Site-directed mutagenesis in hemoglobin: test of functional homology of the F9 amino acid residues of hemoglobin alpha and beta chains

The cysteine residue at F9(93) of the human hemoglobin (Hb A) beta chain, conserved in mammalian and avian hemoglobins, is located near the functionally important alpha1-beta2 interface and C-terminal region of the beta chain and is reactive to sulfhydryl reagents. The functional roles of this residue are still unclear, although regulation of local blood flow through allosteric S-nitrosylation of this residue is proposed. To clarify the role of this residue and its functional homology to F9(88) of the alpha chain, we measured oxygen equilibrium curves, UV-region derivative spectra, Soret-band absorption spectra, the number of titratable -SH groups with p-mercuribenzoate and the rate of reaction of these groups with 4, 4'-dipyridine disulfide for three recombinant mutant Hbs with single amino acid substitutions: Ala-->Cys at 88alpha (rHb A88alphaC), Cys-->Ala at 93beta (rHb C93betaA) and Cys-->Thr at 93beta (rHb C93betaT). These Hbs showed increased oxygen affinities and impaired allosteric effects. The spectral data indicated that the R to T transition upon deoxygenation was partially restricted in these Hbs. The number of titratable -SH groups of liganded form was 3.2-3.5 for rHb A88alphaC compared with 2.2 for Hb A, whereas those for rHb C93betaA and rHb C93betaT were negligibly small. The reduction of rate of reaction with 4,4'-dipyridine disulfide upon deoxygenation in rHb A88alphaC was smaller than that in Hb A. Our experimental data have shown that the residues at 88alpha and 93beta have definite roles but they have no functional homology. Structure-function relationships in our mutant Hbs are discussed.     

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.     

Allosteric effects on oxidative and nitrosative reactions of cell-free hemoglobins

A review of the oxidative and nitrosative reactions of cell-free hemoglobin-based oxygen carriers (HBOCs) shows that these reactions are intimately linked and are subject to allosteric control. Cross-linking reactions used to produce HBOCs introduce conformational constraints and result in Hbs with reduced responses to heterotropic and homotropic allosteric effectors. The Nernst plots of heme oxidation of cross-linked HBOCs are shifted to higher potentials relative to unmodified Hb in the absence of allosteric effectors, in accord with their T-state stabilization and right-shifted Hill plots of O(2) binding. They exhibit enhanced rates of autoxidation and nitrite-induced oxidation, features that appear due to their having more solvent-accessible heme pockets. The stability of their NO-Hb derivatives varies as a result of allosteric effects on the extent of formation of pentacoordinate NO-heme geometry by alpha chains and subsequent oxidation of partner beta chains. The physiological implications of these findings on the safety, efficacy and design of second generation HBOCs are discussed in the framework of a reaction scheme showing linkages between Hb-mediated redox reactions. These redox reactions can drive formation of SNO-Hb and other reactive species and are of significance for the use of cell-free Hbs in vivo.     

Combining the influence of two low O2 affinity-inducing chemical modifications of the central cavity of hemoglobin

HexaPEGylated hemoglobin (Hb), a non-hypertensive Hb, exhibits high O2 affinity, which makes it difficult for it to deliver the desired levels of oxygen to tissues. The PEGylation of very low O2 affinity Hbs is now contemplated as the strategy to generate PEGylated Hbs with intermediate levels of O2 affinity. Toward this goal, a doubly modified Hb with very low O2 affinity has been generated. The amino terminal of the beta-chain of HbA is modified by 2-hydroxy, 3-phospho propylation first to generate a low oxygen affinity Hb, HPPr-HbA. The oxygen affinity of this Hb is insensitive to DPG and IHP. Molecular modeling studies indicated potential interactions between the covalently linked phosphate group and Lys-82 of the trans beta-chain. To further modulate the oxygen affinity of Hb, the alpha alpha-fumaryl cross-bridge has been introduced into HPPr-HbA in the mid central cavity. The doubly modified HbA (alpha alpha-fumaryl-HPPr-HbA) exhibits an O2 affinity lower than that of either of the singly modified Hbs, with a partial additivity of the two modifications. The geminate recombination and the visible resonance Raman spectra of the photoproduct of alpha alpha-fumaryl-HPPr-HbA also reflect a degree of additive influence of each of these modifications. The two modifications induced a synergistic influence on the chemical reactivity of Cys-93(beta). It is suggested that the doubly modified Hb has accessed the low affinity T-state that is non-responsive to effectors. The doubly modified Hb is considered as a potential candidate for generating PEGylated Hbs with an O2 affinity comparable to that of erythrocytes for developing blood substitutes.     

