Nitrite reductase activity of hemoglobin S (sickle) provides insight into contributions of heme redox potential versus ligand affinity

Hemoglobin A (HbA) is an allosterically regulated nitrite reductase that reduces nitrite to NO under physiological hypoxia. The efficiency of this reaction is modulated by two intrinsic and opposing properties: availability of unliganded ferrous hemes and R-state character of the hemoglobin tetramer. Nitrite is reduced by deoxygenated ferrous hemes, such that heme deoxygenation increases the rate of NO generation. However, heme reactivity with nitrite, represented by its bimolecular rate constant, is greatest when the tetramer is in the R quaternary state. The mechanism underlying the higher reactivity of R-state hemes remains elusive. It can be due to the lower heme redox potential of R-state ferrous hemes or could reflect the high ligand affinity geometry of R-state tetramers that facilitates nitrite binding. We evaluated the nitrite reductase activity of unpolymerized sickle hemoglobin (HbS), whose oxygen affinity and cooperativity profile are equal to those of HbA, but whose heme iron has a lower redox potential. We now report that HbS exhibits allosteric nitrite reductase activity with competing proton and redox Bohr effects. In addition, we found that solution phase HbS reduces nitrite to NO significantly faster than HbA, supporting the thesis that heme electronics (i.e. redox potential) contributes to the high reactivity of R-state deoxy-hemes with nitrite. From a pathophysiological standpoint, under conditions where HbS polymers form, the rate of nitrite reduction is reduced compared with HbA and solution-phase HbS, indicating that HbS polymers reduce nitrite more slowly.     

A recombinant human hemoglobin with anti-sickling properties greater than fetal hemoglobin

A new recombinant, human anti-sickling beta-globin polypeptide designated beta(AS3) (betaGly(16) --> Asp/betaGlu(22) --> Ala/betaThr(87) --> Gln) was designed to increase affinity for alpha-globin. The amino acid substitutions at beta22 and beta87 are located at axial and lateral contacts of the sickle hemoglobin (HbS) polymers and strongly inhibit deoxy-HbS polymerization. The beta16 substitution confers the recombinant beta-globin subunit (beta(AS3)) with a competitive advantage over beta(S) for interaction with the alpha-globin polypeptide. Transgenic mouse lines that synthesize high levels of HbAS3 (alpha(2)beta(AS3)(2)) were established, and recombinant HbAS3 was purified from hemolysates and then characterized. HbAS3 binds oxygen cooperatively and has an oxygen affinity that is comparable with fetal hemoglobin. Delay time experiments demonstrate that HbAS3 is a potent inhibitor of HbS polymerization. Subunit competition studies confirm that beta(AS3) has a distinct advantage over beta(S) for dimerization with alpha-globin. When equal amounts of beta(S)- and beta(AS3)-globin monomers compete for limiting alpha-globin chains up to 82% of the tetramers formed is HbAS3. Knock-out transgenic mice that express exclusively human HbAS3 were produced. When these mice were bred with knock-out transgenic sickle mice the beta(AS3) polypeptides corrected all hematological parameters and organ pathology associated with the disease. Expression of beta(AS3)-globin should effectively lower the concentration of HbS in erythrocytes of patients with sickle cell disease, especially in the 30% percent of these individuals who coinherit alpha-thalassemia. Therefore, constructs expressing the beta(AS3)-globin gene may be suitable for future clinical trials for sickle cell disease.     

Molecular insights into the irreversible mechanical behavior of sickle hemoglobin

Sickle cell disease is caused by the amino acid substitution of glutamic acid to valine, which leads to the polymerization of deoxygenated sickle hemoglobin (HbS) into long strands. These strands are responsible for the sickling of red blood cells (RBCs), making blood hyper-coagulable leading to an increased chance of vaso-occlusive crisis. The conformational changes in sickled RBCs traveling through narrow blood vessels in a highly viscous fluid are critical in understanding; however, there are few studies that investigate the origins of the molecular mechanical behavior of sickled RBCs. In this work, we investigate the molecular mechanical properties of HbS molecules. A mechanical model was used to estimate the directional stiffness of an HbS molecule and the results were compared to adult human hemoglobin (HbA). The comparison shows a significant difference in strength between HbS and HbA, as well as anisotropic behavior of the hemoglobin molecules. The results also indicated that the HbS molecule experienced more irreversible mechanical behavior than HbA under compression. Further, we have characterized the elastic and compressive properties of a double stranded sickle fiber using six HbS molecules, and it shows that the HbS molecules are bound to each other through strong inter-molecular forces.     

