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.     

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.     

Roles of the beta 146 histidyl residue in the molecular basis of the Bohr effect of hemoglobin: a proton nuclear magnetic resonance study

Assessment of the roles of the carboxyl-terminal beta 146 histidyl residues in the alkaline Bohr effect in human normal adult hemoglobin by high-resolution proton nuclear magnetic resonance spectroscopy requires assignment of the resonances corresponding to these residues. Previous resonance assignments in low ionic strength buffers for the beta 146 histidyl residue in the carbonmonoxy form of hemoglobin have been controversial [see Ho and Russu (1987) Biochemistry 26, 6299-6305; and references therein]. By a careful spectroscopic study of human normal adult hemoglobin, enzymatically prepared des(His146 beta)-hemoglobin, and the mutant hemoglobins Cowtown (beta 146His----Leu) and York (beta 146His----Pro), we have resolved some of these conflicting results. By a close incremental variation of pH over a wide range in chloride-free 0.1 M N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid buffer, a single resonance has been found to be consistently missing in the proton nuclear magnetic resonance spectra of these hemoglobin variants. The spectra of each of these variants show additional perturbations; therefore, the assignment has been confirmed by an incremental titration of buffer conditions to benchmark conditions, i.e., 0.2 M phosphate, where the assignment of this resonance is unambiguous. The strategy of incremental titration of buffer conditions also allows extension of this resonance assignment to spectra taken in 0.1 M [bis(2-hydroxyethyl)amino]tris(hydroxymethyl)methane buffer. Participation of the beta 146 histidyl residues in the Bohr effect has been calculated from the pK values determined for the assigned resonances in chloride-free 0.1 M N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid buffer. Our results indicate that the contribution of the beta 146 histidyl residues is 0.52 H+/hemoglobin tetramer at pH 7.6, markedly less than the 0.8 H+/hemoglobin tetramer estimated by study of the mutant hemoglobin Cowtown (beta 146His----Leu) by Shih and Perutz [(1987) J. Mol. Biol. 195, 419-422]. We have found that at least two histidyl residues in the carbonmonoxy form of this mutant have pK values that are perturbed, and we suggest that these pK differences may in part account for this discrepancy. Furthermore, summation of the positive contribution of the beta 146 histidyl residues and the negative contribution of the beta 2 histidyl residues to the maximum Bohr effect measured in 0.1 M N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid buffer suggests that additional sites in the hemoglobin molecule account for proton release upon ligation greater than the contribution of the beta 146 histidyl residues.(ABSTRACT TRUNCATED AT 400 WORDS)     

Novel recombinant hemoglobin, rHb (beta N108Q), with low oxygen affinity, high cooperativity, and stability against autoxidation

Using our Escherichia coli expression system, we have constructed rHb (beta N108Q), a new recombinant hemoglobin (rHb), with the amino acid substitution located in the alpha(1)beta(1) subunit interface and in the central cavity of the Hb molecule. rHb (beta N108Q) exhibits low oxygen affinity, high cooperativity, enhanced Bohr effect, and slower rate of autoxidation of the heme iron atoms from the Fe(2+) to the Fe(3+) state than other low-oxygen-affinity rHbs developed in our laboratory, e.g., rHb (alpha V96W) and rHb (alpha V96W, beta N108K). It has been reported by Olson and co-workers [Carver et al. (1992) J. Biol. Chem. 267, 14443-14450; Brantley et al. (1993) J. Biol. Chem. 268, 6995-7010] that the substitution of phenylalanine for leucine at position 29 of myoglobin can inhibit autoxidation in myoglobin and at position 29 of the alpha-chain of hemoglobin can lower NO reaction in both the deoxy and the oxy forms of human normal adult hemoglobin. Hence, we have further introduced this mutation, alpha L29F, into beta N108Q. rHb (alpha L29F, beta N108Q) is stabilized against auto- and NO-induced oxidation as compared to rHb (beta N108Q), but exhibits lower oxygen affinity at pH below 7.4 and good cooperativity as compared to Hb A. Proton nuclear magnetic resonance (NMR) studies show that rHb (beta N108Q) has similar tertiary structure around the heme pockets and quaternary structure in the alpha(1)beta(1) and alpha(1)beta(2) subunit interfaces as compared to those of Hb A. The tertiary structure of rHb (alpha L29F, beta N108Q) as measured by (1)H NMR, especially the alpha-chain heme pocket region (both proximal and distal histidyl residues), is different from that of CO- and deoxy-Hb A, due to the amino acid substitution at alpha L29F. (1)H NMR studies also demonstrate that rHb (beta N108Q) can switch from the R quaternary structure to the T quaternary structure without changing ligation state upon adding an allosteric effector, inositol hexaphosphate, and reducing the temperature. On the basis of its low oxygen affinity, high cooperativity, and stability against autoxidation, rHb (beta N108Q) is considered a potential candidate for the Hb-based oxygen carrier in a blood substitute system.     

