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.     

Melatonin nitrosation promoted by NO*2; comparison with the peroxynitrite reaction

N-nitroso species have recently been detected in animal tissues. Protein N-nitrosotryptophan is the best candidate for this N-nitroso pool. N-nitrosation of N-blocked trytophan derivatives like melatonin (MelH) by N2O3 or peroxynitrite (ONOOH/ONOO- ) has been observed under conditions of pH and reagent concentrations similar to in vivo conditions. We studied the reaction of NO*2 with MelH. When NO*2 was synthesized by gamma-irradiation of aqueous neutral solutions of nitrate under anaerobic conditions, detected oxidation and nitration of MelH were negligible. In the presence of additional nitrite, when NO* was also generated, formation of 1-nitrosomelatonin increased with nitrite concentration. Nitrosation is not due to N2O3 but could proceed via successive additions of NO*2 and NO*. For comparison, peroxynitrite was infused into a solution of MelH under air leading to the same products as those detected in irradiated solutions but in different proportions. In the presence of additional nitrite, the formation of nitroderivatives increased significantly while N-formylkynuramine and 1-nitrosomelatonin were maintained at similar levels. Mechanistic implications are discussed.     

Ascorbate is a potent antioxidant against peroxynitrite-induced oxidation reactions. Evidence that ascorbate acts by re-reducing substrate radicals produced by peroxynitrite

Peroxynitrite (ONOO(((-)))/ONOOH) is expected in vivo to react predominantly with CO(2), thereby yielding NO(2)(.) and CO(3) radicals. We studied the inhibitory effects of ascorbate on both NADH and dihydrorhodamine 123 (DHR) oxidation by peroxynitrite generated in situ from 3-morpholinosydnonimine N-ethylcarbamide (SIN-1). SIN-1 (150 micrometer)-mediated oxidation of NADH (200 micrometer) was half-maximally inhibited by low ascorbate concentrations (61-75 micrometer), both in the absence and presence of CO(2). Control experiments performed with thiols indicated both the very high antioxidative efficiency of ascorbate and that in the presence of CO(2) in situ-generated peroxynitrite exclusively oxidized NADH via the CO(3) radical. This fact is attributed to the formation of peroxynitrate (O(2)NOO(-)/O(2)NOOH) from reaction of NO(2)(.) with O(2), which is formed from reaction of CO(3) with NADH. SIN-1 (25 micrometer)-derived oxidation of DHR was half-maximally inhibited by surprisingly low ascorbate concentrations (6-7 micrometer), irrespective of the presence of CO(2). Control experiments performed with authentic peroxynitrite revealed that ascorbate was in regard to both thiols and selenocompounds much more effective to protect DHR. The present results demonstrate that ascorbate is highly effective to counteract the oxidizing properties of peroxynitrite in the absence and presence of CO(2) by both terminating CO(3)/HO( small middle dot) reactions and by its repair function. Ascorbate is therefore expected to act intracellulary as a major peroxynitrite antagonist. In addition, a novel, ascorbate-independent protection pathway exists: scavenging of NO(2)(.) by O(2) to yield O(2)NOO(-), which further decomposes into NO(2)(-) and O(2).     

Reaction of superoxide and nitric oxide with peroxynitrite. Implications for peroxynitrite-mediated oxidation reactions in vivo

Peroxynitrite (ONOO(-)/ONOOH), the product of the diffusion-limited reaction of nitric oxide (*NO) with superoxide (O(-*)(2)), has been implicated as an important mediator of tissue injury during conditions associated with enhanced *NO and O(-*)(2) production. Although several groups of investigators have demonstrated substantial oxidizing and cytotoxic activities of chemically synthesized peroxynitrite, others have proposed that the relative rates of *NO and production may be critical in determining the reactivity of peroxynitrite formed in situ (Miles, A. M., Bohle, D. S., Glassbrenner, P. A., Hansert, B., Wink, D. A., and Grisham, M. B. (1996) J. Biol. Chem. 271, 40-47). In the present study, we examined the mechanisms by which excess O(-*)(2) or *NO production inhibits peroxynitrite-mediated oxidation reactions. Peroxynitrite was generated in situ by the co-addition of a chemical source of *NO, spermineNONOate, and an enzymatic source of O(-*)(2), xanthine oxidase, with either hypoxanthine or lumazine as a substrate. We found that the oxidation of the model compound dihydrorhodamine by peroxynitrite occurred via the free radical intermediates OH and NO(2), formed during the spontaneous decomposition of peroxynitrite and not via direct reaction with peroxynitrite. The inhibitory effect of excess O(-*)(2) on the oxidation of dihydrorhodamine could not be ascribed to the accumulation of the peroxynitrite scavenger urate produced from the oxidation of hypoxanthine by xanthine oxidase. A biphasic oxidation profile was also observed upon oxidation of NADH by the simultaneous generation of *NO and O(-*)(2). Conversely, the oxidation of glutathione, which occurs via direct reaction with peroxynitrite, was not affected by excess production of *NO. We conclude that the oxidative processes initiated by the free radical intermediates formed from the decomposition of peroxynitrite are inhibited by excess production of *NO or O(-*)(2), whereas oxidative pathways involving a direct reaction with peroxynitrite are not altered. The physiological implications of these findings are discussed.     

