The crystal structure of bar-headed goose hemoglobin in deoxy form: the allosteric mechanism of a hemoglobin species with high oxygen affinity

The crystal structure of a high oxygen affinity species of hemoglobin, bar-headed goose hemoglobin in deoxy form, has been determined to a resolution of 2.8 A. The R and R(free) factor of the model are 0.197 and 0.243, respectively. The structure reported here is a special deoxy state of hemoglobin and indicates the differences in allosteric mechanisms between the goose and human hemoglobins. The quaternary structure of the goose deoxy hemoglobin shows obvious differences from that of human deoxy hemoglobin. The rotation angle of one alphabeta dimer relative to its partner in a tetramer molecule from the goose oxy to deoxy hemoglobin is only 4.6 degrees, and the translation is only 0.3 A, which are much smaller than those in human hemoglobin. In the alpha(1)beta(2) switch region of the goose deoxy hemoglobin, the imidazole ring of His beta(2)97 does not span the side-chain of Thr alpha(1)41 relative to the oxy hemoglobin as in human hemoglobin. And the tertiary structure changes of heme pocket and FG corner are also smaller than that in human hemoglobin. A unique mutation among avian and mammalian Hbs of alpha119 from proline to alanine at the alpha(1)beta(1 )interface in bar-headed goose hemoglobin brings a gap between Ala alpha119 and Leu beta55, the minimum distance between the two residues is 4.66 A. At the entrance to the central cavity around the molecular dyad, some residues of two beta chains form a positively charged groove where the inositol pentaphosphate binds to the hemoglobin. The His beta146 is at the inositol pentaphosphate binding site and the salt-bridge between His beta146 and Asp beta94 does not exist in the deoxy hemoglobin, which brings the weak chloride-independent Bohr effect to bar-headed goose hemoglobin.     

X-ray and functional studies of hemoglobins Nancy and Cochin-Port-Royal.

The mutations in hemoglobin Nancy beta145(HC2) Tyr leads to Asp and hemoglobin Cochin-Portal-Royal beta146(HC3) His leads to Arg involve residues which are thought to be essential for the full expression of allosteric action in hemoglobin. Relative to the structure of deoxyhemoglobin A, our x-ray study of deoxyhemoglobin Nancy shows severe disordering of the beta chain COOH-terminal tetrapeptide and a possible movement of the beta heme iron atom toward the plane of the porphyrin ring. These structural perturbations result in a high oxygen affinity, reduced Bohr effect, and lack of cooperatively in hemoglobin Nancy. In the presence of inositol hexaphosphate (IHP), the Hill constant for hemoglobin Nancy increases from 1.1 to 2.0. But relative to its action on hemoglobin A, IHP is much less effective in reducing the oxygen affinity and in increasing the Bohr effect of hemoglobin Nancy. This indicates that IHP does not influence the R in equilibrium T equilibrium as much in hemoglobin Nancy as in hemoglobin A, and this probably is due to the disordering of His 143beta which is known to be part of the IHP binding site. IHP is also known to produce large changes in the absorption spectrum of methemoglobin A, but we find that it has no effect on the spectrum of methemoglobin Nancy. In contrast to the large structural changes in deoxyhemoglobin Nancy, the structure of deoxyhemoglobin Cochin-Port-Royal differs from deoxyhemoglobin A only in the position of the side chain of residue 146beta. The intrasubunit salt bridge between His 146beta and Asp 94beta in deoxyhemoglobin A is lost in deoxyhemoglobin Cochin-Portal-Royal with the guanidinium ion of Arg 146beta floating freely in solution. This small difference in structure results in a reduced Bohr effect, but does not cause a change in the Hill coefficient, the response to 2,3-diphosphoglycerate, or the oxygen affinity at physiological pH.     

Assessment of roles of surface histidyl residues in the molecular basis of the Bohr effect and of beta 143 histidine in the binding of 2,3-bisphosphoglycerate in human normal adult hemoglobin.