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.     

Effects of amino acid substitutions at beta 131 on the structure and properties of hemoglobin: evidence for communication between alpha 1 beta 1- and alpha 1 beta 2-subunit interfaces

Substitutions of Asn, Glu, and Leu for Gln at the beta131 position of the hemoglobin molecule result in recombinant hemoglobins (rHbs) with moderately lowered oxygen affinity and high cooperativity compared to human normal adult hemoglobin (Hb A). The mutation site affects the hydrogen bonds present at the alpha(1)beta(1)-subunit interface between alpha103His and beta131Gln as well as that between alpha122His and beta35Tyr. NMR spectroscopy shows that the hydrogen bonds are indeed perturbed; in the case of rHb (beta131Gln --> Asn) and rHb (beta131Gln --> Leu), the perturbations are propagated to the other alpha(1)beta(1)-interface H-bond involving alpha122His and beta35Tyr. Proton exchange measurements also detect faster exchange rates for both alpha(1)beta(1)-interface histidine side chains of the mutant rHbs in 0.1 M sodium phosphate buffer at pH 7.0 than for those of Hb A under the same conditions. In addition, the same measurements in 0.1 M Tris buffer at pH 7.0 show a much slower exchange rate for mutant rHbs and Hb A. One of the mutants, rHb (beta131Gln --> Asn), shows the conformational exchange of its interface histidines, and exchange rate measurements have been attempted. We have also conducted studies on the reactivity of the SH group of beta93Cys (a residue located in the region of the alpha(1)beta(2)-subunit interface) toward p-mercuribenzoate, and our results show that low-oxygen-affinity rHbs have a more reactive beta93Cys than Hb A in the CO form. Our results indicate that there is communication between the alpha(1)beta(1)- and alpha(1)beta(2)-subunit interfaces, and a possible communication pathway for the cooperative oxygenation of Hb A that allows the alpha(1)beta(1)-subunit interface to modulate the functional properties in conjunction with the alpha(1)beta(2) interface is proposed.     

An additional H-bond in the alpha 1 beta 2 interface as the structural basis for the low oxygen affinity and high cooperativity of a novel recombinant hemoglobin (beta L105W)

Site-directed mutagenesis has been used to construct three recombinant mutant hemoglobins (rHbs), rHb(beta L105W), rHb(alpha D94A/betaL105W), and rHb(alpha D94A). rHb(beta L105W) is designed to form a new hydrogen bond from beta 105Trp to alpha 94Asp in the alpha(1)beta(2) subunit interface to lower the oxygen binding affinity by stabilizing the deoxy quaternary structure. We have found that rHb(beta L105W) does indeed possess a very low oxygen affinity and maintains normal cooperativity (P(50) = 28.2 mmHg, n(max) = 2.6 in 0.1 M sodium phosphate at pH 7.4) compared to those of Hb A (P(50) = 9.9 mmHg, n(max) = 3.2 at pH 7.4). rHb(alpha D94A/beta L105W) and rHb(alpha D94A) are expressed to provide evidence that rHb(betaL 105W) does form a new H-bond from beta 105Trp to alpha 94Asp in the alpha(1)beta(2) subunit interface of the deoxy quaternary structure. Our multinuclear, multidimensional nuclear magnetic resonance (NMR) studies on (15)N-labeled rHb(beta L105W) have identified the indole nitrogen-attached (1)H resonance of beta 105Trp for rHb(beta L105W). (1)H NMR studies on Hb A and mutant rHbs have been used to investigate the structural basis for the low O(2) affinity of rHb(beta L105W). Our NMR results provide evidence that rHb(beta L105W) forms a new H-bond from beta 105Trp to alpha 94Asp in the alpha(1)beta(2) subunit interface of the deoxy quaternary structure. The NMR results also show that these three rHbs can switch from the R quaternary structure to the T quaternary structure in their ligated state upon addition of an allosteric effector, inositol hexaphosphate. We propose that the low O(2) affinity of rHb(beta L105W) is due to the formation of a new H-bond between alpha 105Trp and alpha 94Asp in the deoxy quaternary structure.     