A biochemical--biophysical study of hemoglobins from woolly mammoth, Asian elephant, and humans

This study is aimed at investigating the molecular basis of environmental adaptation of woolly mammoth hemoglobin (Hb) to the harsh thermal conditions of the Pleistocene ice ages. To this end, we have carried out a comparative biochemical-biophysical characterization of the structural and functional properties of recombinant hemoglobins (rHb) from woolly mammoth (rHb WM) and Asian elephant (rHb AE) in relation to human hemoglobins Hb A and Hb A(2) (a minor component of human blood). We have obtained oxygen equilibrium curves and calculated O(2) affinities, Bohr effects, and the apparent heat of oxygenation (ΔH) in the presence and absence of allosteric effectors [inorganic phosphate and inositol hexaphosphate (IHP)]. Here, we show that the four Hbs exhibit distinct structural properties and respond differently to allosteric effectors. In addition, the apparent heat of oxygenation (ΔH) for rHb WM is less negative than that of rHb AE, especially in phosphate buffer and the presence of IHP, suggesting that the oxygen affinity of mammoth blood was also less sensitive to temperature change. Finally, (1)H NMR spectroscopy data indicates that both α(1)(β/δ)(1) and α(1)(β/δ)(2) interfaces in rHb WM and rHb AE are perturbed, whereas only the α(1)δ(1) interface in Hb A(2) is perturbed compared to that in Hb A. The distinct structural and functional features of rHb WM presumably facilitated woolly mammoth survival in the Arctic environment.     

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.     

Heme Orientation of Cavity Mutant Hemoglobins (His F8 → Gly) in Either α or β Subunits: Circular Dichroism, 1H NMR,and Resonance Raman Studies

Native human adult hemoglobin (Hb A) has mostly normal orientation of heme, whereas recombinant Hb A (rHb A) expressed in E. coli contains both normal and reversed orientations of heme. Hb A with the normal heme exhibits positive circular dichroism (CD) bands at both the Soret and 260‐nm regions, while rHb A with the reversed heme shows a negative Soret and decreased 260‐nm CD bands. In order to examine involvement of the proximal histidine (His F8) of either α or β subunits in determining the heme orientation, we prepared two cavity mutant Hbs, rHb(αH87G) and rHb(βH92G), with substitution of glycine for His F8 in the presence of imidazole. CD spectra of both cavity mutant Hbs did not show a negative Soret band, but instead exhibited positive bands with strong intensity at the both Soret and 260‐nm regions, suggesting that the reversed heme scarcely exists in the cavity mutant Hbs. We confirmed by ¹H NMR and resonance Raman (RR) spectroscopies that the cavity mutant Hbs have mainly the normal heme orientation in both the mutated and native subunits. These results indicate that the heme Fe‐His F8 linkage in both α and β subunits influences the heme orientation, and that the heme orientation of one type of subunit is related to the heme orientation of the complementary subunits to be the same. The present study showed that CD and RR spectroscopies also provided powerful tools for the examination of the heme rotational disorder of Hb A, in addition to the usual ¹H NMR technique. Chirality 28:585–592, 2016. © 2016 Wiley Periodicals, Inc.     

The role of beta93 Cys in the inhibition of Hb S fiber formation

Recent studies have suggested that nitric oxide (NO) binding to hemoglobin (Hb) may lead to the inhibition of sickle cell fiber formation and the dissolution of sickle cell fibers. NO can react with Hb in at least 3 ways: 1) formation of Hb(II)NO, 2) formation of methemoglobin, and 3) formation of S-nitrosohemoglobin, through nitrosylation of the beta93 Cys residue. In this study, the role of beta93 Cys in the mechanism of sickle cell fiber inhibition is investigated through chemical modification with N-ethylmaleimide. UV resonance Raman, FT-IR and electrospray ionization mass spectroscopic methods in conjunction with equilibrium solubility and kinetic studies are used to characterize the effect of beta93 Cys modification on Hb S fiber formation. Both FT-IR spectroscopy and electrospray mass spectrometry results demonstrate that modification can occur at both the beta93 and alpha104 Cys residues under relatively mild reaction conditions. Equilibrium solubility measurements reveal that singly-modified Hb at the beta93 position leads to increased amounts of fiber formation relative to unmodified or doubly-modified Hb S. Kinetic studies confirm that modification of only the beta93 residue leads to a faster onset of polymerization. UV resonance Raman results indicate that modification of the alpha104 residue in addition to the beta93 residue significantly perturbs the alpha(1)beta(2) interface, while modification of only beta93 does not. These results in conjunction with the equilibrium solubility and kinetic measurements are suggestive that modification of the alpha104 Cys residue and not the beta93 Cys residue leads to T-state destabilization and inhibition of fiber formation. These findings have implications for understanding the mechanism of NO binding to Hb and NO inhibition of Hb S fiber formation.     

