence of IHP and BZF stabilizes the hemoglobin T quaternary structure at very low O2 pressures, but as oxygenation proceeds it does not impair the transition toward the R structure, which indeed occurs also under these conditions; (iii) under room air pressure (i.e. pO2 = 150 torr), IHP and BZF together induce the formation of an asymmetric dioxygenated hemoglobin tetramer, whose features appear reminiscent of those suggested for transition state species (i.e. T- and R-like tertiary conformation(s) within a quaternary R-like structure).