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.     

Proton nuclear magnetic resonance studies of hemoglobins Osler (beta145HC2 Tyr replaced by Asp) and McKee Rocks (beta145HC2 Tyr replaced by term): an assignment for an important tertiary structural probe in hemoglobin

High-resolution proton nuclear magnetic resonance studies of deoxyhemoglobins Osler (beta145HC2 Tyr replaced by Asp) and McKees Rocks (beta 145HC2 Tyr replaced by term) indicate that these hemoglobins are predominately in the oxy quaternary structure in 0.1 M [bis(2-hydroxyethyl)imino]-tris(hydroxymethyl) methane buffer at pH 7. Upon the addition of inositol hexaphosphate, the proton nuclear magnetic resonance spectra of these hemoglobins become similar to those characteristic of a hemoglobin molecule in the deoxy quaternary structure. The exchangeable proton resonance which is found at -6.4 ppm from H2O in the spectrum of normal human adult deoxyhemoglobin is absent in the spectra of these two mutant hemoglobins. Consequently we believe the hydrogen bond between the hydroxyl group of tyrosine-beta145HC2 and the carboxyl oxygen of valine-beta98FG5 gives rise to this resonance. This assignment allows us to use the -6.4ppm resonance as an important tertiary structural probe in the investigation of the cooperative oxygenation of hemoglobin.     

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.     

Proton nuclear magnetic resonance studies of hemoglobin Providence (beta82EF6 Lys replaced by Asn or Asp): a residue involved in anion binding

High-resolution proton nuclear magnetic resonance studies of hemoglobins Providence-Asn (beta82EF6 Lys replaced by Asn) and Providence-Asp (beta82EF6 Lys replaced by Asp) show that different amino acid substitutions at the same position in the hemoglobin molecule have different effects on the structure of the protein molecule. Hemoglobin Providence-Asp appears to be in a low-affinity tertiary structure in both the deoxy and carbonmonoxy forms. Deoxyhemoglobin Providence-Asn has its beta heme resonance shifted downfield slightly from its position in normal adult hemoglobin; however, the tertiary structures of the heme pocket of hemoglobins A and Providence-Asn are very similar when both proteins are in the carbonmonoxy form. These results are consistent with the oxygen equilibrium measurements of Bonaventura, J., et al. [(1976) J. Biol. Chem. 251, 7563] which show that both Hb Providence-Asn and Hb Providence-Asp have oxygen affinities lower than normal adult hemoglobin, with Hb Providence-Asp having the lowest. Our studies of the effects of sodium chloride on the hyperfine shifted proton resonances of deoxyhemoglobins A, Providence-Asn, and Providence-Asp indicate that the beta82EF6 lysine is probably one, but not the only binding site for chloride ions.     

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.     

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.     

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.     

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.     

Ruthenium-iron hybrid hemoglobins as a model for partially liganded hemoglobin: oxygen equilibrium curves and resonance Raman spectra

The structure and function of iron(II)-ruthenium(II) hybrid hemoglobins alpha(Ru-CO)2 beta(Fe)2 and alpha(Fe)2 beta(Ru-CO)2, which can serve as models for the intermediate species of the oxygenation step in native human adult hemoglobin, were investigated by measuring oxygen equilibrium curves and the Fe(II)-N epsilon (His F8) stretching resonance Raman lines. The oxygen equilibrium properties indicated that these iron-ruthenium hybrid hemoglobins are good models for the half-liganded hemoglobin. The pH dependence of the oxygen binding properties and the resonance Raman line revealed that the quaternary and tertiary structural transition was induced by pH changes. When the pH was lowered, both the iron-ruthenium hybrid hemoglobins exhibited relatively higher cooperativity and a Raman line typical of normal deoxy structure, suggesting that their structure is stabilized at a "T-like" state. However, the oxygen affinity of alpha(Fe)2 beta(Ru-CO)2 was lower than that of alpha(Ru-CO)2 beta(Fe)2, and the transition to the "deoxy-type" Fe-N epsilon stretching Raman line of alpha(Fe2)beta(Ru-CO)2 was completed at pH 7.4, while that of the complementary counterpart still remained in an "oxy-like" state under the same condition. These observations clearly indicate that the beta-liganded hybrid has more "T"-state character than the alpha-liganded hybrid. In other words, the ligation to the alpha subunit induces more pronounced changes in the structure and function in Hb than the ligation to the beta subunit. This feature agrees with our previous observations by NMR and sulfhydryl reactivity experiments. The present results are discussed in relation to the molecular mechanism of the cooperative stepwise oxygenation in native human adult hemoglobin.     

