A microscopic study of the deoxyhemoglobin-catalyzed generation of nitric oxide from nitrite anion

There is recent evidence suggesting that nitrite anion (NO 2 (-)) represents the major intravascular NO storage molecule whose transduction to NO is facilitated by a reduction mechanism catalyzed by deoxygenated hemoglobin (deoxy-Hb). In this work, we provide a detailed microscopic study of deoxy-Hb nitrite reductase (NIR) activity by combining classical molecular dynamics and hybrid quantum mechanical-molecular mechanical simulations. Our results point out that two alternative mechanisms could be operative and suggest that the most energetic barriers should stem from either reprotonation of the distal histidine or NO dissociation from the ferric heme. In the first proposed mechanism, which is similar to that proposed for bacterial NIRs, nitrite anion or nitrous acid coordinates to the heme through the N atom. This pathway involves HisE7 in a one or two proton transfer process, depending on whether the active species is nitrite anion or nitrous acid, to yield an intermediate Fe(III)NO species which eventually dissociates leading to NO and methemoglobin. In the second mechanism, the nitrite anion coordinates to the heme through the O atom. This pathway requires only one proton transfer from HisE7 and leads directly to the formation of a hydroxo Fe(III) complex and NO.     

Immuno-spin trapping of hemoglobin and myoglobin radicals derived from nitrite-mediated oxidation

The reaction of nitrite with hemoglobin has become of increasing interest due to the realization that plasma nitrite may act as an NO congener that is activated by interaction with red blood cells. Using a combination of spectrophotometry, immuno-spin trapping, and EPR, we have examined the formation of radicals during the oxidation of oxyhemoglobin (oxyHb) and oxymyoglobin (oxyMb) by inorganic nitrite. The proposed intermediacy of ferryl species during this oxidation was confirmed by spectrophotometry using multiple linear regression analysis of kinetic data. Using EPR/spin trapping, a protein radical was observed in the case of oxyMb, but not oxyHb, and was inhibited by catalase. When DMPO spin trapping was combined with Western blot analysis using an anti-DMPO-nitrone antibody, globin/DMPO adducts of both oxyHb and oxyMb were detected, and their formation was inhibited by catalase. Catalase effects confirm the intermediacy of hydrogen peroxide as a heme oxidant in this system. Spectrophotometric kinetic studies revealed that the presence of DMPO elongated the lag phase and decreased the maximal rate of oxidation of both oxyHb and oxyMb, which suggests that the globin radical plays an active role in the mechanism of autocatalysis. Interestingly, the oxidation of oxyHb or oxyMb by nitrite, but not by hydrogen peroxide, produced a diffusible radical that was able to generate spin adducts on a bystander protein. This indicates that the oxidation of oxyhemeproteins by nitrite may cause more widespread oxidative damage than the corresponding oxidation by hydrogen peroxide. The immuno-spin trapping technique represents an important new development for the study of the range and extent of protein oxidation by free radicals and oxidants.     

The reaction of Pseudomonas nitrite reductase and nitrite. A stopped-flow and EPR study

The reaction between reduced Pseudomonas nitrite reductase and nitrite has been studied by stopped-flow and rapid-freezing EPR spectroscopy. The interpretation of the kinetics at pH 8.0 is consistent with the following reaction mechanism (where k1 and k3 much greater than k2). [formula: see text] The bimolecular step (Step 1) is very fast, being lost in the dead time of a rapid mixing apparatus; the stoichiometry of the complex has been estimated to correspond to one NO2- molecule/heme d1. The final species is the fully reduced enzyme with NO bound to heme d1; and at all concentrations of nitrite, there is no evidence for dissociation of NO or for further reduction of NO to N2O. Step 2 is assigned to an internal electron transfer from heme c to reduced NO-bound heme d1 occurring with a rate constant of 1 s-1; this rate is comparable to the rate of internal electron transfer previously determined when reducing the oxidized enzyme with azurin or cytochrome c551. When heme d1 is NO-bound, the rate at which heme c can accept electrons from ascorbate is remarkably increased as compared to the oxidized enzyme, suggesting an increase in the redox potential of the latter heme.     