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.     

A biophysical investigation of recombinant hemoglobins with aromatic B10 mutations in the distal heme pockets

This study examines the structural and functional effects of amino acid substitutions in the distal side of both the alpha- and beta-chain heme pockets of human normal adult hemoglobin (Hb A). Using our Escherichia coli expression system, we have constructed four recombinant hemoglobins: rHb(alphaL29F), rHb(alphaL29W), rHb(betaL28F), and rHb(betaL28W). The alpha29 and beta28 residues are located in the B10 helix of the alpha- and beta-chains of Hb A, respectively. The B10 helix is significant because of its proximity to the ligand-binding site. Previous work showed the ability of the L29F mutation to inhibit oxidation. rHb(alphaL29W), rHb(betaL28F), and rHb(betaL28W) exhibit very low oxygen affinity and reduced cooperativity compared to those of Hb A, while the previously studied rHb(alphaL29F) exhibits high oxygen affinity. Proton nuclear magnetic resonance spectroscopy indicates that these mutations in the B10 helix do not significantly perturb the alpha(1)beta(1) and alpha(1)beta(2) subunit interfaces, while as expected, the tertiary structures near the heme pockets are affected. Experiments in which visible spectrophotometry was utilized reveal that rHb(alphaL29F) has equivalent or slower rates of autoxidation and azide-induced oxidation than does Hb A, while rHb(alphaL29W), rHb(betaL28F), and rHb(betaL28W) have increased rates. Bimolecular rate constants for NO-induced oxidation have been determined using a stopped-flow apparatus. These findings indicate that amino acid residues in the B10 helix of the alpha- and beta-chains can play different roles in regulating the functional properties and stability of the hemoglobin molecule. These results may provide new insights for designing a new generation of hemoglobin-based oxygen carriers.     

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.     

Interspecies hybrid HbS: complete neutralization of Val6(beta)-dependent polymerization of human beta-chain by pig alpha-chains

Interspecies hybrid HbS (alpha(2)(P)beta(2)(S)), has been assembled in vitro from pig alpha-globin and human beta(S)-chain. The alpha(2)(P)beta(2)(S) retains normal tetrameric structure (alpha(2)beta(2)) of human Hb and an O(2) affinity comparable to that of HbS in 50 mM Hepes buffer; but, its O(2) affinity is slightly higher than that of HbS in the presence of allosteric effectors (chloride, DPG and phosphate). The (1)H-NMR spectroscopy detected distinct differences between the heme environments and alpha(1)beta(1) interfaces of pig Hb and HbS, while their alpha(1)beta(2) interfaces appear very similar. The interspecies hybrid alpha(2)(H)beta(2)(P) resembles pig Hb; the pig beta-chain dictated the conformation of the heme environment of the human alpha-subunit, and to the alpha(1)beta(1) interfaces of the hybrid. In the alpha(2)(P)beta(2)(S) hybrid, beta(S)-chain dictated the conformation of human heme environment to the pig alpha-chain in the hybrid; but the conformation of alpha(1)beta(1) interface of this hybrid is close to, but not identical to that of HbS. On the other hand, the alpha(1)beta(2) interface conformation is identical to that of HbS. More important, the alpha(2)(P)beta(2)(S) does not polymerize when deoxygenated; pig alpha-chain completely neutralizes the beta(S)-chain dependent polymerization. The polymerization inhibitory propensity of pig alpha-chain is higher when it is present in the cis alpha(P)beta(S) dimer relative to that in a trans alpha(P)beta(A) dimer. The semisynthetically generated chimeric pig-human and human-pig alpha-chains by exchanging the alpha(1-30) segments of human and pig alpha-chains have established that the sequence differences of pig alpha(31-141) segment can also completely neutralize the polymerization. Comparison of the electrostatic potential energy landscape of the alpha-chain surfaces of HbS and alpha(2)(P)beta(2)(S) suggests that the differences in electrostatic potential energy surfaces on the alpha-chain of alpha(2)(P)beta(2)(S) relative to that in HbS, particularly the ones involving CD region, E-helix and EF-corner of pig alpha-chain are responsible for the polymerization neutralization activity. The pig and human-pig chimeric alpha-chains can serve as blueprints for the design of a new generation of variants of alpha-chain(s) suitable for the gene therapy of sickle cell disease.     