Sickle Cell Hemoglobin in the Ferryl State Promotes βCys-93 Oxidation and Mitochondrial Dysfunction in Epithelial Lung Cells (E10)

Polymerization of intraerythrocytic deoxyhemoglobin S (HbS) is the primary molecular event that leads to hemolytic anemia in sickle cell disease (SCD). We reasoned that HbS may contribute to the complex pathophysiology of SCD in part due to its pseudoperoxidase activity. We compared oxidation reactions and the turnover of oxidation intermediates of purified human HbS and HbA. Hydrogen peroxide (H₂O₂) drives a catalytic cycle that includes the following three distinct steps: 1) initial oxidation of ferrous (oxy) to ferryl Hb; 2) autoreduction of the ferryl intermediate to ferric (metHb); and 3) reaction of metHb with an additional H₂O₂ molecule to regenerate the ferryl intermediate. Ferrous and ferric forms of both proteins underwent initial oxidation to the ferryl heme in the presence of H₂O₂ at equal rates. However, the rate of autoreduction of ferryl to the ferric form was slower in the HbS solutions. Using quantitative mass spectrometry and the spin trap, 5,5-dimethyl-1-pyrroline-N-oxide, we found more irreversibly oxidized βCys-93in HbS than in HbA. Incubation of the ferric or ferryl HbS with cultured lung epithelial cells (E10) induced a drop in mitochondrial oxygen consumption rate and impairment of cellular bioenergetics that was related to the redox state of the iron. Ferryl HbS induced a substantial drop in the mitochondrial transmembrane potential and increases in cytosolic heme oxygenase (HO-1) expression and mitochondrial colocalization in E10 cells. Thus, highly oxidizing ferryl Hb and heme, the product of oxidation, may be central to the evolution of vasculopathy in SCD and may suggest therapeutic modalities that interrupt heme-mediated inflammation.     

Nitric oxide-mediated heme oxidation and selective beta-globin nitrosation of hemoglobin from normal and sickle erythrocytes

Nitric oxide (NO) has been reported to modulate the oxygen affinity of blood from sickle cell patients (SS), but not that of normal adult blood (AA), with little or no heme oxidation. However, we had found that the NO donor compounds 2-(N, N-diethylamino)-diazenolate-2-oxide (DEANO) and S-nitrosocysteine (CysNO) caused increased oxygen affinity of red cells from both AA and SS individuals and also caused significant methemoglobin (metHb) formation. Rapid kinetic experiments in which HbA(0), AA, or SS erythrocytes were mixed with CysNO or DEANO showed biphasic time courses indicative of initial heme oxidation followed by reductive heme nitrosylation, respectively. Hemolysates treated with CysNO showed by electrospray mass spectrometry a peak corresponding to a 29 mass unit increase (consistent with NO binding) of both the beta(A) and beta(S) chains but not of the alpha chains. Therapeutic use of NO in sickle cell disease may ultimately require further optimization of these competing reactions, i.e., heme reactivity (nitrosylation or oxidation) versus direct S-nitrosation of hemoglobin on the beta-globin.     