Scavenging of reactive nitrogen species by oxygenated hemoglobin: globin radicals and nitrotyrosines distinguish nitrite from nitric oxide reaction

The reaction of *NO and NO2- with hemoglobin (Hb) is of pivotal importance to blood vessel function. Both species show at least two different reactions with Fe2+ Hb: one with deoxygenated Hb, in which the biological properties of *NO are preserved, and another with oxygenated hemoglobin (oxyHb), in which both species are oxidizes to NO3-. In this study we compared the oxidative reactions of *NO and NO2- and, in particular, the radical intermediates formed during transformation to NO3-. The reaction of NO2- with oxyHb was accelerated at high heme concentrations and produced stoichiometric amounts of NO3-. Direct EPR and spin trapping studies showed that NO2-, but not *NO, induced the formation of globin Tyr-, Trp-, and Cys-centered radicals. MS studies provided evidence of the formation of approximately 2% nitrotyrosine in both the alpha and beta subunits, suggesting that *NO2 diffuses in part away from the heme and reacts with Tyr radicals. No nitrotyrosines were detected in the reaction of *NO with oxyHb. Collectively, these results indicate that NO2- reaction with oxyHb causes an oxidative challenge not observed with *NO. The differences in oxidation mechanisms of *NO and NO2- are discussed.     

Potential of peroxynitrite to promote the conversion of oxyhemoglobin to methemoglobin

Superoxide anion and NO can react to form the highly oxidizing species peroxynitrite (ONOO-)which can react directly with hemoglobin (Hb) even in the presence of physiological concentration CO:. Thisresearch was to determine the ONOO--mediated oxidation damage to the heme of oxyhemoglobin (oxyHb)under conditions expected in blood. Results showed that 8-10 mol ONOO- was needed to quickly andcompletely convert 1 mol oxyHb to methemoglobin (metHb). ONOO- (20-140 μM) caused raoid andextensive formation of metHb from oxyHb (50 μM) mainly occurring within first 5-20 min of incubation.The conversion efficiency reached 16%, 48%, 60%, 79% and 88% output of metHb after 90 min ofincubation at 0, 20, 40, 100, and 140 μM ONOO- respectively. 1 mM CO2 caused a small decrease in theability of ONOO- to oxidize oxyHb, and ONOO--promoted conversion of oxyHb to metHb increased whenpH decreased from 8.0 to 6.0. Relatively lower temperature in blood condition will inhibit this reaction insome degree. We postulate that ONOO- can mediate oxidation damage to the heme, and cause heme lossfrom the hydrophobic cavity of Hb when its concentration exceeded 90 μM. These results indicated thatONOO- could convert oxyHb to metHb under the conditions expected in blood, and this reaction wasregulated by CO2 concentration, reaction time, temperature and pH value.     

Isotope labeling studies on the mechanism of N-N bond formation in denitrification

The mechanism of the denitrification and nitrosation reactions catalyzed by the heme cd-containing nitrite reductase from Pseudomonas stutzeri JM 300 has been studied with whole cell suspensions using H2(18)O, 15NO, and 15NO-2. The extent of H2(18)O exchange with the enzyme-bound nitrosyl intermediate, as determined by the 18O content of product N2O, decreased with increasing nitrite concentration, which is consistent with production of N2O by sequential reaction of two nitrite ions with the enzyme. Reaction of NO with whole cells in H2(18)O gave amounts of 18O in the N2O product consistent with equilibration of nitric oxide with a small pool of free nitrite. Using 15NO and NH2OH, competition between denitrification and nitrosation reactions was demonstrated, as is required if the enzyme-nitrosyl complex is an intermediate in both nitrosation and denitrification reactions. The first evidence for exchange of 18O between H2(18)O and a nitrosation intermediate occurring after the enzyme-nitrosyl complex, presumably an enzyme-bound nitrosamine, has been obtained. The collective results are most consistent with denitrification N2O originating via attack of NO-2 on a coordinated nitrosyl, as proposed earlier (Averill, B. A., and Tiedje, J. M. (1982) FEBS Lett. 138, 8-11).     

