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.     

X-ray diffraction study of di and tetra-ligated T-state hemoglobin from high salt crystals.

X-ray diffraction difference electron density maps at 3 A resolution obtained from di and tetra-ligated T-state hemoglobin (Hb) crystals are reported. Crystals isomorphous with native deoxyhemoglobin were obtained from ammonium sulfate solutions incubated with the synthetic allosteric effector RSR-56. RSR-56 binds at two symmetry-related Hb central water cavity sites and each molecule has major interactions with three different subunit side-chains; one effector with Arg141 alpha 2 HC3, Lys99 alpha 1 G6 and Asn108 beta 1 and the other with the symmetry related residues, Arg141 alpha 1 Lys99 alpha 2 and Asn108 beta 2. Crystals mounted in a nitrogen filled glove box were di-ligated as previously found with polyethyleneglycol Hb crystals. Crystals mounted in air under a layer of mother liquor were bright red and showed all four heme groups ligated. The difference electron density from the di-ligated crystals showed atomic movements to be restricted to the immediate neighborhood of the heme groups and the allosteric effector. By contrast, the tetra-ligated structure showed extended difference electron density near amino acid residues around both alpha and beta heme groups and along the alpha 1/beta 2 interface. Ligation of the beta heme group appears to magnify the difference density around the alpha heme groups. There is no evidence of breakage of the Bohr salt bridge, His146 beta HC3----Asp94 beta FG1, in the crystal. The observed difference electron density maps may help to clarify the way the allosteric mechanism is triggered.     

The assignment of carbon monoxide association rate constants to the alpha and beta subunits in native and mutant human deoxyhemoglobin tetramers

The association kinetics of CO binding to site-directed mutants of human deoxyhemoglobin were measured by stopped-flow rapid mixing techniques at pH 7.0, 20 degrees C. Hemoglobin tetramers were constructed from one set of native subunits and one set of mutated partners containing His(E7) to Gly, Val(E11) to Ala, or Val(E11) to Ile substitutions. The reactivity of beta Cys93 with p-hydroxymercuribenzoate was measured to ensure that the mutant deoxyhemoglobins were capable of forming T-state quaternary conformations. Time courses for the complete binding of CO were measured by mixing the deoxygenated proteins with a 5-fold excess of ligand in the absence and presence of inositol hexaphosphate. Association rate constants for the individual alpha and beta subunits in the T-state conformation were assigned by measuring time courses for the reaction of a small, limiting amount of CO with a 20-fold excess of deoxyhemoglobin (i.e. Hb4 + CO----Hb4(CO)). The effects of the E7 and E11 mutations in T-state alpha subunits were qualitatively similar to those observed for the same subunit in the R-state (Mathews, A.J., Rohlfs, R.J., Olson, J.S., Tame, J., Renaud, J-P., and Nagai, K. (1989) J. Biol. Chem. 264, 16573-16583). The alpha His58(E7) to Gly and Val62(E11) to Ala substitutions caused 80- and 3-fold increases, respectively, in k'CO for T-state alpha subunits, and the alpha Val62(E11) to Ile mutation caused a 3-fold decrease. The beta His63(E7) to Gly and Val67(E11) to Ala substitutions produced 70- and 8-fold increases, respectively, in k'CO for T-state beta subunits whereas these same mutations caused little effect on the rate of CO binding to R-state beta subunits. The beta Val67(E11) to Ile mutation produced the same large effect, a 23-fold reduction in k'CO, in both quaternary conformations of beta subunits. These kinetic results can be interpreted qualitatively in terms of differences between the alpha and beta subunits in the deoxy and liganded crystal structures of human hemoglobin (Perutz, M.F. (1990) Annu. Rev. Physiol. 52, 1-25). Both the structural and functional data suggest that the distal portion of the beta heme pocket is tightly packed in deoxyhemoglobin whereas the CO binding site in R-state beta subunits is much more open. In contrast, the distal portion of the alpha heme pocket is restricted sterically in both quaternary states.(ABSTRACT TRUNCATED AT 400 WORDS)     

Proton magnetic resonance study of the influence of chemical modification, mutation, quaternary state, and ligation state on dynamic stability of the heme pocket in hemoglobin as reflected in the exchange of the proximal histidyl ring labile proton

