Effects of substitutions of lysine and aspartic acid for asparagine at beta 108 and of tryptophan for valine at alpha 96 on the structural and functional properties of human normal adult hemoglobin: roles of alpha 1 beta 1 and alpha 1 beta 2 subunit interfaces in the cooperative oxygenation process.

Using our Escherichia coli expression system, we have produced five mutant recombinant (r) hemoglobins (Hbs): r Hb (alpha V96 W), r Hb Presbyterian (beta N108K), r Hb Yoshizuka (beta N108D), r Hb (alpha V96W, beta N108K), and r Hb (alpha V96W, beta N108D). These r Hbs allow us to investigate the effect on the structure-function relationship of Hb of replacing beta 108Asn by either a positively charged Lys or a negatively charged Asp as well as the effect of replacing alpha 96Val by a bulky, nonpolar Trp. We have conducted oxygen-binding studies to investigate the effect of several allosteric effectors on the oxygenation properties and the Bohr effects of these r Hbs. The oxygen affinity of these mutants is lower than that of human normal adult hemoglobin (Hb A) under various experimental conditions. The oxygen affinity of r Hb Yoshizuka is insensitive to changes in chloride concentration, whereas the oxygen affinity of r Hb Presbyterian exhibits a pronounced chloride effect. r Hb Presbyterian has the largest Bohr effect, followed by Hb A, r Hb (alpha V96W), and r Hb Yoshizuka. Thus, the amino acid substitution in the central cavity that increases the net positive charge enhances the Bohr effect. Proton nuclear magnetic resonance studies demonstrate that these r Hbs can switch from the R quaternary structure to the T quaternary structure without changing their ligation states upon the addition of an allosteric effector, inositol hexaphosphate, and/or by reducing the temperature. r Hb (alpha V96W, beta N108K), which has the lowest oxygen affinity among the hemoglobins studied, has the greatest tendency to switch to the T quaternary structure. The following conclusions can be derived from our results: First, if we can stabilize the deoxy (T) quaternary structure of a hemoglobin molecule without perturbing its oxy (R) quaternary structure, we will have a hemoglobin with low oxygen affinity and high cooperativity. Second, an alteration of the charge distribution by amino acid substitutions in the alpha 1 beta 1 subunit interface and in the central cavity of the hemoglobin molecule can influence the Bohr effect. Third, an amino acid substitution in the alpha 1 beta 1 subunit interface can affect both the oxygen affinity and cooperativity of the oxygenation process. There is communication between the alpha 1 beta 1 and alpha 1 beta 2 subunit interfaces during the oxygenation process. Fourth, there is considerable cooperativity in the oxygenation process in the T-state of the hemoglobin molecule.     

Allosteric properties of haemoglobin beta 41 (C7) Phe-->Tyr: a stable, low-oxygen-affinity variant synthesized in Escherichia coli.

In human deoxy haemoglobin, the alpha 42(C7)Tyr-residue is hydrogen-bonded to beta 99(G1)Asp which stabilizes the low-oxygen-affinity deoxy conformation. We engineered a haemoglobin with Tyr for Phe at the homologous C7 position in beta-chains. The oxygen affinity of the variant is decreased about two-fold relative to Hb A while keeping similar KR and KT values. This mutant may be a candidate for the development of an artificial oxygen carrier, as it would not require an external effector for significant oxygen unloading in vivo.     

Hemoglobin Saint Mandé [beta 102 (G4) Asn----Tyr]. Functional studies and structural modeling reveal an altered T state

Oxygen equilibrium studies of purified hemoglobin Saint Mandé (Hb SM) [beta 102 (G4) Asn----Tyr] reveal a decreased oxygen affinity and cooperativity but to a lesser extent than found for Hb Kansas (beta 102 Thr). The low affinity of Hb SM depends on environmental conditions: eliminating chloride or raising the pH greatly elevated the ratio of p50 of Hb SM to that of Hb A. The alkaline Bohr effect is reduced by about 40%. The effects of anions (chloride, organophosphates) binding to deoxy Hb SM are also reduced. These data indicate that the functional properties of Hb SM are intermediary between Hb A and Hb Kansas. In addition, molecular graphics modeling of Hb SM in the oxy and deoxy structures indicate the possibility of a new hydrogen bond in the T state between beta(1)102 Tyr and alpha(2)42 Tyr. Stabilisation of the T state in this manner is a plausible explanation for several of the effects observed.     