Mechanistic studies of the oxidation of oxyhemoglobin by peroxynitrite

The strong oxidizing and nitrating agent peroxynitrite has been shown to diffuse into erythrocytes and oxidize oxyhemoglobin (oxyHb) to metHb. Because the value of the second-order rate constant for this reaction is on the order of 10(4) M(-)(1) s(-)(1) and the oxyHb concentration is about 20 mM (expressed per heme), this process is rather fast and oxyHb is considered a sink for peroxynitrite. In this work, we showed that the reaction of oxyHb with peroxynitrite, both in the presence and absence of CO(2), proceeds via the formation of oxoiron(iv)hemoglobin (ferrylHb), which in a second step is reduced to metHb and nitrate by its reaction with NO(2)(*). In the presence of physiological relevant amounts of CO(2), ferrylHb is generated by the reaction of NO(2)(*) with the coordinated superoxide of oxyHb (HbFe(III)O(2)(*)(-)). This reaction proceeds via formation of a peroxynitrato-metHb complex (HbFe(III)OONO(2)), which decomposes to generate the one-electron oxidized form of ferrylHb, the oxoiron(iv) form of hemoglobin with a radical localized on the globin. CO(3)(*)(-), the second radical formed from the reaction of peroxynitrite with CO(2), is also scavenged efficiently by oxyHb, in a reaction that finally leads to metHb production. Taken together, our results indicate that oxyHb not only scavenges peroxynitrite but also the radicals produced by its decomposition.     

H218O isotope exchange studies on the mechanism of reduction of nitric oxide and nitrite to nitrous oxide by denitrifying bacteria. Evidence for an electrophilic nitrosyl during reduction of nitric oxide

Reduction of NO and NO2-by whole cells of eight strains of denitrifying bacteria known to contain either heme cd1 or copper-containing nitrite reductases (NiRs) has been examined in the presence of H218O. All organisms containing heme cd1 NiRs exhibited relatively large extents of exchange between NO2- and H218O (39-100%), as monitored by the 18O content of product N2O. Organisms containing copper NiRs gave highly variable results, with Achromobacter cycloclastes and Pseudomonas aureofaciens exhibiting no 18O incorporation and Rhodopseudomonas sphaeroides and Alcaligenes entrophus exhibiting complete exchange between NO2- and H218O. Organisms containing heme cd1 NiRs exhibited significant but lower levels of exchange between NO and H218O than between NO2- and H218O, while organisms containing copper NiRs gave significantly higher amounts of 18O incorporation than observed for the heme cd1 organisms. These results demonstrate the existence of an NO-derived species capable of undergoing O-atom exchange with H218O during the reduction of NO. Trapping experiments with 15NO, 14N3-, and crude extracts of R. sphaeroides support the electrophilic nature of this intermediate and suggest its formulation as an enzyme nitrosyl, E-NO+, analogous to that observed during reduction of NO2-. The observation of lower levels of 18O incorporation with NO2- than with NO as substrate for A. cycloclastes and P. aureofaciens indicates that, for these organisms at least, a sequential pathway involving free NO as an intermediate is significantly less important than a direct pathway in which N2O is formed via reaction of two NO2- ions on a single enzyme.     

Reactions of nitrite with hemoglobin measured by membrane inlet mass spectrometry

Membrane inlet mass spectrometry was used to observe nitric oxide in the well-studied reaction of nitrite with hemoglobin. The membrane inlet was submerged in the reaction solutions and measured NO in solution via its flux across a semipermeable membrane leading to the mass spectrometer detecting the mass-to-charge ratio m/z 30. This method measures NO directly in solution and is an alternate approach compared with methods that purge solutions to measure NO. Addition to deoxy-Hb(Fe(II)) (near 38 microM heme concentration) of nitrite in a range of 80 microM to 16 mM showed no accumulation of either NO or N(2)O(3) on a physiologically relevant time scale with a sensitivity near 1 nM. The addition of nitrite to oxy-Hb(Fe(II)) and met-Hb(Fe(III)) did not accumulate free NO to appreciable extents. These observations show that for several minutes after mixing nitrite with hemoglogin, free NO does not accumulate to levels exceeding the equilibrium level of NO. The presence of cyanide ions did not alter the appearance of the data; however, the presence of 2 mM mercuric ions at the beginning of the experiment with deoxy-Hb(Fe(II)) shortened the initial phase of NO accumulation and increased the maximal level of free, unbound NO by about twofold. These experiments appear consistent with no role of met-Hb(Fe(III)) in the generation of NO and an increase in nitrite reductase activity caused by the presumed binding of mercuric to cysteine residues. These results raise questions about the ability of reduction of nitrite mediated by deoxy-Hb(Fe(II)) to play a role in vasodilation.     