Site-directed mutagenesis has been used to construct two mutant recombinant hemoglobins (rHbs), rHb(betaH116Q) and rHb(betaH143S). Purified rHbs were used to assign the C2 proton resonances of beta116His and beta143His and to resolve the ambiguous assignments made over the past years. In the present work, we have identified the C2 proton resonances of two surface histidyl residues of the beta chain, beta116His and beta143His, in both the carbonmonoxy and deoxy forms, by comparing the proton nuclear magnetic resonance (NMR) spectra of human normal adult hemoglobin (Hb A) with those of rHbs. Current assignments plus other previous assignments complete the assignments for all 24 surface histidyl residues of human normal adult hemoglobin. The individual pK values of 24 histidyl residues of Hb A were also measured in deuterium oxide (D(2)O) in 0.1 M N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid (HEPES) buffer in the presence of 0.1 M chloride at 29 degrees C by monitoring the shifts of the C2 proton resonances of the histidyl residues as a function of pH. Among those surface histidyl residues, beta146His has the biggest contribution to the alkaline Bohr effect (63% at pH 7.4), and beta143His has the biggest contribution to the acid Bohr effect (71% at pH 5.1). alpha20His, alpha112His, and beta117His have essentially no contribution; alpha50His, alpha72His, alpha89His, beta97His, and beta116His have moderate positive contributions; and beta2His and beta77His have a moderate negative contribution to the Bohr effect. The sum of the contributions from 24 surface histidyl residues accounted for 86% of the alkaline Bohr effect at pH 7.4 and about 55% of the acid Bohr effect at pH 5.1. Although beta143His is located in the binding site for 2,3-bisphosphoglycerate (2,3-BPG) according to the crystal structure of deoxy-Hb A complexed with 2, 3-BPG, beta143His is not essential for the binding of 2,3-BPG in the neutral pH range according to the proton NMR and oxygen affinity studies presented here. With the accurately measured and assigned individual pK values for all surface histidyl residues, it is now possible to evaluate the Bohr effect microscopically for novel recombinant Hbs with important functional properties, such as low oxygen affinity and high cooperativity. The present study further confirms the importance of a global electrostatic network in regulating the Bohr effect of the hemoglobin molecule.     

Hydrogen-tritium exchange survey of allosteric effects in hemoglobin

The oxy and deoxy forms of hemoglobin display major differences in H-exchange behavior. Hydrogen-tritium exchange experiments on hemoglobin were performed in the low-resolution mode to observe the dependence of these differences on pH (Bohr effect), organic phosphates, and salt. Unlike a prior report, increasing pH was found to decrease the oxy-deoxy difference monotonically, in general accordance with the alkaline Bohr effect. A prior report that the H-exchange difference between oxy- and deoxyhemoglobin vanishes at pH 9, and thus appears to reflect the Bohr effect alone, was found to be due to the borate buffer used, which at high pH tends to abolish the oxy-deoxy difference in a limited region of the H-exchange curve. Effects on hemoglobin H exchange due to organic phosphates parallel the differential binding of these agents (inositol hexaphosphate more than diphosphoglycerate, deoxy more than oxy, at low pH more than at high pH). Added salt slows H exchange of deoxyhemoglobin and has no effect on the oxy form. These results display the sensitivity of simple H-exchange measurements for finding and characterizing effects on structure and dynamics that may occur anywhere in the protein and help to define conditions for higher resolution approaches that can localize the changes observed.     

A high resolution NMR study of localized dynamic and structural perturbations in human hemoglobin modified with thiol reagents

The hydrogen exchange kinetics of the N delta H proton in His F8 of iodoacetamide- and N-ethylmaleimide-treated human deoxyhemoglobins were studied using a NMR method. Comparison with unmodified hemoglobin shows that the reagents, covalently bound to Cys beta 93, significantly increase (about one order of magnitude) the exchange kinetics in beta chains only. This effect was partially reversed by the strong allosteric effector inositol hexaphosphate. Study of the high resolution 400-MHz NMR spectra of modified oxy- and deoxy-hemoglobins permitted localization of the extent of chemically induced structural perturbations. The resonances corresponding to hydrogen bonds specific to the deoxy conformation are not changed, in accord with the preserved cooperativity. Under the experimental conditions (0.1 M bis-Tris, 10 mM Cl-, pH 7.2), the salt bridge at the C terminus of the beta chain in the deoxy state (His beta 146-Asp beta 94) is perturbed by both modifications. The His beta 146 appears to be rendered more immobilized by the reagents in the oxy conformation. From the resonances corresponding to heme pocket protons of oxyhemoglobin it is deduced that the perturbations do not extend over the distal side of the heme pocket but are limited to the FG, F, and HC segments of the beta chain.     

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.     