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.     

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.     

Site-directed mutations of human hemoglobin at residue 35beta: a residue at the intersection of the alpha1beta1, alpha1beta2, and alpha1alpha2 interfaces

Because Tyr35beta is located at the convergence of the alpha1beta1, alpha1beta2, and alpha1alpha2 interfaces in deoxyhemoglobin, it can be argued that mutations at this position may result in large changes in the functional properties of hemoglobin. However, only small mutation-induced changes in functional and structural properties are found for the recombinant hemoglobins betaY35F and betaY35A. Oxygen equilibrium-binding studies in solution, which measure the overall oxygen affinity (the p50) and the overall cooperativity (the Hill coefficient) of a hemoglobin solution, show that removing the phenolic hydroxyl group of Tyr35beta results in small decreases in oxygen affinity and cooperativity. In contrast, removing the entire phenolic ring results in a fourfold increase in oxygen affinity and no significant change in cooperativity. The kinetics of carbon monoxide (CO) combination in solution and the oxygen-binding properties of these variants in deoxy crystals, which measure the oxygen affinity and cooperativity of just the T quaternary structure, show that the ligand affinity of the T quaternary structure decreases in betaY35F and increases in betaY35A. The kinetics of CO rebinding following flash photolysis, which provides a measure of the dissociation of the liganded hemoglobin tetramer, indicates that the stability of the liganded hemoglobin tetramer is not altered in betaY35F or betaY35A. X-ray crystal structures of deoxy betaY35F and betaY35A are highly isomorphous with the structure of wild-type deoxyhemoglobin. The betaY35F mutation repositions the carboxyl group of Asp126alpha1 so that it may form a more favorable interaction with the guanidinium group of Arg141alpha2. The betaY35A mutation results in increased mobility of the Arg141alpha side chain, implying that the interactions between Asp126alpha1 and Arg141alpha2 are weakened. Therefore, the changes in the functional properties of these 35beta mutants appear to correlate with subtle structural differences at the C terminus of the alpha-subunit.     

Contribution of alpha140Tyr and beta37Trp to the near-UV CD spectra on quaternary structure transition of human hemoglobin A

The CD band of human adult hemoglobin (Hb A) at 280 approximately 290 nm shows a pronounced change from a small positive band to a definite negative band on the oxy (R) to deoxy (T) structural transition. This change has been suggested to be due to environmental alteration of Tyrs (alpha42, alpha140, and beta145) or beta37 Trp residues located at the alpha1beta2 subunit interface by deoxygenation. In order to evaluate contributions of alpha140Tyr and beta37Trp to this change of CD band, we compared the CD spectra of two mutant Hbs, Hb Rouen (alpha140Tyr-->His) and Hb Hirose (beta37Trp-->Ser) with those of Hb A. Both mutant Hbs gave a distinct, but smaller negative CD band at 287nm in the deoxy form than that of deoxyHb A. Contributions of alpha140Tyr and beta37Trp to the negative band at the 280 approximately 290 nm region were estimated from difference spectra to be 30% and 26%, respectively. These results indicate that the other aromatic amino acid residues, alpha42Tyr and beta145Tyr, at the alpha1beta2 interface, are also responsible for the change of the CD band upon the R-->T transition of Hb A.     

Hemoglobin Warsaw (Phe beta 42(CD1)----Val), an unstable variant with decreased oxygen affinity. Characterization of its synthesis, functional properties, and structure