Intramolecular electron transfer between tyrosyl radical and cysteine residue inhibits tyrosine nitration and induces thiyl radical formation in model peptides treated with myeloperoxidase, H2O2, and NO2-: EPR SPIN trapping studies

We investigated the effects of a cysteine residue on tyrosine nitration in several model peptides treated with myeloperoxidase (MPO), H(2)O(2), and nitrite anion (NO(2)(-)) and with horseradish peroxidase and H(2)O(2). Sequences of model peptides were acetyl-Tyr-Cys-amide (YC), acetyl-Tyr-Ala-Cys-amide (YAC), acetyl-Tyr-Ala-Ala-Cys-amide (YAAC), and acetyl-Tyr-Ala-Ala-Ala-Ala-Cys-amide (YAAAAC). Results indicate that nitration and oxidation products of tyrosyl residue in YC and other model peptides were barely detectable. A major product detected was the corresponding disulfide (e.g. YCysCysY). Spin trapping experiments with 5,5'-dimethyl-1-pyrroline N-oxide (DMPO) revealed thiyl adduct (e.g. DMPO-SCys-Tyr) formation from peptides (e.g. YC) treated with MPO/H(2)O(2) and MPO/H(2)O(2)/NO(2)(-). The steady-state concentrations of DMPO-thiyl adducts decreased with increasing chain length of model peptides. Blocking the sulfydryl group in YC with methylmethanethiosulfonate (that formed YCSSCH(3)) totally inhibited thiyl radical formation as did substitution of Tyr with Phe (i.e. FC) in the presence of MPO/H(2)O(2)/NO(2)(-). However, increased tyrosine nitration, tyrosine dimerization, and tyrosyl radical formation were detected in the MPO/H(2)O(2)/NO(2)(-)/YCSSCH(3) system. Increased formation of S-nitrosated YC (YCysNO) was detected in the MPO/H(2)O(2)/(*)NO system. We conclude that a rapid intramolecular electron transfer reaction between the tyrosyl radical and the Cys residue impedes tyrosine nitration and induces corresponding thiyl radical and nitrosocysteine product. Implications of this novel intramolecular electron transfer mechanism in protein nitration and nitrosation are discussed.     

Mechanistic studies of the oxygen-mediated oxidation of nitrosylhemoglobin

Nitrosylhemoglobin (HbFe(II)NO) has been shown to be generated in vivo from the reaction of deoxyHb with NO(*) as well as with nitrite. Despite the physiological importance attributed to this form of Hb, its reactivity has not been investigated in detail. In this study, we showed that the rate of oxidation of HbFe(II)NO by O(2) does not depend on the O(2) concentration. The reaction time courses had to be fitted to a two-exponential expression, and the obtained rates were approximately 2 x 10(-)(4) and 1 x 10(-)(4) s(-)(1), respectively. In the presence of the allosteric effector inositol hexaphosphate (IHP), the value for the fast component of the rate was significantly larger (44 x 10(-)(4) s(-)(1)) whereas that for the slow step was only slightly higher (2.5 x 10(-)(4) s(-)(1)). Moreover, we found that both in the absence and in the presence of IHP the rate of the O(2)-mediated oxidation of HbFe(II)NO is essentially identical to that of NO(*) dissociation from HbFe(II)NO, determined under analogous conditions by replacement of NO(*) with CO in the presence of an excess of dithionite. Taken together, our data show that the reaction between O(2) and HbFe(II)NO proceeds in three steps via dissociation of NO(*) (rate-determining step), binding of O(2) to deoxyHb, and NO(*)-mediated oxidation of oxyHb to metHb and nitrate.     

Transmembrane nitration of hydrophobic tyrosyl peptides. Localization,characterization, mechanism of nitration,and biological implications