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.     

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)     

Site mutations disrupt inter-helical H-bonds (alpha14W-alpha67T and beta15W-beta72S) involved in kinetic steps in the hemoglobin R-->T transition without altering the free energies of oxygenation

Three recombinant mutant hemoglobins (rHbs) of human normal adult hemoglobin (Hb A), rHb (alphaT67V), rHb (betaS72A), and rHb (alphaT67V, betaS72A), have been constructed to test the role of the tertiary intra-subunit H-bonds between alpha67T and alpha14W and between beta72S and beta15W in the cooperative oxygenation of Hb A. Oxygen-binding studies in 0.1 M sodium phosphate buffer at 29 degrees C show that rHb (alphaT67V), rHb (betaS72A), and rHb (alphaT67V, betaS72A) exhibit oxygen-binding properties similar to those of Hb A. The binding of oxygen to these rHbs is highly cooperative, with a Hill coefficient of approximately 2.8, compared to approximately 3.1 for Hb A. Proton nuclear magnetic resonance (NMR) studies show that rHb (alphaT67V), rHb (betaS72A), rHb (alphaT67V, betaS72A), and Hb A have similar quaternary structures in the alpha(1)beta(2) subunit interfaces. In particular, the inter-subunit H-bonds between alpha42Tyr and beta99Asp and between beta37Trp and alpha94Asp are maintained in the mutants in the deoxy form. There are slight perturbations in the distal heme pocket region of the alpha- and beta-chains in the mutants. A comparison of the exchangeable 1H resonances of Hb A with those of these three rHbs suggests that alpha67T and beta72S are H-bonded to alpha14W and beta15W, respectively, in the CO and deoxy forms of Hb A. The absence of significant free energy changes for the oxygenation process of these three rHbs compared to those of Hb A, even though the inter-helical H-bonds are abolished, indicates that these two sets of H-bonds are of comparable strength in the ligated and unligated forms of Hb A. Thus, the mutations at alphaT67V and betaS72A do not affect the overall energetics of the oxygenation process. The preserved cooperativity in the binding of oxygen to these three mutants also implies that there are multiple interactions involved in the oxygenation process of Hb A.     

Changes of near-UV CD spectrum of human hemoglobin upon oxygen binding: a study of mutants at alpha 42, alpha 140, beta 145 tyrosine or beta 37 tryptophan

The deoxy-form of human adult hemoglobin (Hb A) exhibits a distinct negative CD band at 287 nm that disappears in the oxy-form. It has been suggested that the negative CD band is due to the environmental alteration of Tyr-alpha 42 or Trp-beta 37 at the alpha(1)beta(2) contact upon deoxygenation. To evaluate the contributions of the aromatic residues at the alpha(1)beta(2) contact and the penultimate tyrosine residues of the alpha and beta subunits (alpha 140 and beta 145) to the negative CD band, three recombinant (r) Hbs (rHb Ser-alpha 42, rHb His-beta 37, and rHb Thr-beta 145) were produced in Escherichia coli, and we compared the near-uv CD spectra of these three rHbs and Hb Rouen (Tyr-alpha 140-->His) with the spectra of Hb A under the condition in which all mutant Hbs were able to undergo the T-->R transition (Hill's n > 2.0). The contributions of Tyr-alpha 42, Trp-beta 37, Tyr-alpha 140, and Tyr-beta 145 to the negative CD band were estimated from changes in the ellipticity of the negative CD band at 287 nm to be 4, 18, 32, and 27%, respectively. These results indicate that environmental alteration of the penultimate tyrosine residues caused by the formation of salt bridges upon the R-->T transition is primarily responsible for the negative CD band.     