Enhanced inhibition of polymerization of sickle cell hemoglobin in the presence of recombinant mutants of human fetal hemoglobin with substitutions at position 43 in the gamma-chain

Four recombinant mutants of human fetal hemoglobin [Hb F (alpha2gamma2)] with amino acid substitutions at the position 43 of the gamma-chain, rHb (gammaD43L), rHb (gammaD43E), rHb (gammaD43W), and rHb (gammaD43R), have been expressed in our Escherichia coli expression system and used to investigate their inhibitory effect on the polymerization of deoxygenated sickle cell hemoglobin (Hb S). Oxygen-binding studies show that rHb (gammaD43E), rHb (gammaD43W), and rHb (gammaD43R) exhibit higher oxygen affinity than human normal adult hemoglobin (Hb A), Hb F, or rHb (gammaD43L), and all four rHbs are cooperative in binding O2. Proton nuclear magnetic resonance (NMR) studies of these four rHbs indicate that the quaternary and tertiary structures around the heme pockets are similar to those of Hb F in both deoxy (T) and liganded (R) states. Solution light-scattering experiments indicate that these mutants remain mostly tetrameric in the liganded (R) state. In equimolar mixtures of Hb S and each of the four rHb mutants (gammaD43L, gammaD43E, gammaD43R, and gammaD43W), the solubility (Csat) of each of the pairs of Hbs is higher than that of a similar mixture of Hb S and Hb A, as measured by dextran-Csat experiments. Furthermore, the Csat values for Hb S/rHb (gammaD43L), Hb S/rHb (gammaD43E), and Hb S/rHb (gammaD43R) mixtures are substantially higher than that for Hb S/Hb F. The results suggest that these three mutants of Hb F are more effective than Hb F in inhibiting the polymerization of deoxy-Hb S in equimolar mixtures.     

Oxygen binding to partially nitrosylated hemoglobin

Reactions of nitric oxide (NO) with hemoglobin (Hb) are important elements in protection against nitrosative damage. NO in the vasculature is depleted by the oxidative reaction with oxy Hb or by binding to deoxy Hb to generate partially nitrosylated Hb (Hb–NO). Many aspects of the formation and persistence of Hb–NO are yet to be clarified. In this study, we used a combination of EPR and visible absorption spectroscopy to investigate the interactions of partially nitrosylated Hb with O₂. Partially nitrosylated Hb samples had predominantly hexacoordinate NO–heme geometry and resisted oxidation when exposed to O₂ in the absence of anionic allosteric effectors. Faster oxidation occurred in the presence of 2,3-diphosphoglycerate (DPG) or inositol hexaphosphate (IHP), where the NO–heme derivatives had higher levels of pentacoordinate heme geometry. The anion-dependence of the NO–heme geometry also affected O₂ binding equilibria. O₂-binding curves of partially nitrosylated Hb in the absence of anions were left-shifted at low saturations, indicating destabilization of the low O₂ affinity T-state of the Hb by increasing percentages of NO–heme, much as occurs with increasing levels of CO–heme. Samples containing IHP showed small decreases in O₂ affinity, indicating shifts toward the low-affinity T-state and formation of inert α-NO/β-met tetramers. Most remarkably, O₂-equilibria in the presence of the physiological effector DPG were essentially unchanged by up to 30% NO–heme in the samples. As will be discussed, under physiological conditions the interactions of Hb with NO provide protection against nitrosative damage without impairing O₂ transport by Hb's unoccupied heme sites. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.     

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)     

Control of heme reactivity by diffusion: structural basis and functional characterization in hemoglobin mutants.

The effect of mutagenesis on O(2), CO, and NO binding to mutants of human hemoglobin, designed to modify some features of the reactivity that hinder use of hemoglobin solutions as blood substitute, has been extensively investigated. The kinetics may be interpreted in the framework of the Monod-Wyman-Changeux two-state allosteric model, based on the high-resolution crystallographic structures of the mutants and taking into account the control of heme reactivity by the distal side mutations. The mutations involve residues at topological position B10 and E7, i.e., Leu (B10) to Tyr and His (E7) to Gln, on either the alpha chains alone (yielding the hybrid tetramer Hbalpha(YQ)), the beta chains alone (hybrid tetramer Hbbeta(YQ)), or both types of chains (Hb(YQ)). Our data indicate that the two mutations affect ligand diffusion into the pocket, leading to proteins with low affinity for O(2) and CO, and especially with reduced reactivity toward NO, a difficult goal to achieve. The observed kinetic heterogeneity between the alpha(YQ) and beta(YQ) chains in Hb(YQ) has been rationalized on the basis of the three-dimensional structure of the active site. Furthermore, we report for the first time an experiment of partial CO binding, selective for the beta chains, to high salt crystals of the mutant Hb(YQ) in the T-state; these crystallographic data may be interpreted as "snapshots" of the initial events possibly occurring on ligand binding to the T-allosteric state of this peculiar mutant Hb.     