Proton nuclear magnetic resonance spectroscopy has been utilized to investigate the rates of exchange with deuterium of the proximal histidyl ring protons in a series of chemically modified and mutated forms of Hb A. Differences in rates of exchange are related to differences in the stability of the deformed or partially unfolded intermediates from which exchange with bulk solvent takes place. Each modified/mutated Hb exhibited kinetic subunit heterogeneity in the reduced ferrous state, with the alpha subunit exhibiting faster exchange than the beta subunit. Modification or mutation resulted in significant increases in the His F8 ring NH exchange rates primarily for the affected subunit and only if the modification/mutation occurs at the allosterically important alpha 1 beta 2 subunit interface. Moreover, this enhancement in exchange rate is observed primarily in that quaternary state of the modified/mutated Hb in which the modified/substituted residue makes the intersubunit contact. This confirms the importance of allosteric constraints in determining the dynamic properties of the heme pocket. Using modified or mutated Hbs that can switch between the alternate quaternary states within a given ligation state or ligate within a given quaternary state, we show that the major portion of the enhanced exchange rate in R-state oxy Hb relative to T-state deoxy Hb originates from the quaternary switch rather than from ligation. However, solely ligation effects are not negligible. The exchange rates of the His F8 ring labile protons increase dramatically upon oxidizing the iron to the ferric state, and both the subunit kinetic heterogeneity and the allosteric sensitivity to the quaternary state are essentially abolished.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effector-induced structural fluctuation regulates the ligand affinity of an allosteric protein: binding of inositol hexaphosphate has distinct dynamic consequences for the T and R states of hemoglobin

The present study reports distinct dynamic consequences for the T- and R-states of human normal adult hemoglobin (Hb A) due to the binding of a heterotropic allosteric effector, inositol hexaphosphate (IHP). A nuclear magnetic resonance (NMR) technique based on modified transverse relaxation optimized spectroscopy (TROSY) has been used to investigate the effect of conformational exchange of Hb A in both deoxy and CO forms, in the absence and presence of IHP, at 14.1 and 21.1 T, and at 37 degrees C. Our results show that the majority of the polypeptide backbone amino acid residues of deoxy- and carbonmonoxy-forms of Hb A in the absence of IHP is not mobile on the micros-ms time scale, with the exception of several amino acid residues, that is, beta109Val and beta132Lys in deoxy-Hb A, and alpha40Lys in HbCO A. The mobility of alpha40Lys in HbCO A can be explained by the crystallographic data showing that the H-bond between alpha40Lys and beta146His in deoxy-Hb A is absent in HbCO A. However, the conformational exchange of beta109Val, which is located in the intradimer (alpha 1beta 1 or alpha 2beta 2) interface, is not consistent with the crystallographic observations that show rigid packing at this site. IHP binding appears to rigidify alpha40Lys in HbCO A, but does not significantly affect the flexibility of beta109Val in deoxy-Hb A. In the presence of IHP, several amino acid residues, especially those at the interdimer (alpha 1beta 2 or alpha 2beta 1) interface of HbCO A, exhibit significant conformational exchange. The affected residues include the proximal beta92His in the beta-heme pocket, as well as some other residues located in the flexible joint (betaC helix-alphaFG corner) and switch (alphaC helix-betaFG corner) regions that play an important role in the dimer-dimer rotation of Hb during the oxygenation process. These findings suggest that, upon IHP binding, HbCO A undergoes a conformational fluctuation near the R-state but biased toward the T-state, apparently along the trajectory of its allosteric transition, accompanied by structural fluctuations in the heme pocket of the beta-chain. In contrast, no significant perturbation of the dynamic features on the ms-micros time scale has been observed upon IHP binding to deoxy-Hb A. We propose that the allosteric effector-induced quaternary structural fluctuation may contribute to the reduced ligand affinity of ligated hemoglobin. Conformational exchange mapping of the beta-chain of HbCO A observed at 21.1 T shows significantly increased scatter in the chemical exchange contribution to the transverse relaxation rate ( R ex) values, relative to those at lower fields, due to the enhanced effect of the local chemical shift anisotropy (CSA) fluctuation. A spring-on-scissors model is proposed to interpret the dynamic phenomena induced by the heterotropic effector, IHP.     

NMR study of human mutant hemoglobins synthesized in Escherichia coli. Consequences of tyrosine alpha 42 substitutions

The hydroxyl group of Tyr alpha 42 in human hemoglobin forms a hydrogen bond with the carboxylate of Asp beta 99 which is considered to be one of the most important hydrogen bonds for stabilizing the "T-state." However, no spontaneous mutation at position 42 of the alpha subunit has been reported, and the role of the tyrosine has not been tested experimentally. Two artificial human mutant hemoglobins in which Tyr alpha 42 was replaced by phenylalanine or histidine were synthesized in Escherichia coli, and their proton NMR spectra were studied with particular attention to the hyperfine-shifted and hydrogen-bonded proton resonances. The site-directed mutagenesis of the Tyr alpha 42----Phe removes the hydrogen bond described above and prevents transition to the T-state so that the mutant Hb is rather similar to the "R-state" even when deoxygenated. On the other hand, the mutation from tyrosine to histidine causes less drastic structural changes, and its quaternary and tertiary structures are almost the same as native deoxy-Hb A. This may be attributed to the formation of a new hydrogen bond between His alpha 1(42) and Asp beta 2(99). These observations indicate that the hydrogen bond formed between Tyr alpha 42 and Asp beta 99 is required to convert unliganded Hb to the T-state.     