Identification of an allosterically sensitive unfolding unit in hemoglobin

Hydrogen-exchange studies locate a set of seven allosterically sensitive amide NH protons side by side around two turns of the F-FG helical segment in the hemoglobin beta chain. Some of these protons are on the aqueous protein surface and some deeply inside, yet they all exchange with solvent protons at similar rates. Further, they move in unison to a new common rate when hemoglobin changes its allosteric form. These observations and analogous results for other proteins appear to be inconsistent with penetration-dependent models which relate H-exchange rate to solvent accessibility in the native state. Rather, these results point to sizeable fluctuational distortions that make small sets of protons more or less equally accessible in some transient H-exchange transition state, as visualized in the local unfolding model. The set of allosterically sensitive protons studied here exchanges 30-fold faster in liganded hemoglobin than in the deoxy form. In terms of the unfolding model, this means that the F-FG structure is relatively destabilized in oxyhemoglobin, so that the allosterically linked change in structural free energy at F-FG favors the deoxy state. The 30-fold change in H-exchange rate suggests a contribution to the allosteric free energy by this segment of 2 kcal (1 cal = 4.184 J). These experiments utilized a labeling technique, described earlier, that selectively places tritium on sites whose H-exchange rates are sensitive to the protein functional state, and used a method introduced by Rosa & Richards (1979,1981) to locate this label in the protein. The latter method, which rapidly separates protein fragments under conditions that can preserve exchangeable label, was here brought to a more quantitative level. Taken together, these techniques provide a "functional labeling" method capable of selectively labeling and identifying protein segments that participate in functional interactions.     

Hemoglobin Cochin-Port-Royal: consequences of the replacement of the beta chain C-terminal by an arginine.

Hemoglobin Cochin Port-Royal beta 146 (HC3) His yields Arg is the second example in which the beta C-terminal residue is replaced. Owing to the known importance of His beta 146 in the co-operative effects of hemoglobin, the functional properties of this variant were carefully studied. It had a normal Hill coefficient but a reduced alkaline Bohr effect. However, the reduction in Bohr effect is less than the halving predicted from previous mutants and modified hemoglobins.     

Role of Bohr group salt bridges in cooperativity in hemoglobin.

Possible problems in measuring the first Adair constant, K1, from accurate oxygen equilibrium curves have been investigated. Of these only the presence of small amounts of CO-hemoglobin or hemoglobin dimers had a significant effect. The former can be eliminated by treatment with oxygen, the latter by measuring the concentration-dependence of K1 or working at high protein concentrations. K1 values have been measured for normal hemoglobin at pH 7 and 9, hemoglobin specifically reacted with cyanate at Val 1alpha (alphac2beta2) and des(His 146beta) hemoglobin at pH 7. K1 is equal to KT, the oxygen affinity of the T state of hemoglobin, for all these hemoglobins and was increased in all of them when compared to normal hemoglobin at pH 7. This shows that the breakage of the Bohr group salt bridges by increasing pH or specific modification changes KT. Hence the Bohr group salt bridges break on ligation of the T state and are partially responsible for the free energy of cooperativity.     

Minimal structural requirements for root effect: crystal structure of the cathodic hemoglobin isolated from the antarctic fish Trematomus newnesi

The cathodic hemoglobin component of the Antarctic fish Trematomus newnesi (HbCTn) is a Root-effect protein. The interpretation of its functional properties in relation to its sequence is puzzling. Indeed, HbCTn sequence is characterized by an extremely low histidyl content, and in particular by the lack of His146beta and His69beta, which are believed to be important in Bohr and Root effects, respectively. Furthermore, previous analyses suggested that the local environment of Asp95alpha, Asp99beta, and Asp101beta should not be appropriate for the formation of Asp-Asp interactions, which are important for the Root effect. Here, we report the high-resolution crystal structure of the deoxy form of HbCTn. Our data provide a structural interpretation for the very low oxygen affinity of the protein and insights into the structural determinants of the Root effect protein. The structure demonstrates that the presence of Ile41alpha and Ser97alpha at the alpha1beta2 interface does not prevent the formation of the inter-Asp interactions in HbCTn, as previous studies had suggested. The present data indicate that the hydrogen bond formed between Asp95alpha and Asp101beta, which is stabilized by Asp99beta, is per se sufficient to generate the Root effect, and it is the minimal structural requirement needed for the design of Root-effect Hbs.     

Heats of carbon monoxide binding by hemoglobin M Iwate.