Potential of isoquercitrin as antisickling agent: a multi-spectroscopic,thermophoresis and molecular modeling approach

Abstract

Sickle cell disease is an inherited disease caused by point mutation in hemoglobin (β-globin gene). Under oxygen saturation, sickle hemoglobin form polymers, leading to rigid erythrocytes. The transition of the blood vessels is altered and initiated by the adhesion of erythrocytes, neutrophils and endothelial cells. Sickle Hemoglobin (HbS) polymerization is a major cause in red blood cells (RBC), promoting sickling and destruction of RBCs. Isoquercitrin, a medicinal bioactive compound found in various medicinal plants, has multiple health benefits. The present study examines the potential of isoquercitrin as an anti-sickle agent, showing a significant decrease in the rate of polymerization as well as sickling of RBCs. Isoquercitrin-induced graded alteration in absorbance and fluorescence of HbS, confirmed their interaction. A negative value of ΔG° strongly suggests that it is a spontaneous exothermic reaction induced by entropy. Negative ΔH° and positive ΔS° predicted that hydrogen and hydrophobic binding forces interfered with a hydrophobic microenvironment of β6Val leading to polymerization inhibition of HbS. HbS-Isoquercitrin complex exhibits helical structural changes leading to destabilization of the HbS polymer as confirmed by CD spectroscopy. MST and DSC results indicate greater changes in thermophoretic mobility and thermal stability of sickle hemoglobin in the presence of isoquercitrin, respectively. These findings were also supported by molecular simulation studies using DOCK6 and GROMACS. Hence, we can conclude that isoquercitrin interacts with HbS through hydrogen bonding, which leads to polymerization inhibition. Consequently, isoquercitrin could potentially be used as a medication for the treatment of sickle cell disease.

Communicated by Ramaswamy H. Sarma     

Hydroxy-urea protects erythrocytes against oxidative damage

Hydroxy-urea (OH-U) is used to treat sickle cell anemia by increasing hemoglobin fetal-fraction. It has been suggested that the sickle cell mutations lead to the formation of unstable HbS and release of iron, which can result in lipid peroxidation (LPO), and eventual cell damage. Since oxidative processes might be involved in pathogenesis of sickle cell disease, we investigated the antioxidant property of OH-U in a red blood cell (RBC) model. Intact RBCs or RBC membranes were exposed to t-butyl hydroperoxide (t-BHP, 0.75 mM) or iron (ferrous sulfate; 100 microM) at 37 degrees C for 60 min in the presence or absence of OH-U (1.25 mM). The extent of oxidative damage was measured by LPO (as thiobarbituric acid reactive substances, TBARS), hemoglobin oxidation (as percent of methemoglobin, metHb %), and decrease in the activities of membrane-bound Na+/K+-ATPase and Ca2+-ATPases. Our results show that OH-U inhibited t-BHP-induced LPO in fresh RBC membranes significantly (P <0.01). OH-U significantly inhibited t-BHP-mediated LPO (P <0.01) and metHb formation (P <0.01) in intact RBC. Also, OH-U inhibited iron-induced LPO and metHb formation in intact RBC (P <0.01). In addition, OH-U blocked t-BHP-mediated changes in membrane ATPase activities. Furthermore, OH-U blocked iron-mediated hydroxyl radical generation in a dose-dependent fashion. In conclusion, the observed antioxidant properties of OH-U might contribute to its therapeutic action in sickle cell disease.     

The reactions of myoglobin, normal adult hemoglobin, sickle cell hemoglobin and hemin with hydroxyurea

The kinetics of the reaction of hydroxyurea (HU) with myoglobin (Mb), hemin, sickle cell hemoglobin (HbS), and normal adult hemoglobin (HbA) were determined using optical absorption spectroscopy as a function of time, wavelength, and temperature. Each reaction appeared to follow pseudo-first order kinetics. Electron paramagnetic resonance spectroscopy (EPR) experiments indicated that each reaction produced an FeNO product. Reactions of hemin and the ferric forms of HbA, HbS, and myoglobin with HU also formed the NO adduct. The formation of methemoglobin and nitric oxide-hemoglobin from these reactions may provide further insight into the mechanism of how HU benefits sickle cell patients.     