In Hb Warsaw Val replaces the Phe normally present at the heme contact position beta 42 (CD1). This variant is unstable, and it readily undergoes methemoglobin formation. In DEAE-cellulose chromatography, the variant hemoglobin co-eluted with Hb A; a partially heme-depleted fraction of the variant, representing 5-6% of the total hemoglobin, eluted separately and in pure form. The heme replete form of Hb Warsaw exhibited decreased oxygen affinity with a normal Bohr effect and normal cooperativity and interaction with 2,3-diphosphoglycerate (DPG). The heme-depleted Hb Warsaw had a higher oxygen affinity than that of Hb A, decreased cooperativity and 2,3-DPG interaction, and a very low alkaline Bohr effect. Gel filtration of the heme-depleted form showed it to exist entirely as alpha beta dimers. Globin chain synthesis by Hb Warsaw-containing reticulocytes followed a balanced alpha/beta ratio. In short-term synthesis experiments, a major portion of incorporated radiolabeled L-leucine was recovered from the dimeric, heme-depleted Hb Warsaw fraction, suggesting that subunit association precedes the incorporation of heme into the beta subunits in the post-synthetic assembly of this hemoglobin. Structural analysis of deoxyhemoglobin containing roughly equal proportions of normal and variant beta chains showed that the replacement leaves a cavity next to the heme that is large enough to hold a water molecule, which may account for the instability of Hb Warsaw. The heme and the pyrrol nearest to ValCD1 tilt into the cavity. The resulting increase in the tilt of the proximal histidine relative to the heme plane, coupled with a possible stretching of the Fe-N epsilon bond may account for the low oxygen affinity.     

Intersubunit interactions associated with Tyr42 alpha stabilize the quaternary-T tetramer but are not major quaternary constraints in deoxyhemoglobin

Previous mutational studies on Tyr42alpha variants as well as the current studies on the mutant hemoglobin alphaY42A show that the intersubunit interactions associated with Tyr42alpha significantly stabilize the alpha1beta2 interface of the quaternary-T deoxyhemoglobin tetramer. However, crystallographic studies, UV and visible resonance Raman spectroscopy, CO combination kinetic measurements, and oxygen binding measurements on alphaY42A show that the intersubunit interactions formed by Tyr42alpha have only a modest influence on the structural properties and ligand affinity of the deoxyhemoglobin tetramer. Therefore, the alpha1beta2 interface interactions associated with Tyr42alpha do not contribute significantly to the quaternary constraints that are responsible for the low oxygen affinity of deoxyhemoglobin. The slight increase in the ligand affinity of deoxy alphaY42A correlates with small, mutation-induced structural changes that perturb the environment of Trp37beta, a critical region of the quaternary-T alpha1beta2 interface that has been shown to be the major source of quaternary constraint in deoxyhemoglobin.     

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.     

Site-directed mutagenesis in hemoglobin: functional and structural role of inter- and intrasubunit hydrogen bonds as studied with 37 beta and 145 beta mutations.

In order to clarify the functional and structural role of intra- and intersubunit hydrogen bonds in human hemoglobin (Hb A), we prepared two artificial beta chain mutant hemoglobins by site-directed mutagenesis. The mutant Hb Phe-37 beta, in which Trp-37 beta is replaced by Phe to remove the intersubunit hydrogen bond between Asp-94 alpha and Trp-37 beta at the alpha 1-beta 2 interface in deoxy Hb A, showed a markedly increased oxygen affinity and almost completely diminished Bohr effect and cooperativity. However, 1H-NMR data indicated that the structure of deoxy Hb Phe-37 beta is rather similar to that of deoxy Hb A. The enhanced tetramer-to-dimer dissociation previously observed in Hb Hirose (Trp-37 beta----Ser) together with our observation of the effects of organic phosphate on the structure and function of Hb Phe-37 beta suggested that a large part of the abnormal properties of Hb Phe-37 beta observed for dilute solutions appears to result from partial dissociation into alpha beta dimers rather than direct destabilization of the T-quaternary structure in the deoxygenated state. Thus, the primary and direct role of the hydrogen bond between Asp-94 alpha and Trp-37 beta is to stabilize the tetrameric assembly, and thereby this hydrogen bond indirectly contributes to stabilization of the T-quaternary structure. The other mutant Hb Phe-145 beta has a Phe residue at the 145 beta site and lacks the intrasubunit hydrogen bond formed between Tyr-145 beta and the carbonyl group of Val-98 beta in deoxy Hb A.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of oxygen affinity by quaternary enhancement: Does hemoglobin ypsilanti represent an allosteric intermediate?