The heat of reaction of CO gas with the alpha2Mmetbeta2 and alpha2Mbeta2 species of the alpha-chain mutant hemoglobin M Iwate has been studied in buffers with different heats of ionization of 25degrees and in the absence of organic phosphates. For the alpha2Mmetbeta2deoxy species we find a small Bohr effect (0.12 mol of H+/mol of CO) which is in correspondence with that found in equilibrium studies. The heat of reaction, when corrected for proton reaction with buffer, is -18.4 +/- 0.3 kcal/mol of CO at pH 7.4 At pH 9 the same value is observed within experimental error. This value compares closely with heats of reaction of CO with myoglobin and with van't Hoff determinations of the heat of oxygen binding to isolated hemoglobin alpha and beta chains after correction for the heat of replacement of O2 by CO. Furthermore, an analysis of the differential heat of ligand binding as a function of the extent of reaction indicated that, within experimental error, the heat of reaction with the first beta-chain heme in alpha2Mmetbeta2deoxy is the same as the second. Since the quaternary Tleads to R transition is blocked in this mutant hemoglobin, we compared it with Hb A to estimate the enthalpic component of the allosteric T leads to R transition in Hb A. The heats of reaction with CO(g) and Hb A are -15.7 +/- 0.5 and -20.9 +/- 0.5 kcal/mol at pH 7.4 and 9.0, respectively. In going from the T to the R state we find an enthalpy of transition of 9 +/- 2.5 kcal at pH 7.4 and -12 +/- 2.5 kcal at pH 9.0. From published free energies of transsition we conclude the T leads to R transition is enthalpically controlled at p/ 7.4 but entropically controlled at pH 9.0 A near normal Bohr effect is estimated from heats of reaction of CO with alpha2Mdeoxybeta2deoxy in various buffers. A large than normal heat of reaction (-21.6 +/- 0.5 kcal/mol of CO) is attributed to the abnormal alpha chains in Hb M Iwate.     

The CO and NO Bohr effect of human hemoglobin with and without inositolhexaphosphate.

Using NO and CO as ligands the Bohr effect of human hemoglobin has been measured with and without inositolhexophosphate. It appears that in the absence and presence of inositolhexaphosphate hemoglobin shows a distinct ligand specificity with respect to the Bohr effect. Ligation with NO is accompanied by release of a larger number of Bohr effect. It is shown that this latter result is due to the fact that the number of protons taken up upon binding of inositolhexaphosphate to ligated hemoglobin is larger for HbNO than for HbCO. It is suggested that this additional proton uptake is partially due to a restoration of the saltbridge between His 146beta and Asp 94beta upon addition of IHP.     

Structure-function relationships in hemoglobin Kariya, Lys-40(C5) alpha----Glu, with high oxygen affinity. Functional role of the salt bridge between Lys-40 alpha and the beta chain COOH terminus

The epsilon-amino group of Lys-40 alpha forms a salt bridge with the alpha-carboxyl group of beta chain in deoxyhemoglobin and is considered to impose a constraint upon hemoglobin tetramer, stabilizing the T quaternary structure. Hb Kariya, in which Lys-40 alpha is replaced by Glu, provides a unique opportunity to investigate the functional role of this salt bridge. Hb Kariya showed oxygen binding properties characterized by a high affinity, diminished cooperativity, a reduced alkaline Bohr effect, and a decreased effect of phosphates upon oxygen affinity. In deoxyHb Kariya the reactivity of the sulfhydryl groups of cysteins-93 beta with 4,4'-dipyridine disulfide was profoundly enhanced, being comparable to that for normal oxyhemoglobin (oxyHb A). The Soret band spectra, UV derivative spectra, and UV oxyminus-deoxy difference spectra indicated that oxyHb Kariya assumes a quaternary structure similar to that of oxyHb A whereas the T structure of deoxyHb Kariya is destabilized, and Hb Kariya remains predominantly in the R state upon deoxygenation. Resonance Raman scattering by deoxyHb Kariya showed that the Fe-N epsilon(proximal His) bond is less stretched than that of deoxyHb A. These experimental results provide structural basis for explaining the oxygen binding characteristics of Hb Kariya and further give direct evidence that the intersubunit salt bridge between Lys-40 alpha and the beta chain COOH terminus actually contributes to stabilization of the T quaternary structure, thereby playing a key role in cooperative oxygen binding by hemoglobin. The nature of another salt bridge between Asp-94 beta and the COOH-terminal His of beta chain was also discussed in comparison with the salt bridge involving Lys-40 alpha.     