We have shown previously that peroxynitrite-induced nitration of a hydrophobic tyrosyl probe is greater than that of tyrosine in the aqueous phase (Zhang, H., Joseph, J., Feix, J., Hogg, N., and Kalyanaraman, B. (2001) Biochemistry 40, 7675-7686). In this study, we have tested the hypothesis that the extent of tyrosine nitration depends on the intramembrane location of tyrosyl probes and on the nitrating species. To this end, we have synthesized membrane spanning 23-mer containing a single tyrosyl residue at positions 4, 8, and 12. The location of the tyrosine residues in the phospholipid membrane was determined by fluorescence and electron spin resonance techniques. Nitration was initiated by slow infusion of peroxynitrite, co-generated superoxide and nitric oxide ((.)NO), or a myeloperoxidase/hydrogen peroxide/nitrite anion (MPO/H(2)O(2)/NO(2)(-)) system. Results indicate that with slow infusion of peroxynitrite, nitration of transmembrane tyrosyl peptides was much higher (10-fold or more) than tyrosine nitration in aqueous phase. Peroxynitrite-dependent nitration of tyrosyl-containing peptides increased with increasing depth of the tyrosyl residue in the bilayer. In contrast, MPO/H(2)O(2)/ NO(2)(-)-induced tyrosyl nitration decreased with increasing depth of tyrosyl residues in the membrane. Transmembrane nitrations of tyrosyl-containing peptides induced by both peroxynitrite and MPO/H(2)O(2)/NO(2)(-) were totally inhibited by (.)NO that was slowly released from spermine NONOate. Nitration of peptides in both systems was concentration-dependently inhibited by unsaturated fatty acid. Concomitantly, an increase in lipid oxidation was detected. A mechanism involving (.)NO(2) radical is proposed for peroxynitrite and MPO/H(2)O(2)/NO(2)(-)-dependent transmembrane nitration reactions.     

Role of heme in intracellular trafficking of thyroperoxidase and involvement of H2O2 generated at the apical surface of thyroid cells in autocatalytic covalent heme binding

Thyroperoxidase (TPO) is a glycosylated hemoprotein that plays a key role in thyroid hormone synthesis. We previously showed that in CHO cells expressing human TPO (hTPO) only 2% of synthesized hTPO reaches the cell surface. Herein, we investigated the role of heme moiety insertion in the exit of hTPO from the endoplasmic reticulum. Peroxidase activity at the cell surface and cell surface expression of hTPO were decreased by approximately 30 and approximately 80%, respectively, with succinyl acetone, an inhibitor of heme biosynthesis, and were increased by 20% with holotransferrin and aminolevulinic acid, precursors of heme biosynthesis. Results were similar with holotransferrin plus aminolevulinic acid or hemin, but hemin increased cell surface activity more efficiently (+120%) relative to the control. It had been suggested (DePillis, G., Ozaki, S., Kuo, J. M., Maltby, D. A., and Ortiz de Montellano, P. R. (1997) J. Biol. Chem. 272, 8857-8960) that covalent attachment of heme to mammalian peroxidases could be an H2O2-dependent autocatalytic processing. In our study, heme associated intracellularly with hTPO, and we hypothesized that there was insufficient exposure to H2O2 in Chinese hamster ovary cells before hTPO reached the cell surface. After a 10-min incubation, 10 microM H2O2 led to a 65% increase in cell surface activity. In contrast, in thyroid cells, H2O2 was synthesized at the apical cell surface and allowed covalent attachment of heme. Two-day incubation of primocultures of thyroid cells with catalase led to a 30% decrease in TPO activity at the cell surface. In conclusion, we provide compelling evidence for an essential role of 1) heme incorporation in the intracellular trafficking of hTPO and of 2) H2O2 generated at the apical pole of thyroid cells in the autocatalytic covalent heme binding to the TPO molecule.     

Critical role of His369 in the reactivity of Pseudomonas aeruginosa cytochrome cd1 nitrite reductase with oxygen

In the denitrification pathway, Pseudomonas aeruginosa cytochrome cd1 nitrite reductase catalyzes the reduction of nitrite to nitric oxide; in vitro, this enzyme is also competent in the reduction of O2 to 2H2O. In this article, we present a comparative kinetic study of the O2 reaction in the wild-type nitrite reductase and in three site-directed mutants (Tyr10-->Phe, His369-->Ala and His327-->Ala/His369-->Ala) of the amino acid residues close to the d1 heme on the distal side. The results clearly indicate that His369 is the key residue in the control of reactivity, as its substitution with Ala, previously shown to affect the reduction of nitrite, also impairs the reaction with O2, affecting both the properties and lifespan of the intermediate species. Our findings allow the presentation of an overall picture for the reactivity of cytochrome cd1 nitrite reductase and extend our previous conclusion that the conserved distal histidines are essential for the binding to reduced d1 heme of different anions, whether a substrate such as nitrite, a ligand such as cyanide, or an intermediate in the O2 reduction. Moreover, we propose that His369 also exerts a protective role against degradation of the d1 heme, by preventing the formation and adverse effects of the reactive O2 species (never present in significant amounts in wild-type cytochrome cd1 nitrite reductase), a finding with clear physiological implications.     