Recent crystallographic studies on the mutant human hemoglobin Ypsilanti (beta 99 Asp-->Tyr) have revealed a previously unknown quaternary structure called "quaternary Y" and suggested that the new structure may represent an important intermediate in the cooperative oxygenation pathway of normal hemoglobin. Here we measure the oxygenation and subunit assembly properties of hemoglobin Ypsilanti and five additional beta 99 mutants (Asp beta 99-->Val, Gly, Asn, Ala, His) to test for consistency between their energetics and those of the intermediate species of normal hemoglobin. Overall regulation of oxygen affinity in hemoglobin Ypsilanti is found to originate entirely from 2.6 kcal of quaternary enhancement, such that the tetramer oxygenation affinity is 85-fold higher than for binding to the dissociated dimers. Equal partitioning of this regulatory energy among the four tetrameric binding steps (0.65 kcal per oxygen) leads to a noncooperative isotherm with extremely high affinity (pmedian = .14 torr). Temperature and pH studies of dimer-tetramer assembly and sulfhydryl reaction kinetics suggest that oxygenation-dependent structural changes in hemoglobin Ypsilanti are small. These properties are quite different from the recently characterized allosteric intermediate, which has two ligands bound on the same side of the alpha 1 beta 2 interface (see ref. 1 for review). The combined results do, however, support the view that quaternary Y may represent the intermediate cooperativity state of normal hemoglobin that binds the last oxygen.     

Oxygen equilibrium and EPR studies on alpha1beta1 hemoglobin dimer

We undertook this project to clarify whether hemoglobin (Hb) dimers have a high affinity for oxygen and cooperativity. For this, we prepared stable Hb dimers by introducing the mutation Trp-->Glu at beta37 using our Escherichia coli expression system at the alpha1beta2 interface of Hb, and analyzed their molecular properties. The mutant hybrid Hbs with a single oxygen binding site were prepared by substituting Mg(II) protoporphyrin for ferrous heme in either the alpha or beta subunit, and the oxygen binding properties of the free dimers were investigated. Molecular weight determination of both the deoxy and CO forms showed all these molecules to be dimers in the absence of IHP at different protein concentrations. Oxygen equilibrium measurements showed high affinity and non-cooperative oxygen binding for all mutant Hb and hybrid Hb dimers. However, EPR results on the [alpha(N)(Fe-NO)beta(M)(Mg)] hybrid showed some alpha1beta1 interactions. These results provide some clues as to the properties of Hb dimers, which have not been studied extensively owing to practical difficulties in their preparation.     

UV resonance Raman studies of alpha-nitrosyl hemoglobin derivatives: relation between the alpha 1-beta 2 subunit interface interactions and the Fe-histidine bonding of alpha heme.

Human alpha-nitrosyl beta-deoxy hemoglobin A, alpha(NO)beta(deoxy), is considered to have a T (tense) structure with the low O(2) affinity extreme and the Fe-histidine (His87) (Fe-His) bond of alpha heme cleaved. The Fe-His bonding of alpha heme and the intersubunit interactions at the alpha 1-beta 2 contact of alpha(NO)-Hbs have been examined under various conditions with EPR and UV resonance Raman (UVRR) spectra excited at 235 nm, respectively. NOHb at pH 6.7 gave the UVRR spectrum of the R structure, but in the presence of inositol-hexakis-phosphate (IHP) for which the Fe-His bond of the alpha heme is broken, UVRR bands of Trp residues behaved half-T-like while Tyr bands remained R-like. The half-ligated nitrosylHb, alpha(NO)beta(deoxy), in the presence of IHP at pH 5.6, gave T-like UVRR spectra for both Tyr and Trp, but binding of CO to its beta heme (alpha(NO)beta(CO)) changed the UVRR spectrum to half-T-like. Binding of NO to its beta heme (NOHb) changed the UVRR spectrum to 70% T-type for Trp but almost R-type for Tyr. When the pH was raised to 8.2 in the presence of IHP, the UVRR spectrum of NOHb was the same as that of COHb. EPR spectra of these Hbs indicated that the Fe-His bond of alpha(NO) heme is partially cleaved. On the other hand, the UVRR spectra of alpha(NO)beta(deoxy) in the absence of IHP at pH 8.8 showed the T-like UVRR spectrum, but the EPR spectrum indicated that 40-50% of the Fe-His bond of alpha hemes was intact. Therefore, it became evident that there is a qualitative correlation between the cleavage of the Fe-His bond of alpha heme and T-like contact of Trp-beta 37. We note that the behaviors of Tyr and Trp residues at the alpha 1-beta 2 interface are not synchronous. It is likely that the behaviors of Tyr residues are controlled by the ligation of beta heme through His-beta 92(F8)-->Val-beta 98(FG5)-->Asp-beta 99(G1 )-->Tyr-alpha 42(C7) or Tyr-beta 145(HC2).     