Effects of iron nitrosylation on sickle cell hemoglobin solubility

One mechanism by which nitric oxide (NO) has been proposed to benefit patients with sickle cell disease is by reducing intracellular polymerization of sickle hemoglobin (HbS). In this study we have examined the ability of nitric oxide to inhibit polymerization by measuring the solubilizing effect of iron nitrosyl sickle hemoglobin (HbS-NO). Electron paramagnetic resonance spectroscopy was used to confirm that, as found in vivo, the primary type of NO ligation produced in our partially saturated NO samples is pentacoordinate alpha-nitrosyl. Linear dichroism spectroscopy and delay time measurements were used to confirm polymerization. Based on sedimentation studies we found that, although fully ligated (100% tetranitrosyl) HbS is very soluble, the physiologically relevant, partially ligated species do not provide a significant solubilizing effect. The average solubilizing effect of 26% NO saturation was 0.045; much less than the 0.15 calculated for the effect of 26% oxygen saturation. Given the small amounts of NO-ligated hemoglobin achievable through any kind of NO therapy, we conclude that NO therapy does not benefit patients through any direct solubilizing effect.     

O2 dependence of K+ transport in sickle cells: the effect of different cell populations and the substituted benzaldehyde 12C79.

The molecular basis of sickle cell disease (SCD) is well known but the pathophysiology is poorly understood. It remains intractable to therapy. Hyperactivity of several membrane transport systems, including the K+-Cl- cotransporter (termed KCC), cause HbS-containing red cells (termed HbS cells) to dehydrate and sickle, leading to the development of sickle cell crises (SCCs). Contrary to normal red cells (HbA cells), KCC in HbS cells is active at low O2 tensions (PO2s), remaining responsive to low pH or urea. Since these stimuli are usually encountered in hypoxic regions, the abnormal O2 dependence increases the contribution of KCC to dehydration, and hence development of SCCs. These differences with HbA cells may be due to the younger population of cells or to polymerization of HbS. We used 86Rb+ as a K+ congener to investigate the activity of KCC at different PO2s, and density gradient separation to investigate different red cell fractions. We found no correlation of O2 dependence with cell fractions. We also used the substituted benzaldehyde 12C79 to increase the O2 affinity of HbS and found that its effect on HbS O2 saturation and cell sickling correlated with that on both Cl--independent and Cl--dependent K+ transport, implying that, at low PO2s, KCC activity correlated with HbS polymerization. The importance of these results to understanding the pathophysiology of SCD, and for the design of chemotherapeutic agents to ameliorate or prevent SCC, is discussed.     

Solution-active structural alterations in liganded hemoglobins C (beta6 Glu --> Lys) and S (beta6 Glu --> Val).

Based upon existing crystallographic evidence, HbS, HbC, and HbA have essentially the same molecular structure. However, important areas of the molecule are not well defined crystallographically (e.g. the N-terminal nonhelical portion of the alpha and beta chains), and conformational constraints differ in solution and in the crystalline state. Over the years, our laboratory and others have provided evidence of conformational changes in HbS and, more recently, in HbC. We now present data based upon allosteric perturbation monitored by front-face fluorescence, ultraviolet resonance Raman spectroscopy, circular dichroism, and oxygen equilibrium studies that confirm and significantly expand previous findings suggesting solution-active structural differences in liganded forms of HbS and HbC distal to the site of mutation and involving the 2,3-diphosphoglycerate binding pocket. The liganded forms of these hemoglobins are of significant interest because HbC crystallizes in the erythrocyte in the oxy form, and oxy HbS exhibits increased mechanical precipitability and a high propensity to oxidize. Specific findings are as follows: 1) differences in the intrinsic fluorescence indicate that the Trp microenvironments are more hydrophobic for HbS > HbC > HbA, 2) ultraviolet resonance Raman spectroscopy detects alterations in Tyr hydrogen bonding, in Trp hydrophobicity at the alpha1beta2 interface (beta37), and in the A-helix (alpha14/beta15) of both chains, 3) displacement by inositol hexaphosphate of the Hb-bound 8-hydroxy-1,3,6-pyrenetrisulfonate (the fluorescent 2,3-diphosphoglycerate analog) follows the order HbA > HbS > HbC, and 4) oxygen equilibria measurements indicate a differential allosteric effect by inositol hexaphosphate for HbC approximately HbS > HbA.     