Ligand binding properties and structural studies of recombinant and chemically modified hemoglobins altered at beta 93 cysteine

To investigate the roles of beta93 cysteine in human normal adult hemoglobin (Hb A), we have constructed four recombinant mutant hemoglobins (rHbs), rHb (betaC93G), rHb (betaC93A), rHb (betaC93M), and rHb (betaC93L), and have prepared two chemically modified Hb As, Hb A-IAA and Hb A-NEM, in which the sulfhydryl group at beta93Cys is modified by sulfhydryl reagents, iodoacetamide (IAA) and N-ethylmaleimide (NEM), respectively. These variants at the beta93 position show higher oxygen affinity, lower cooperativity, and reduced Bohr effect relative to Hb A. The response of some of these Hb variants to allosteric effectors, 2,3-bisphosphoglycerate (2,3-BPG) and inositol hexaphosphate (IHP), is decreased relative to that of Hb A. The proton nuclear magnetic resonance (NMR) spectra of these Hb variants show that there is a marked influence on the proximal heme pocket of the beta-chain, whereas the environment of the proximal heme pocket of the alpha-chain remains unchanged as compared to Hb A, suggesting that higher oxygen affinity is likely to be determined by the heme pocket of the beta-chain rather than by that of the alpha-chain. This is further supported by NO titration of these Hbs in the deoxy form. For Hb A, NO binds preferentially to the heme of the alpha-chain relative to that of the beta-chain. In contrast, the feature of preferential binding to the heme of the alpha-chain becomes weaker and even disappears for Hb variants with modifications at beta93Cys. The effects of IHP on these Hbs in the NO form are different from those on HbNO A, as characterized by (1)H NMR spectra of the T-state markers, the exchangeable resonances at 14 and 11 ppm, reflecting that these Hb variants have more stability in the R-state relative to Hb A, especially rHb (betaC93L) and Hb A-NEM in the NO form. The changes of the C2 proton resonances of the surface histidyl residues in these Hb variants in both the deoxy and CO forms, compared with those of Hb A, indicate that a mutation or chemical modification at beta93Cys can result in conformational changes involving several surface histidyl residues, e.g., beta146His and beta2His. The results obtained here offer strong evidence to show that the salt bridge between beta146His and beta94Asp and the binding pocket of allosteric effectors can be affected as the result of modifications at beta93Cys, which result in the destabilization of the T-state and a reduced response of these Hbs to allosteric effectors. We further propose that the impaired alkaline Bohr effect can be attributed to the effect on the contributions of several surface histidyl residues which are altered because of the environmental changes caused by mutations and chemical modifications at beta93Cys.     

Influence of allosteric effectors and temperature on oxygen binding properties and the Bohr effect of bovine hemoglobin

The O2 binding properties of bovine Hb were examined. The increase in Cl- and DPG concentration enhanced P50. A reduction in n(max) was observed at high Cl- concentration, while DPG had little effect on n(max). An increase in Cl- concentration enhanced the Bohr effect, the magnitude of which reached a maximum at 0.1 M Cl- and 20 degrees C. This concentration is nearly equal to that at the highest slope of the log P50 vs. log [Cl-] plot, and also equal to the physiological Cl- concentration (0.1 M) of bovine blood. Furthermore, the influence of Cl- concentration on the Bohr effect is independent of temperature. On the other hand, in the absence of Cl-, bovine Hb is sensitive to DPG; an increase in DPG concentration enhanced the Bohr effect, which reached a maximum at 3 mM DPG and 20 degrees C. This concentration is nearly equal to that at the highest slope of the log P50 vs. log [DPG] plot. At low DPG concentrations, the DPG effect on the Bohr effect became small with increasing temperature, whereas at high DPG concentrations, the DPG effect was insensitive to temperature changes. At the physiological concentration of DPG (0.5 mM), increases in both Cl- concentration and temperature diminished the DPG effect. At the physiological concentrations of Cl- and DPG, the Bohr effect was -0.36 at 37 degrees C. The deltaH value at the physiological concentrations of Cl- and DPG was approximately -5.8 kcal/mol at pH 7.4. These results indicate that Cl- and temperature are important determinants of the O2 binding properties of bovine Hb.     

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.     