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.     

NMR investigation of the dynamics of tryptophan side-chains in hemoglobins

NMR relaxation measurements of 15N spin-lattice relaxation rate (R(1)), spin-spin relaxation rate (R(2)), and heteronuclear nuclear Overhauser effect (NOE) have been carried out at 11.7T and 14.1T as a function of temperature for the side-chains of the tryptophan residues of 15N-labeled and/or (2H,15N)-labeled recombinant human normal adult hemoglobin (Hb A) and three recombinant mutant hemoglobins, rHb Kempsey (betaD99N), rHb (alphaY42D/betaD99N), and rHb (alphaV96W), in the carbonmonoxy and the deoxy forms as well as in the presence and in the absence of an allosteric effector, inositol hexaphosphate (IHP). There are three Trp residues (alpha14, beta15, and beta37) in Hb A for each alphabeta dimer. These Trp residues are located in important regions of the Hb molecule, i.e. alpha14Trp and beta15Trp are located in the alpha(1)beta(1) subunit interface and beta37Trp is located in the alpha(1)beta(2) subunit interface. The relaxation experiments show that amino acid substitutions in the alpha(1)beta(2) subunit interface can alter the dynamics of beta37Trp. The transverse relaxation rate (R(2)) for beta37Trp can serve as a marker for the dynamics of the alpha(1)beta(2) subunit interface. The relaxation parameters of deoxy-rHb Kemspey (betaD99N), which is a naturally occurring abnormal human hemoglobin with high oxygen affinity and very low cooperativity, are quite different from those of deoxy-Hb A, even in the presence of IHP. The relaxation parameters for rHb (alphaY42D/betaD99N), which is a compensatory mutant of rHb Kempsey, are more similar to those of Hb A. In addition, TROSY-CPMG experiments have been used to investigate conformational exchange in the Trp residues of Hb A and the three mutant rHbs. Experimental results indicate that the side-chain of beta37Trp is involved in a relatively slow conformational exchange on the micro- to millisecond time-scale under certain experimental conditions. The present results provide new dynamic insights into the structure-function relationship in hemoglobin.     

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.     

Recombinant hemoglobins with low oxygen affinity and high cooperativity

By introducing an additional H-bond in the alpha(1)beta(2) subunit interface or altering the charge properties of the amino acid residues in the alpha(1)beta(1) subunit interface of the hemoglobin molecule, we have designed and expressed recombinant hemoglobins (rHbs) with low oxygen affinity and high cooperativity. Oxygen-binding measurements of these rHbs under various experimental conditions show interesting properties in response to pH (Bohr effect) and allosteric effectors. Proton nuclear magnetic resonance studies show that these rHbs can switch from the oxy (or CO) quaternary structure (R) to the deoxy quaternary structure (T) without changing their ligation states upon addition of an allosteric effector, inositol hexaphosphate, and/or reduction of the ambient temperature. These results indicate that if we can provide extra stability to the T state of the hemoglobin molecule without perturbing its R state, we can produce hemoglobins with low oxygen affinity and high cooperativity. Some of these rHbs are also quite stable against autoxidation compared to many of the known abnormal hemoglobins with altered oxygen affinity and cooperativity. These results have provided new insights into the structure-function relationship in hemoglobin.     

The binding of chloride ions to ligated and unligated human hemoglobin and its influence on the Bohr effect.