Covalent binding of glutathione to hemoglobin. I. Inhibition of hemoglobin S polymerization

Thiol reagents react with cysteine beta 93 of hemoglobin and as a result increase the oxygen affinity of hemoglobin. In the present studies we have used a thiol-disulfide exchange between mixed disulfides of hemoglobin and reduced glutathione to attach intracellular glutathione to hemoglobin and to study its antisickling properties. The rates of production of glutathionyl hemoglobin (G-Hb) depend on the structure of the thiol reagent linked to cysteine beta 93. Up to 25% G-Hb can be produced in normal and sickle red cells because of the high intracellular concentration of reduced glutathione. This high level of G-Hb in normal cells increases the oxygen affinity by about 35% and reduces heme-heme interactions. In sickle cells the increased oxygen affinity is associated with an inhibition of sickling of about 70% at 21 mm Hg. Inhibition of polymerization of deoxy HbS is also due to a direct inhibition of intermolecular contacts in the fibers as demonstrated by the increased solubility and the increased delay time of G-HbS compared to deoxy HbS.     

Hemoglobin S-nitrosation on oxygenation of nitrite/deoxyhemoglobin incubations is attenuated by methemoglobin

Nitrite is present in red blood cells (RBCs) and is proposed to be the largest intravascular storage pool of vasoactive NO. The mechanism by which nitrite exerts NO vasoactivity remains unclear but deoxyHb exhibits nitrite reductase activity. NitrosylHb (HbFe(II)NO) is formed on nitrite reduction by excess deoxyHb, and S-nitrosated Hb (HbSNO) has also been detected in nitrite/deoxyHb incubations. We report data consistent with efficient HbSNO generation from a nitrosylHb intermediate on oxygenation of anaerobic deoxyHb incubations containing physiologically revelant levels of nitrite, whereas previously a labile nitrosylmetHb (HbFe(III)NO) transient was proposed. The HbSNO yield as a function of the initial nitrite concentration varies with the nitrite/deoxyHb ratio, the incubation time, the concentration of added metHb (a nitrite trap), and the concentration of added cyanide (a strong metHb ligand). Our results reveal that metHb strongly attenuates HbSNO formation, which suggests that the met protein may play a regulatory role by limiting the amount of free (or non-Hb-bound) nitrite within RBCs to prevent hypotension.     

Cation Modulation of Hemoglobin Interaction with Sodium <Emphasis Type="Italic">n</Emphasis>-Dodecyl Sulfate (SDS). I: Calcium Modulation at pH 7.20

A comparative denaturation of HbA and HbS in the R states using sodium n-dodecyl sulfate (SDS) was carried out at pH 7.20 in the presence and absence of Calcium (0–40 μM) and monitored by UV–Vis spectrophotometry in the range of 250–650 nm. In the HbS spectra, the calcium alone caused little or no perturbation of the aromatic region but caused a decrease in oxygen affinity when compared to the HbA. The combinations of [SDS] and [Ca] perturbed the HbS the most, relative to the individual spectra of the [SDS] and [Ca]. However, the presence of Ca appeared to diminish the adverse effects of the SDS on HbA. The denaturation pathway of the HbA involved mainly the formation of heme dimers and some ferryl heme species. For the HbS, heme monomers and a large amount of ferryl species were formed. It is suggested that the greater monomer species formed by the HbS denaturation pathway would result in both Fenton and enhanced enzymatic reactions, compared to the dimer. This could lead ultimately to the formation of ferryl radicals. Thus, at physiological pH for the HbS, the Ca–SDS interaction increases the tendency for protein denaturation in comparison to the HbA.     

Sickle hemoglobin polymer stability probed by triple and quadruple mutant hybrids.