Site-directed mutagenesis in haemoglobin. Functional role of tyrosine-42(C7) alpha at the alpha 1-beta 2 interface

To clarify the functional role of Tyr-42(C7) alpha, which forms a hydrogen bond with Asp-99(G1) beta at the alpha 1-beta 2 interface of human deoxyhaemoglobin, we engineered two artificial mutant haemoglobins (Hb), in which Tyr-42 alpha was replaced by Phe (Hb Phe-42 alpha) or His (Hb His-42 alpha), and investigated their oxygen binding properties together with structural consequences of the mutations by using various spectroscopic probes. Like most of the natural Asp-99 beta mutants, Hb Phe-42 alpha showed a markedly increased oxygen affinity, a reduced Bohr effect and diminished co-operativity. Structural probes such as ultraviolet-region derivative and oxy-minus-deoxy difference spectra, resonance Raman scattering and proton nuclear magnetic resonance spectra indicate that, in Hb Phe-42 alpha, the deoxy T quaternary structure is highly destabilized and the strain imposed on the Fe-N epsilon (proximal His) bond is released, stabilizing the oxy tertiary structure. In contrast with Hb Phe-42 alpha, Hb His-42 alpha showed an intermediately impaired function and only moderate destabilization of the T-state, which can be explained by the formation of a new, weak hydrogen bond between His-42 alpha and Asp-99 beta. Spectroscopic data were consistent with this assumption. The present study proves that the hydrogen bond between Tyr-42 alpha and Asp-99 beta plays a key role in stabilizing the deoxy T structure and consequently in co-operative oxygen binding.     

A biochemical and biophysical characterization of recombinant mutants of fetal hemoglobin and their interaction with sickle cell hemoglobin.

Three recombinant mutants of human fetal hemoglobin (Hb F) have been constructed to determine what effects specific amino acid residues in the gamma chain have on the biophysical and biochemical properties of the native protein molecule. Target residues in these recombinant fetal hemoglobins were replaced with the corresponding amino acids in the beta chain of human normal adult hemoglobin (Hb A). The recombinant mutants of Hb F included rHb F (gamma 112Thr --> Cys), rHb F (gamma 130Trp --> Tyr), and rHb F (gamma 112Thr --> Cys/gamma 130Trp --> Tyr). Specifically, the importance of gamma 112Thr and gamma 130Trp to the stability of Hb F against alkaline denaturation and in the interaction with sickle cell hemoglobin (Hb S) was investigated. Contrary to expectations, these rHbs were found to be as stable against alkaline denaturation as Hb F, suggesting that the amino acid residues mentioned above are not responsible for the stability of Hb F against the alkaline denaturation as compared to that of Hb A. Sub-zero isoelectric focusing (IEF) was employed to investigate the extent of hybrid formation in equilibrium mixtures of Hb S with these hemoglobins and with several other hemoglobins in the carbon monoxy form. Equimolar mixtures of Hb A and Hb S and of Hb A(2) and Hb S indicate that 48-49% of the Hb exists as the hybrid tetramer, which is in agreement with the expected binomial distribution. Similar mixtures of Hb F and Hb S contain only 44% hybrid tetramer. The results for two of our recombinant mutants of Hb F were identical to the results for mixtures of Hb F and Hb S, while the other mutant, rHb F (gamma 130Trp --> Tyr), produced 42% hybrid tetramer. The sub-zero IEF technique discussed here is more convenient than room-temperature IEF techniques, which require Hb mixtures in the deoxy state. These recombinant mutants of Hb F were further characterized by equilibrium oxygen binding studies, which indicated no significant differences from Hb F. While these mutants of Hb F did not have tetramer-dimer dissociation properties significantly altered from those of Hb F, future mutants of Hb F may yet prove useful to the development of a gene therapy for the treatment of patients with sickle cell anemia.     

Hemoglobin Fort Gordon or alpha2beta2145 Tyr replaced by Asp, a new high-oxygen-affinity hemoglobin variant.