The contribution of the interaction of chloride ions with deoxy and oxyhemoglobin to the Bohr effect can be described by a simple binding model. Applying this model to experiment data reveals that at physiological pH and ionic strength about half of the release of Bohr protons is due to a difference in chloride ion binding to deoxy- and oxyhemoglobin. The chloride-independent part of the Bohr effect corresponds with the shift in pK which His-146 beta shows upon oxygenation. The proton absorptioon by hemoglobin observed upon oxygenation below pH 6 is apparently due to a chloride-ion-induced proton uptake, which is larger for oxyhemoglobin than for deoxyhemoglobin. The analysis of the experimental data indicates the existence of only two oxygen-linked chloride ion binding sites in both deoxy and oxyhemoglobin. In deoxyhemoglobin the binding sites most likely consist of Val-1 alpha of one chain and Arg-141 alpha of the partner chain. The sites in oxyhemoglobin consist of groups with a pK value in the neutral pH range; they do not contain lysyl or arginyl residues.     

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.     

Contribution of arginine (HC3) 141 alpha to the Bohr effect of the fourth binding step in the reaction of ligand with human hemoglobin

A few years ago we reported that histidine (HC3) 146 beta plays a major role in the pH-dependent properties of the R-state of human hemoglobin, accounting for close to 50% of the R-state Bohr effect. We have extended these studies by examining the role of arginine 141 alpha, another group known to affect the overall Bohr effect. We have compared the pH dependencies of the rate constants for the dissociation and combination of the fourth carbon monoxide molecule, l4 and l'4, respectively, for native hemoglobin A (HbA) and a control reconstituted HbA, and des-(Arg 141 alpha) HbA, the hemoglobin molecule resulting from the enzymatic removal of the C-terminal arginine of the alpha-chain of human Hb. From these kinetic constants the pH dependence of L4, the affinity constant for the fourth carbon monoxide molecule, has been estimated. We find that the removal of arginine 141 alpha reduces the pH dependence of log L4 by about 80% between pH 6 and 8, where the alkaline Bohr effect normally occurs. The sum of the effects of the removal of His 146 beta and of Arg 141 alpha is greater than 100%. This suggests that at least one of these modifications alters the contributions of other residues of this Bohr effect.     

Probing the importance of the amino-terminal sequence of the beta- and gamma-chains to the properties of normal adult and fetal hemoglobins

A recombinant mutant of human fetal hemoglobin (Hb F), named rHb Oscar, has been constructed to explore the importance of the sequence of the amino-terminal region of the gamma-chain to the structural and functional properties of Hb F as compared to human normal adult hemoglobin (Hb A). Substitutions in the N-terminal region of Hb A have shown this region to be important to its structural and functional properties. Recent studies of recombinant mutants of Hb A with gamma-chain mutations have been used to probe the significance of the N-terminal sequence to the properties of Hb F. One of these mutants of Hb A, called rHb Felix, contains eight substitutions in the N-terminal region of the beta-chain corresponding to the sequence of the gamma-chain in that region [Dumoulin et al. (1998) J. Biol. Chem. 273, 35032-35038]. rHb Felix exhibits a 2,3-bisphosphoglycerate (2,3-BPG) response like that of Hb A, but its tetramer-dimer dissociation constant is similar to that of Hb F. In contrast, rHb Oscar contains a gamma-chain with eight mutations at the N-terminal end corresponding to the sequence of the beta-chain of Hb A in that region. (1)H NMR studies of rHb Oscar indicate a global structure like that of Hb F. rHb Oscar is not as stable against alkaline denaturation as Hb F but is more stable than Hb A, and it exhibits a stronger response to 2,3-BPG and inositol hexaphosphate as compared to Hb F. The 2,3-BPG effect in rHb Oscar also appears to be slightly enhanced compared to that in Hb A. Subzero isoelectric focusing experiments suggest that rHb Oscar does not have dissociation properties like those of Hb A. These results along with those of rHb Felix illustrate that the effects of the N-terminal region on structure and function of the Hb molecule are complicated by interactions with the rest of the molecule that are not yet well defined. However, studies of complementary mutations of Hb A and Hb F may eventually help to define such interactions and lead to a better understanding of the relationship between the amino acid sequence and the properties of the Hb molecule.