A proton nuclear magnetic resonance investigation of the anion Bohr effect of human normal adult hemoglobin

High-resolution proton nuclear magnetic resonance spectroscopy has been used to investigate the molecular mechanism of the Bohr effect of human normal adult hemoglobin in the presence of two allosteric effectors, i.e., chloride and inorganic phosphate ions. The individual hydrogen ion equilibria of 22-26 histidyl residues of hemoglobin have been measured in anion-free 0.1 M HEPES buffer and in the presence of 0.18 M chloride or 0.1 M inorganic phosphate ions in both deoxy and carbonmonoxy forms. The results indicate that the beta 2-histidyl residues are strong binding sites for chloride and inorganic phosphate ions in hemoglobin. The affinity of the beta 2-histidyl residues for these anions is larger in the deoxy than in the carbonmonoxy form. Nevertheless, the contribution of these histidyl residues to the anion Bohr effect is small due to their low pK value in deoxyhemoglobin in anion-free solvents. The interactions of chloride and inorganic phosphate ions with the hemoglobin molecule also result in lower pK values and/or changes in the shapes of the hydrogen ion binding curves for several other surface histidyl residues. These results suggest that long-range electrostatic interactions between individual ionizable sites in hemoglobin could play an important role in the molecular mechanism of the anion Bohr effect.     

The mutation beta 99 Asp-Tyr stabilizes Y--a new, composite quaternary state of human hemoglobin

Carbonmonoxy hemoglobin Ypsilanti (beta 99 Asp-Tyr) exhibits a quaternary form distinctly different from any structures previously observed for human hemoglobins. The relative orientation of alpha beta dimers in the new quaternary form lies well outside the range of values observed for normal unliganded and liganded tetramers (Baldwin, J., Chothia, C., J. Mol. Biol. 129:175-220, 1979). Despite this large quaternary structural difference between carbonmonoxy hemoglobin Ypsilanti and the two canonical structures, the new quaternary structure's hydrogen bonding interactions in the "switch" region, and packing interactions in the "flexible joint" region, show noncovalent interactions characteristic of the alpha 1 beta 2 contacts of both unliganded and liganded normal hemoglobins. In contrast to both canonical structures, the beta 97 histidine residue in carbonmonoxy hemoglobin Ypsilanti is disengaged from quaternary packing interactions that are generally believed to enforce two-state behavior in ligand binding. These features of the new quaternary structure, denoted Y, may therefore be representative of quaternary states that occur transiently along pathways between the normal unliganded, T, and liganded, R, hemoglobin structures.     

Conformation of bovine nitrosylhemoglobins: an ESR study

The structural properties of nitrosylhemoglobins from two bovine species, namely cow and buffalo, have been investigated using electron spin resonance spectroscopy. Bovine hemoglobins show sensitivity to the presence of chloride ions and organic phosphates. ESR spectral features indicate a stable deoxy quaternary conformation of the molecule when compared to normal adult human hemoglobin A. Amino acid substitutions at the amino terminal end of the beta chain and at other sites of the alpha and beta chains seems to shift the allosteric equilibrium towards the T state in bovine hemoglobins. The results also confirm the intrinsically low oxygen affinity of bovine hemoglobins under physiological conditions.     