Covalent binding of glutathione to hemoglobin. II. Functional consequences and structural changes reflected in NMR spectra

Binding of glutathione by disulfide linkage to Cys-beta 93 of hemoglobin tetramers within sickle cells increases the oxygen affinity and significantly inhibits sickling at low partial oxygen pressure (Garel, M-C., Domenget, C., Caburi-Martin, J., Prehu, C., Galacteros, F., and Beuzard, Y. (1986) J. Biol. Chem. 261, 14704-14709). This article reports a characterization of the oxygen-binding properties of glutathionyl hemoglobin (G-Hb) in solution in the presence or absence of allosteric effectors. The studies reveal a nearly 6-fold increase in oxygen affinity compared to native HbA and a Hill coefficient at half-saturation (n50) of 1.50 compared to n50 of approximately 2.9 for HbA. The oxygen Bohr effect measured in the alkaline pH range is reduced by 38%. Addition of 2,3-diphosphoglycerate decreases the oxygen affinity of G-Hb and HbA to a similar extent and increases the Bohr effect, indicating that the binding sites for organic phosphates are not perturbed in G-Hb. The rate of autooxidation of G-HbO2 is slower than of HbAO2. Oxidation by ferricyanide of G-HbCO is also reduced and is biphasic, demonstrating a heterogeneous susceptibility of the hemes in G-Hb. Flash photolysis experiments indicate that the tetramer-dimer dissociation constant is 1 order of magnitude greater for G-HbCO than for HbACO. High resolution NMR spectra at 400 MHz show that in G-Hb: the tertiary structure of the beta heme pocket is significantly perturbed, particularly in the F helix and the EF corner; the formation of the salt bridge between His-beta 146 and Asp-beta 94, a feature of the deoxy state, is precluded; and a deoxy interchain (alpha 1 beta 2) contact between Asp beta 2 99 and Tyr alpha 1 42 is appreciably destabilized. The NMR data provide a structural basis for interpreting the high oxygen affinity, reduced cooperativity, and diminished polymerization of G-HbS.     

High resolution NMR studies of histidine-substituted and histidine-perturbed hemoglobin variants. Histidine assignments, electrostatic interactions at the protein surface, and implications for hemoglobin S polymerization

The aromatic region of the proton NMR spectrum of human adult hemoglobin (HbA) contains resonances from at least 11 titratable histidine residues. Assignments for five beta chain histidines have previously been proposed. In order to further characterize the aromatic spectra of HbA we studied 11 histidine-substituted and -perturbed hemoglobin variants in oxy and deoxy states and at different pH values by 400 MHz NMR spectroscopy. We propose assignments for the resonances corresponding to the C2 protons of His alpha 20, His alpha 72, His alpha 112, and His beta 77 in oxy and deoxy spectra and of His beta 97 and His beta 117 in deoxy spectra. Our assignments for His beta 2 and His beta 117 in the oxy state agree with those previously reported for the CO form, but in the deoxy state our spectra suggest a different assignment. Studies with Hb variants in which a histidine is perturbed by a neighboring substitution suggest additional assignments for His alpha 50 and His alpha 89 and demonstrate a strong dependence of the imidazole ring pK on hydrogen bond interactions and on the net charge of neighboring residues. Some of the newly proposed assignments of histidine resonances are used to discuss specific intermolecular interactions implicating His alpha 20, His beta 77, and His beta 117 in deoxy HbS polymers.     

Hemoglobin function in the vertebrates: An evolutionary model

Comparative data on quaternary structure, cooperativity, Bohr effect and regulation by organic phosphates are reviewed for vertebrate hemoglobins. A phylogeny of hemoglobin function in the vertebrates is deduced. It is proposed that from the monomeric hemoglobin of the common ancestor of vertebrates, a deoxy dimer, as seen in the lamprey, could have originated with a single amino acid substitution. The deoxy dimer has a Bohr effect, cooperativity and a reduced oxygen affinity compared to the monomer. One, or two, additional amino acid substitutions could have resulted in the origin of a tetrameric deoxy hemoglobin which dissociated to dimers on oxygenation. Gene duplication, giving incipient alpha and beta genes, probably preceded the origin of a tetrameric oxyhemoglobin. The origin of an organic phosphate binding site on the tetrameric hemoglobin of an early fish required only one, or two, amino acid substitutions. ATP was the first organic phosphate regulator of hemoglobin function. The binding of ATP by hemoglobin may have caused the original elevation in the concentration of ATP in the red blood cells by relieving end product inhibition of ATP synthesis. The switch from regulation of hemoglobin function by ATP to regulation by DPG may have been a consequence of the curtailment of oxidative phosphorylation in the red blood cell. The basic mechanisms by which ATP and DPG concentrations can respond to strss on the oxygen transport system were present before the origin of an organic phosphate binding site on hemoglobin. A switch from ATP regulation to IP5 regulation occurred in the common ancestor of birds.