As part of an effort to understand the interactions in HbS polymerization, we have produced and studied a recombinant triple mutant, D6A(alpha)/D75Y(alpha)/E121R(beta), and a quadruple mutant comprising the preceding mutation plus the natural genetic mutation of sickle hemoglobin, E6V(beta). These recombinant hemoglobins expressed in yeast were extensively characterized, and their structure and oxygen binding cooperativity were found to be normal. Their tetramer-dimer dissociation constants were within a factor of 2 of HbA and HbS. Polymerization of these mutants mixed with HbS was investigated by a micromethod based on volume exclusion by dextran. The elevated solubility of mixtures of HbS with HbA and HbF in dextran could be accurately predicted without any variable parameters. Relative to HbS, the copolymerization probability of the quadruple mutant/HbS hybrid was found to be 6.2, and the copolymerization probability for the triple mutant/HbS hybrid was 0.52. The pure quadruple mutant had a solubility slightly above that of its hybrid with HbS. One way to explain these results is to require significant cis-trans differences in the polymer and that HbA assemble above 42.5 g/dl. A second way to explain these data is by the modification of motional freedom, thereby changing vibrational entropy in the polymer.     

Cryopreservation of cyanate-treated sickle erythrocytes

Metabolic features and in vivo recovery of cryopreserved cyanate-treated erythrocytes from patients with sickle cell anemia were studied. Red cells were treated with the anti-sickling agent sodium cyanate, glycerolized, and frozen at ?80 °C. Cyanate increased post-thaw hemolysis of both normal and sickle erythrocytes. The thawed carbamylated sickle erythrocytes maintained high oxygen affinity but lost more than half of their ATP content. Addition of the metabolic nutrients adenine, pyruvate, and inosine (rejuvenation) during cyanate incubation prevented ATP loss. Rejuvenation also increased red cell 2,3-DPG and opposed the cyanate effect by lowering oxygen affinity. Yet cyanate improved by nearly 50% the intravascular recovery of thawed rejuvenated sickle erythrocytes in a rat transfusion model. Cryopreservation of autologous cyanate-treated erythrocytes could lead to their use as an extracorporeal treatment of sickle cell disease.     

Sickle cell anemia and the affinity of the blood for oxygen]

Blood affinity for oxygen is reduced in patients with homozygous HbS disease. The mechanisms were related to polymerisation and sickling process. In the HbS blood, P50 values were related to the percentage of sickling, the effect of prior deoxygenation on P50 measurement was established. This work underlines the influences of experimental conditions on the determination of sickle cell blood affinity and the difficulties inherent in the evaluation of in vivo oxygen transport in sickle cell disease.     

Human erythrocyte membrane band 3 protein influences hemoglobin cooperativity. Possible effect on oxygen transport

Hemoglobin function can be modulated by the red cell membrane but some mechanistic details are incomplete. For example, the 43-kDa chymotryptic fragment of the cytoplasmic portion of red cell membrane Band 3 protein and its corresponding N-terminal 11-residue synthetic peptide lower the oxygen affinity of hemoglobin but effects on cooperativity are unclear. Using highly purified preparations, we also find a lowered Hill coefficient (n values <2) at subequivalent ratios of Band 3 fragment or of synthetic peptide to Hb, resulting in an oxygen affinity that is moderately decreased and a partially hyperbolic shape for the O2 binding curve. Both normal HbA and sickle HbS display this property. Thus, the determinant responsible for the Hb cooperativity decreases by the 43-kDa fragment resides within its first 11 N-terminal residues. This effect is observed in the absence of chloride and is reversed by its addition. As effector to Hb ratios approach equivalence or with saturating chloride normal cooperativity is restored, and oxygen affinity is further lowered because the shape of the oxygen binding curve becomes completely sigmoidal. The relative efficiencies of 2,3-diphosphoglycerate (DPG), the 43-kDa Band 3 fragment, and the 11-residue synthetic peptide in lowering cooperativity are very similar. The findings are explained based on the stereochemical mechanism of cooperativity because of two populations of T-state hemoglobin tetramers, one with bound effector and the other with free (Perutz, M. F. (1989) Q. Rev. Biophys. 22, 139-237). As a result of this property, hemoglobin at the membrane inner surface in contact with the N-terminal region of Band 3 could preferentially bind O2 at low oxygen tension and then release it upon saturation with 2,3-diphosphoglycerate in the interior of the red cell. Membrane modulation of hemoglobin oxygen affinity has particularly interesting implications for the polymerization of hemoglobin S in the sickle red cell.