Hemoglobin Fort Gordon, alpha2beta2145 Tyr replaced by Asp (HC2), has been observed in a 20-year-old black male with compensatory erythrocytosis. The variant was readily identified by electrophoresis and chromatography, and comprised about 30% of the red cell hemoglobin. The substitution was identified through analyses of tryptic peptides of various digests of the isolated beta chain. The oxygen affinity of whole blood was increased; two components were observed one of which had a greatly increased affinity for oxygen and a markedly reduced subunit cooperativity. It appears that the Tyr replaced by Asp substitution resembles the Tyr replaced by His substitution in hemoglobin Bethesda (Bunn, H. F. et al. (1972) J. Clin. Invest. 51, 2299-2309; Olson, J. S. and Gibson, G. H. (1972) J Biol. Chem. 247, 3662-3670; Adamson et al. (1972) J. Clin. Invest. 51, 2883-2888) in that both inhibit the quarternary change of the oxy to the deoxy conformation, resulting in greatly altered functional properties. Studies of a few members of the family were negative.     

Co-binding studies on Hb M Iwate. Allostery of a T state haemoglobin.

The mutant haemoglobin Hb M Iwate alpha 2Mmet87His leads to Tyr beta 2, is characterized by a stable T structure and a low ligand affinity. Sigmoidal CO-binding isotherms of symmetrical shape with Hill coefficients of n = 1.4 at pH 6 to n = 1.9 at pH 10 and the differences in the mean affinity (PCO(1/2)) and the affinity of the first ligand-binding beta subunit (1/L1 greater than Pco(1/2)) are the evidence for the cooperativity. The comparison of the Bohr effects of the two valency hybrid states (alpha 2Mmet beta met beta deoxy alpha 2Mmet beta 2deoxy) in the absence of and in the presence of polyphosphates leads to an indirect proof of pH-dependent subunit-subunit interaction. Inositol hexaphosphate-binding suppresses cooperativity in the pH range 5.5-8 (n = 1). Above pH 8 hte cooperativity increases to a final value of n = 1.9 at pH greater than 10, which is identical to that of stripped Hb M Iwate. The CO binding to the first binding site exhibits a Bohr effect. Polyphosphate anions have no influence on the CO binding of the first binding site. The heterotropic effects are discussed as intrachain effects (Bohr effect of the first binding site) and interchain effects (Bohr effect of Pco(1/2); influence of polyphosphates).     

Differences in changes of the alpha1-beta2 subunit contacts between ligand binding to the alpha and beta subunits of hemoglobin A: UV resonance raman analysis using Ni-Fe hybrid hemoglobin

The alpha1-beta2 subunit contacts in the half-ligated hemoglobin A (Hb A) have been explored with ultraviolet resonance Raman (UVRR) spectroscopy using the Ni-Fe hybrid Hb under various solution conditions. Our previous studies demonstrated that Trpbeta37, Tyralpha42, and Tyralpha140 are mainly responsible for UVRR spectral differences between the complete T (deoxyHb A) and R (COHb A) structures [Nagai, M., Wajcman, H., Lahary, A., Nakatsukasa, T., Nagatomo, S., and Kitagawa, T. (1999) Biochemistry, 38, 1243-1251]. On the basis of it, the UVRR spectra observed for the half-ligated alpha(Ni)beta(CO) and alpha(CO)beta(Ni) at pH 6.7 in the presence of IHP indicated the adoption of the complete T structure similar to alpha(Ni)beta(deoxy) and alpha(deoxy)beta(Ni). The extent of the quaternary structural changes upon ligand binding depends on pH and IHP, but their characters are qualitatively the same. For alpha(Ni)beta(Fe), it is not until pH 8.7 in the absence of IHP that the Tyr bands are changed by ligand binding. The change of Tyr residues is induced by binding of CO, but not of NO, to the alpha heme, while it was similarly induced by binding of CO and NO to the beta heme. The Trp bands are changed toward R-like similarly for alpha(Ni)beta(CO) and alpha(CO)beta(Ni), indicating that the structural changes of Trp residues are scarcely different between CO binding to either the alpha or beta heme. The ligand induced quaternary structural changes of Tyr and Trp residues did not take place in a concerted way and were different between alpha(Ni)beta(CO) and alpha(CO)beta(Ni). These observations directly indicate that the phenomenon occurring at the alpha1-beta2 interface is different between the ligand binding to the alpha and beta hemes and is greatly influenced by IHP. A plausible mechanism of the intersubunit communication upon binding of a ligand to the alpha or beta subunit to the other subunit and its difference between NO and CO as a ligand are discussed.     

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.