Assessment of role of beta 146-histidyl and other histidyl residues in the Bohr effect of human normal adult hemoglobin

The contribution of the carboxyl-terminal histidines of the beta chains, beta 146(HC3), to the alkaline Bohr effect of human normal adult hemoglobin has been shown by this laboratory to depend upon the solvent composition. Using high-resolution proton nuclear magnetic resonance spectroscopy, we have found that the pKa value of the beta 146-histidine is 8.0 in the deoxy form, while in the carbonmonoxy form it ranges from 7.1 to 7.85 depending upon the concentration of inorganic phosphate and chloride ions present. These conclusions have been questioned by Perutz and co-workers on the basis of biochemical, structural, and proton nuclear magnetic resonance studies of mutant and enzymatically or chemically modified hemoglobins [Perutz, M. F., Kilmartin, J. V., Nishikura, K., Fogg, J. H., Butler, P. J., & Rollema, H. S. (1980) J. Mol. Biol. 138, 649-670; Kilmartin, J. V., Fogg, J. H., & Perutz, M. F. (1980) Biochemistry 19, 3189-3193; Perutz, M. F., Gronenborn, A. M., Clore, G. M., Fogg, J. H., & Shih, D. T.-b. (1985) J. Mol. Biol. 183, 491-498]. In this work, we use proton nuclear magnetic resonance spectroscopy to assess the effects of structural modifications on the histidyl residues and on the overall conformation of the hemoglobin molecule in solution. The structural perturbations investigated all occur within the tertiary domains around the carboxyl-terminal region of the beta chain as follows: Hb Cowtown (beta 146His----Leu); Hb Wood (beta 97His----Leu); Hb Malm? (beta 97His----Gln); Hb Abruzzo (beta 143His----Arg). Our results demonstrate that the conformational effects of single-site structural modifications upon the conformation and dynamics of hemoglobin depend strongly on their location in the three-dimensional structure of the protein molecule and also on their chemical nature. Furthermore, in normal hemoglobin, the spectral properties of several surface histidyl residues are found to depend, in the ligated state, upon the nature of the ligand. Our present findings do not support the recent spectral assignments proposed by Perutz et al. (1985) for the proton resonances of the beta 146- and beta 97-histidines and their suggestion that the enzymatic removal of the carboxyl-terminal beta 146-histidyl residues induces a conformational equilibrium for the beta 97-histidines in the des-beta 146His hemoglobin molecule in the carbonmonoxy form.     

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...     

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).     

A proton nuclear magnetic resonance investigation of histidyl residues in human normal adult hemoglobin

High-resolution proton nuclear magnetic resonance (NMR) spectroscopy at 250 MHz has been used to titrate 22 individual surface histidyl residues (11 per alpha beta dimer) of human normal adult hemoglobin in both the deoxy and the carbon monoxy forms. The proton resonances of beta 2, beta 143, and beta 146 histidyl residues are assigned by a parallel 1H NMR titration of appropriate mutant and chemically modified hemoglobins. The pK values of the 22 histidyl residues investigated are found to range from 6.35 to 8.07 in the deoxy form and from 6.20 to 7.87 in the carbon monoxy form, in the presence of 0.1 M Bis-Tris or 0.1 M Tris buffer in D2O with chloride ion concentrations varying from 5 to 60 mM at 27 degrees C. Four histidyl residues in the deoxy form and one histidyl residue in the carbon monoxy form are found to have proton nuclear magnetic resonance titration curves that deviate greatly from that predicted by the simple proton dissociation equilibrium of a single ionizable group. The proton nuclear magnetic resonance data are used to ascertain the role of several surface histidyl residues in the Bohr effect of hemoglobin under the above-mentioned experimental conditions. Under these experimental conditions, we have found that (i) the beta 146 histidyl residues do not change their electrostatic environments significantly upon binding of ligand to deoxyhemoglobin and, thus, their contribution to the Bohr effect is negligible, (ii) the beta 2 histidyl residues have a negative contribution to the Bohr effect, and (iii) the total contribution of the 22 histidyl residues investigated here to the Bohr effect is, in magnitude, comparable to the Bohr effect observed experimentally. These results suggest that the molecular mechanism of the Bohr effect proposed by Perutz [Perutz, M.F. (1970) Nature (London) 228, 726-739] is not unique and that the detailed mechanism depends on experimental conditions, such as the solvent composition.     