Purification of cytochrome cd1 nitrite reductase from Pseudomonas stutzeri JM300 and reconstitution with native and synthetic heme d1.

Cytochrome cd1 nitrite reductase has been purified from Pseudomonas stutzeri strain JM 300. This enzyme appears to be a dimer with a subunit molecular mass of 54 kDa and its isoelectric point is determined to be 5.4. The N terminus of amino acid sequence has strong homology with that of nitrite reductase from P. aeruginosa. The apoprotein of this enzyme has been reconstituted with native and synthetic heme d1. The nitrite reductase activity measured by NO and N2O gas evolution can be restored to 82% of the activity of the original enzyme when the protein was reconstituted with the native heme d1 and to 77% of the activity when reconstituted with the synthetic heme d1. The absorption spectra of both reconstituted enzymes are essentially identical to that of the original nitrite reductase. These results further substantiate the novel dione structure of heme d1 as proposed. The loss of NO2- reducing activity in the absence of heme d1 and its restoration by addition of heme d1 provides further evidence that heme d1 plays a key role in the conversion of NO2- to NO and N2O.     

The heme pocket geometry of Lucina pectinata hemoglobin II restricts nitric oxide and peroxide entry: model of ligand control for the design of a stable oxygen carrier

Blood pressure elevation has been attributed in large part to the consumption of nitric oxide (NO) by extracellular hemoglobin (Hb) therapeutics following infusion in humans. We studied NO and hydrogen peroxide (H2O2) oxidative reaction kinetics of monomeric Hbs isolated from the clam Lucina pectinata to probe the effects of their distinctive heme pocket chemistries on ligand controls and heme oxidative stability. HbI (Phe43(CD1), Gln64(E7), Phe29(B10), and Phe68(E11)) reacted with high avidity with NO (k'(ox,NO) = 91 microM-1 s-1), whereas HbII (Phe44(CD1), Gln65(E7), Tyr30(B10), and Phe69(E11)) reacted at a much slower rate (k'(ox,NO)= 2.8 microM-1 s-1). However, replacing B10 (Phe) by Tyr in recombinant HbI (HbI PheB10Tyr) produced only a 2-fold reduction in the NO-induced oxidation rate (k'(ox,NO)= 49.9 microM-1 s-1). Among the clam Hbs, HbII exhibited the fastest NO dissociation and the slowest NO association with ferrous iron. Autoxidation, H2O2-mediated ferryl iron (FeIV) formation, and the subsequent heme degradation kinetics were much slower in HbII and HbI PheB10Tyr when compared to those of HbI. The Tyr(B10) residue appears to afford a greater heme oxidative stability advantage toward H2O2, whereas the close proximity of this residue together with Gln(E7) to the heme iron contributes largely to the distal control of NO binding. Engineering of second-generation Hb-based oxygen therapeutics that are resistant to NO/H2O2-driven oxidation may ultimately require further optimization of the heme pocket architecture to limit heme exposure to solvent.     

Mechanism of autocatalytic oxidation of oxyhemoglobin by nitrite. An intermediate detected by electron spin resonance

Oxidation of oxyhemoglobin by nitrite is characterized by the presence of a lag phase followed by the autocatalysis. Just before the autocatalysis begins, an asymmetric ESR signal is detected which is similar to that of the methemoglobin radical generated from methemoglobin and H2O2 in shape, g value (2.005), peak-to-peak width (18 G) and other properties, except the difference in the dependence on temperature. Generation of H2O2 is indicated by the prolongation of the lag phase by the addition of catalase. On the other hand, the oxidation is modified by neither superoxide dismutase nor Nitroblue tetrazolium. The oxidation is prolonged in the presence of KCN. The present results indicate a free-radical mechanism for the oxidation in which the asymmetric radical catalyzes the formation of NO2 from NO2- by a peroxidase action and NO2 oxidizes oxyhemoglobin in the autocatalytic phase.     