Structure and function of the Gondwanian hemoglobin of Pseudaphritis urvillii, a primitive notothenioid fish of temperate latitudes

The suborder Notothenioidei dominates the Antarctic ichthyofauna. The non-Antarctic monotypic family Pseudaphritidae is one of the most primitive families. The characterization of the oxygen-transport system of euryhaline Pseudaphritis urvillii is herewith reported. Similar to most Antarctic notothenioids, this temperate species has a single major hemoglobin (Hb 1, over 95% of the total). Hb 1 has strong Bohr and Root effects. It shows two very uncommon features in oxygen binding: At high pH values, the oxygen affinity is exceptionally high compared to other notothenioids, and subunit cooperativity is modulated by pH in an unusual way, namely the curve of the Hill coefficient is bell-shaped, with values approaching 1 at both extremes of pH. Molecular modeling, electronic absorption and resonance Raman spectra have been used to characterize the heme environment of Hb 1 in an attempt to explain these features, particularly in view of some potentially important nonconservative replacements found in the primary structure. Compared to human HbA, no major changes were found in the structure of the proximal cavity of the alpha-chain of Hb 1, although an altered distal histidyl and heme position was identified in the models of the beta-chain, possibly facilitated by a more open heme pocket due to reduced steric constraints on the vinyl substituent groups. This conformation may lead to the hemichrome form identified by spectroscopy in the Met state, which likely fulfils a potentially important physiological role.     

Autoxidation and Oxygen Binding Properties of Recombinant Hemoglobins with Substitutions at the αVal-62 or βVal-67 Position of the Distal Heme Pocket

The E11 valine in the distal heme pocket of either the α- or β-subunit of human adult hemoglobin (Hb A) was replaced by leucine, isoleucine, or phenylalanine. Recombinant proteins were expressed in Escherichia coli and purified for structural and functional studies. ¹H NMR spectra were obtained for the CO and deoxy forms of Hb A and the mutants. The mutations did not disturb the α₁β₂ interface in either form, whereas the H-bond between αHis-103 and βGln-131 in the α₁β₁ interfaces of the deoxy α-subunit mutants was weakened. Localized structural changes in the mutated heme pocket were detected for the CO form of recombinant Hb (rHb) (αV62F), rHb (βV67I), and rHb (βV67F) compared with Hb A. In the deoxy form the proximal histidyl residue in the β-subunit of rHb (βV67F) has been altered. Furthermore, the interactions between the porphyrin ring and heme pocket residues have been perturbed in rHb (αV62I), rHb (αV62F), and rHb (βV67F). Functionally, the oxygen binding affinity (P₅₀), cooperativity (n₅₀), and the alkaline Bohr Effect of the three α-subunit mutants and rHb (βV67L) are similar to those of Hb A. rHb (βV67I) and rHb (βV67F) exhibit low and high oxygen affinity, respectively. rHb (βV67F) has P₅₀ values lower that those reported for rHb (αL29F), a B10 mutant studied previously in our laboratory (Wiltrout, M. E., Giovannelli, J. L., Simplaceanu, V., Lukin, J. A., Ho, N. T., and Ho, C. (2005) Biochemistry 44, 7207–7217). These E11 mutations do not slow down the autoxidation and azide-induced oxidation rates of the recombinant proteins. Results from this study provide new insights into the roles of E11 mutants in the structure-function relationship in hemoglobin.     

Heterotropic effectors control the hemoglobin function by interacting with its T and R states--a new view on the principle of allostery

Careful analyses of precise oxygenation curves of hemoglobin (Hb) clearly indicate that, contrary to the common belief, allosteric effectors exert a dramatic control of the oxygenation characteristics of the protein by binding not only to the T (unligated), but also to the R (ligated) state, in a process that is proton-driven and involves proton uptake. The most striking functional changes were obtained when the allosteric effectors were bound to the fully ligated Hb: the oxygen affinity decreased dramatically, Bohr effect was enhanced, and cooperativity of oxygen ligation was almost absent, emulating a Root effect-like behavior. However, structural analysis, such as Cys beta 93 sulfhydryl reactivity and ultraviolet circular dichroism, confirmed that the ligated Hb was in fact in the R state, despite its extremely low affinity state features. These findings provide a new global view for allosteric interactions and invoke for a modern interpretation of the role of allosteric effectors and a reformulation of the Monod-Wyman-Changeaux model for control of allosteric systems, and other complementary models as well.