X-ray diffraction studies of a partially liganded hemoglobin, [alpha(FeII-CO)beta(MnII)]2

We have applied single-crystal X-ray diffraction methods to analyze the structure of [alpha(FeII-CO)beta(MnII)]2, a mixed-metal hybrid hemoglobin that crystallizes in the deoxyhemoglobin quaternary structure (the T-state) even though it is half liganded. This study, carried out at a resolution of 3.0 A, shows that (1) the Mn(II)-substituted beta subunits are structurally isomorphous with normal deoxy beta subunits, and (2) CO binding to the alpha subunits induces small, localized changes in the T-state that lack the main directional component of the corresponding larger structural changes in subunit tertiary structure that accompany complete ligand binding to all four subunits and the deoxy to oxy quaternary structure change. Specifically, in the T-state, CO binding to the alpha heme group draws the iron atom toward the heme plane, and this in turn pulls the last turn of the F helix (residues 85 through 89) closer to the heme group. The direction of these small movements is almost perpendicular to the axis of the F helix. In contrast, when the structures of fully liganded and deoxyhemoglobin are compared, extensive structural changes occur throughout the F helix and FG corner, and the main component of the atomic movements in the F helix (in addition to the smaller component toward the heme) is in a direction parallel to the heme plane and toward the alpha 1 beta 2 interface. These findings are discussed in terms of the current stereochemical theories of co-operative ligand binding and the Bohr effect.     

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

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

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.     

Functional and spectroscopic characterization of half-liganded iron-zinc hybrid hemoglobin: evidence for conformational plasticity within the T state

Functionally distinct conformations of HbA (human adult hemoglobin) were probed using deoxy and diliganded derivatives of symmetric Fe-Zn hybrids of HbA. To expand the range of accessible structures, different environments were utilized including solution, sol-gel encapsulation, and crystals. Further structural and functional modulation was achieved by the addition of allosteric effectors. Functional characterization included oxygen affinity measurements, CO combination rates, and geminate and bimolecular CO recombination, after photodissociation. The conformational properties were studied using visible resonance Raman spectroscopy as a probe of local tertiary structure at the iron-containing hemes and UV resonance Raman spectroscopy as a probe of elements of the globin known to be sensitive to quaternary structure. The combined results show a pattern in which there is a progression of conformational and functional properties that are consistent with a picture in which the T quaternary structure can accommodate a range of tertiary conformations (plasticity). At one end of the distribution is the equilibrium deoxy T state conformation that has the lowest ligand reactivity. At the other end of the distribution are T state conformations with higher ligand reactivity that exhibit "loosened" T state constraints within the globin including the alpha(1)beta(2) interface and reduced proximal strain at the heme.     

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.     

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.     

Functional consequences of mutations at the allosteric interface in hetero- and homo-hemoglobin tetramers.

A seminal difference exists between the two types of chains that constitute the tetrameric hemoglobin in vertebrates. While alpha chains associate weakly into dimers, beta chains self-associate into tightly assembled tetramers. While heterotetramers bind ligands cooperatively with moderate affinity, homotetramers bind ligands with high affinity and without cooperativity. These characteristics lead to the conclusion that the beta 4 tetramer is frozen in a quaternary R-state resembling that of liganded HbA. X-ray diffraction studies of the liganded beta 4 tetramers and molecular modeling calculations revealed several differences relative to the native heterotetramer at the "allosteric" interface (alpha 1 beta 2 in HbA) and possibly at the origin of a large instability of the hypothetical deoxy T-state of the beta 4 tetramer. We have studied natural and artificial Hb mutants at different sites in the beta chains responsible for the T-state conformation in deoxy HbA with the view of restoring a low ligand affinity with heme-heme interaction in homotetramers. Functional studies have been performed for oxygen equilibrium binding and kinetics after flash photolysis of CO for both hetero- and homotetramers. Our conclusion is that the "allosteric" interface is so precisely tailored for maintaining the assembly between alpha beta dimers that any change in the side chains of beta 40 (C6), beta 99 (G1), and beta 101 (G3) involved in the interface results in increased R-state behavior. In the homotetramer, the mutations at these sites lead to the destabilization of the beta 4 hemoglobin and the formation of lower affinity noncooperative monomers.     