The nitric oxide congener nitrite inhibits myeloperoxidase/H2O2/ Cl- -mediated modification of low density lipoprotein

Nitric oxide, a pivotal molecule in vascular homeostasis, is converted under aerobic conditions to nitrite. Recent studies have shown that myeloperoxidase (MPO), an abundant heme protein released by activated leukocytes, can oxidize nitrite (NO(2-)) to a radical species, most likely nitrogen dioxide. Furthermore, hypochlorous acid (HOCl), the major strong oxidant generated by MPO in the presence of physiological concentrations of chloride ions, can also react with nitrite, forming the reactive intermediate nitryl chloride. Since MPO and MPO-derived HOCl, as well as reactive nitrogen species, have been implicated in the pathogenesis of atherosclerosis through oxidative modification of low density lipoprotein (LDL), we investigated the effects of physiological concentrations of nitrite (12.5-200 microm) on MPO-mediated modification of LDL in the absence and presence of physiological chloride concentrations. Interestingly, nitrite concentrations as low as 12.5 and 25 microm significantly decreased MPO/H2O2)/Cl- -induced modification of apoB lysine residues, formation of N-chloramines, and increases in the relative electrophoretic mobility of LDL. In contrast, none of these markers of LDL atherogenic modification were affected by the MPO/H2O2/NO2-) system. Furthermore, experiments using ascorbate (12.5-200 microm) and the tyrosine analogue 4-hydroxyphenylacetic acid (12.5-200 microm), which are both substrates of MPO, indicated that nitrite inhibits MPO-mediated LDL modifications by trapping the enzyme in its inactive compound II form. These data offer a novel mechanism for a potential antiatherogenic effect of the nitric oxide congener nitrite.     

Ras regulation by reactive oxygen and nitrogen species

Ras GTPases cycle between inactive GDP-bound and active GTP-bound states to modulate a diverse array of processes involved in cellular growth control. We have previously shown that both NO/O(2) (via nitrogen dioxide, (*)NO(2)) and superoxide radical anion (O(2)(*)(-)) promote Ras guanine nucleotide dissociation. We now show that hydrogen peroxide in the presence of transition metals (i.e., H(2)O(2)/transition metals) and peroxynitrite also trigger radical-based Ras guanine nucleotide dissociation. The primary redox-active reaction species derived from H(2)O(2)/transition metals and peroxynitrite is O(2)(*)(-) and (*)NO(2), respectively. A small fraction of hydroxyl radical (OH(*)) is also present in both. We also show that both carbonate radical (CO(3)(*)(-)) and (*)NO(2), derived from the mixture of peroxynitrite and bicarbonate, facilitate Ras guanine nucleotide dissociation. We further demonstrate that NO/O(2) and O(2)(*)(-) promote Ras GDP exchange with GTP in the presence of a radical-quenching agent, ascorbate, or NO, and generation of Ras-GTP promotes high-affinity binding of the Ras-binding domain of Raf-1, a downstream effector of Ras. S-Nitrosylated Ras (Ras-SNO) can be formed when NO serves as a radical-quenching agent, and hydroxyl radical but not (*)NO(2) or O(2)(*)(-) can further react with Ras-SNO to modulate Ras activity in vitro. However, given the lack of redox specificity associated with the high redox potential of OH(*), it is unclear whether this reaction occurs under physiological conditions.     

Microperoxidase 8 catalyzed nitration of phenol by nitrogen dioxide radicals.

Microperoxidase 8 (MP8) is a heme octapeptide obtained by hydrolytic digestion of horse heart cytochrome c. At pH below 9, the heme iron is axially coordinated to the imidazole side chain of His18 and to a water molecule. Replacement of this weak ligand by H2O2 allows the formation of high-valent iron-oxo species which are responsible for both peroxidase-like and cytochrome P450-like activities of MP8. This paper shows that MP8 is able to catalyze the nitration of phenol by nitrite. The reaction requires H2O2 and is inhibited by ligands having a high affinity for the iron, catalase and radical scavengers. This suggests that the nitrating species could be NO2* radicals formed by the oxidation of nitrite by high-valent iron-oxo species. This new activity of MP8 opens a new access to nitro-aromatic compounds under mild conditions and validates the use of this minienzyme to mimick heme peroxidases, especially in the reactions of NO-derived species with biomolecules under oxidative stress conditions.