1H and 31P nuclear magnetic resonance investigation of the interaction between 2,3-diphosphoglycerate and human normal adult hemoglobin

High-resolution 1H and 31P nuclear magnetic resonance spectroscopy has been used to investigate the binding of 2,3-diphosphoglycerate to human normal adult hemoglobin and the molecular interactions involved in the allosteric effect of the 2,3-diphosphoglycerate molecule on hemoglobin. Individual hydrogen ion NMR titration curves have been obtained for 22-26 histidyl residues of hemoglobin and for each phosphate group of 2,3-diphosphoglycerate with hemoglobin in both the deoxy and carbonmonoxy forms. The results indicate that 2,3-diphosphoglycerate binds to deoxyhemoglobin at the central cavity between the two beta chains and the binding involves the beta 2-histidyl residues. Moreover, the results suggest that the binding site of 2,3-diphosphoglycerate to carbonmonoxyhemoglobin contains the same (or at least some of the same) amino acid residues responsible for binding in the deoxy form. As a result of the specific interactions with 2,3-diphosphoglycerate, the beta 2-histidyl residues make a significant contribution to the alkaline Bohr effect under these experimental conditions (up to 0.5 proton/Hb tetramer). 2,3-Diphosphoglycerate also affects the individual hydrogen ion equilibria of several histidyl residues located away from the binding site on the surface of the hemoglobin molecule, and, possibly, in the heme pockets. These results give the first experimental demonstration that long-range electrostatic and/or conformational effects of the binding could play an important role in the allosteric effect of 2,3-diphosphoglycerate on hemoglobin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of anions on the molecular basis of the Bohr effect of hemoglobin

High-resolution 1H-NMR spectroscopy has been used to investigate the molecular basis of the Bohr effect in human normal adult hemoglobin in the presence of anions which serve as heterotropic effectors, i.e., Cl-, Pi, and 2,3-diphosphoglycerate. The individual H+ equilibria of 22-26 histidyl residues of hemoglobin in both deoxy and carbonmonoxy forms have been measured under buffer conditions chosen to demonstrate the effects of anion binding. The results indicate that beta 2His residues are binding sites for Cl- and Pi in both deoxy and carbonmonoxy forms, and that the affinity of this site for these anions is greater in the deoxy form. Recently assigned, the resonance of beta 146His does not show evidence of involvement in anion binding. The results also indicate that the binding of 2,3-diphosphoglycerate at the central cavity between the two beta-chains in deoxyhemoglobin involves the beta 2His residues, and that the 2,3-diphosphoglycerate-binding site in carbonmonoxyhemoglobin may remain similar to that in deoxyhemoglobin. The interactions of Cl-, Pi and 2,3-diphosphoglycerate also result in changes in the pK values for other surface histidyl residues which vary in both magnitude and direction. The array of pK changes is specific for the interaction of each effector. The participation of beta 2His in the Bohr effect demonstrates that this residue can release or capture protons, depending on its protonation properties and its linkage to anion binding, and therefore provides an excellent illustration of the variable roles of a given amino acid. Although beta 146His does not bind anions, its contributions to the Bohr effect are substantially affected by the presence of anions. These results demonstrate that long-range electrostatic and/or conformational effects of anions binding play significant roles in the molecular basis of the Bohr effect of hemoglobin.     

Circular dichroism and spin-label studies of carp hemoglobin

Circular dichroism (c.d.) spectra were obtained for deoxy, oxy, carboxy, nitrosyl, aquomet and azidomet derivatives of carp hemoglobin. The spectra of the hemolysate and its two major components are virtually identical. Binding of diatomic ligands induces large changes in the 287 nm ellipticity. In the case of oxygen binding this change appears to be proportional to the free energy of co-operation. The changes of L-band ellipticity and Soret rotational strength with ligation reflect tertiary structural alterations and bear no relationship to quaternary transitions. The c.d. results indicate that carp deoxyhemoglobin has very similar tertiary and quaternary structures between pH 6·4 and 8·0, whereas the oxyhemoglobin undergoes continuous conformational adjustment in response to pH changes. The effect of inositol hexaphosphate on c.d. spectra is much smaller than it is on the functional properties. Electron paramagnetic resonance spectra of iodoacetamide nitroxide label are sensitive to ligation, the label is probably attached to Cys142β.