Hb Santa Clara (beta 97His-->Asn), a human haemoglobin variant: functional characterization and structure modelling

This study examines the functional and structural effects of amino acid substitution at alpha(1)beta(2) interface of Hb Santa Clara (beta 97His-->Asn). We have characterized the variation by a combination of electrospray ionisation mass spectrometry and DNA sequence analysis followed by oxygen-binding experiments. Functional studies outlined an increased oxygen affinity, reduced effect of organic phosphates and a reduced Bohr effect with respect to HbA. In view of the primary role of this interface in the cooperative quaternary transition from the T to R conformational state, a theoretical three-dimensional model of Hb Santa Clara was generated. Structural investigations suggest that replacement of Asn for His beta 97 results in a significant stabilization of the high affinity R-state of the haemoglobin molecule with respect to the low affinity T-state. The role of beta FG4 position has been further examined by computational models of known beta FG4 variants, namely Hb Malm? (beta 97His-->Gln), Hb Wood (beta 97His-->Leu), Hb Nagoya (beta 97His-->Pro) and Hb Moriguchi (beta 97His-->Tyr). These findings demonstrate that, among the various residues at the alpha(1)beta(2) (and alpha(2)beta(1)) intersubunit interface, His beta FG4 contributes significantly to the quaternary constraints that are responsible for the low oxygen affinity of human deoxyhaemoglobin.     

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.     

Changes in the abnormal alpha-subunit upon CO-binding to the normal beta-subunit of Hb M Boston: resonance Raman,EPR and CD study

Heme-heme interaction in Hb M Boston (His alpha 58-->Tyr) was investigated with visible and UV resonance Raman (RR), EPR, and CD spectroscopies. Although Hb M Boston has been believed to be frozen in the T quaternary state, oxygen binding exhibited appreciable co-operativity (n=1.4) and the near-UV CD spectrum indicated weakening of the T marker at pH 9.0. Binding of CO to the normal beta-subunit gave no change in the EPR and visible Raman spectra of the abnormal alpha-subunit at pH 7.5, but it caused an increase of EPR rhombicity and significant changes in the Raman coordination markers as well as the Fe(III)-tyrosine related bands of the alpha-subunit at pH 9.0. The UVRR spectra indicated appreciable changes of Trp but not of Tyr upon CO binding to the alpha-subunit at pH 9.0. Therefore, we conclude that the ligand binding to the beta heme induces quaternary structure change at pH 9.0 and is communicated to the alpha heme, presumably through His beta 92-->Trp beta 37-->His alpha 87.     

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.     

Hemoglobin site-mutants reveal dynamical role of interhelical H-bonds in the allosteric pathway: time-resolved UV resonance Raman evidence for intra-dimer coupling

The dynamical effect of eliminating specific tertiary H-bonds in the hemoglobin (Hb) tetramer has been investigated by site-directed mutagenesis and time-resolved absorption and ultraviolet resonance Raman (UVRR) spectroscopy. The Trp alpha 14...Thr alpha 67 and Trp beta 15...Ser beta 72 H-bonds connect the A and E helices in the alpha and beta chains, and are proposed to break in the earliest protein intermediate (Rdeoxy) following photo-deligation of HbCO, along with a second pair of H-bonds involving tyrosine residues. Mutation of the acceptor residues Thr alpha 67 and Ser beta 72 to Val and Ala eliminates the A-E H-bonds, but has been shown to have no significant effect on ligand-binding affinity or cooperativity, or on spectroscopic markers of the T-state quaternary interactions. However, the mutations have profound and unexpected effects on the character of the Rdeoxy intermediate, and on the dynamics of the subsequent steps leading to the T state. Formation of the initial quaternary contact (RT intermediate) is accelerated, by an order of magnitude, but the locking-in of the T state is delayed by a factor of 2. These rate effects are essentially the same for either mutation, or for the double mutation, suggesting that the alpha beta dimer behaves as a mechanically coupled dynamical unit. Further evidence for intra-dimer coupling is provided by the Rdeoxy UVRR spectrum, in which either or both mutations eliminate the tyrosine difference intensity, although only tryptophan H-bonds are directly affected. A possible mechanism for mechanical coupling is outlined, involving transmission of forces through the alpha(1)beta(1) (and alpha(2)beta(2)) interface. The present observations establish that quaternary motions can occur on the approximately 100 ns time-scale. They show also that a full complement of interhelical H-bonds actually slows the initial quaternary motion in Hb, but accelerates the locking in of the T-contacts.