Formation of peroxynitrite from reaction of nitroxyl anion with molecular oxygen.

Peroxynitrite (ONOO(-)/ONOOH) is generally expected to be formed in vivo from the diffusion-controlled reaction between superoxide (O(2)) and nitric oxide ((*)NO). In the present paper we show that under aerobic conditions the nitroxyl anion (NO(-)), released from Angeli's salt (disodium diazen-1-ium-1,2,2-triolate, (-)ON=NO(2)(-)), generated peroxynitrite with a yield of about 65%. Simultaneously, hydroxyl radicals are formed from the nitroxyl anion with a yield of about 3% via a minor, peroxynitrite-independent pathway. Further experiments clearly underline that the chemistry of NO(-) in the presence of oxygen is mainly characterized by peroxynitrite and not by HO( small middle dot) radicals. Quantum-chemical calculations predict that peroxynitrite formation should proceed via intermediary formation of (*)NO and O(2), probably by an electron-transfer mechanism. This prediction is supported by the fact that H(2)O(2) is formed during the decay of NO(-) in the presence of superoxide dismutase (Cu(II),Zn-SOD). Since the nitroxyl anion may be released endogenously by a variety of biomolecules, substantial amounts of peroxynitrite might be formed in vivo via NO(-) in addition to the "classical" ( small middle dot)NO + O(2)() pathway.     

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.     

S-nitrosation of glutathione by nitric oxide,peroxynitrite, and (*)NO/O(2)(*-)

To elucidate potential mechanisms of S-nitrosothiol formation in vivo, we studied nitrosation of GSH and albumin by nitric oxide ((*)NO), peroxynitrite, and (*)NO/O(2)(*)(-). In the presence of O(2), (*)NO yielded 20% of S-nitrosoglutathione (GSNO) at pH 7.5. Ascorbate and the spin trap 4-hydroxy-[2,2,4,4-tetramethyl-piperidine-1-oxyl] (TEMPOL) inhibited GSNO formation by 67%. Electron paramagnetic resonance spectroscopy with 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO) demonstrated intermediate formation of glutathionyl radicals, suggesting that GSNO formation by (*)NO/O(2) is predominantly mediated by (*)NO(2). Peroxynitrite-triggered GSNO formation (0.06% yield) was stimulated 10- and 2-fold by ascorbate and TEMPOL, respectively. Co-generation of (*)NO and O(2)(*)(-) at equal fluxes yielded less GSNO than (*)NO alone, but was 100-fold more efficient (8% yield) than peroxynitrite. Moreover, in contrast to the reaction of peroxynitrite, GSNO formation by (*)NO/O(2)(*)(-) was inhibited by ascorbate. Similar results were obtained with albumin instead of GSH. We propose that sulfhydryl compounds react with O(2)(*)(-) to initiate a chain reaction that forms radical intermediates which combine with (*)NO to yield GSNO. In RAW 264.7 macrophages, S-nitrosothiol formation by (*)NO/O(2) and (*)NO/O(2)(*)(-) occurred with relative efficiencies comparable to those in solution. Our results indicate that concerted generation of (*)NO and O(2)(*)(-) may essentially contribute to nitrosative stress in inflammatory diseases.     

Formation of nitrogen oxides and citrulline upon oxidation of N omega-hydroxy-L-arginine by hemeproteins.

HRP catalyzes the oxidation of N omega-hydroxy-L-arginine (NOHA) by H2O2 with formation of citrulline and NO2- with initial rates of about 0.7 and 0.2 nmol per nmol HRP per min. In the same manner, cytochromes P450 from rat liver microsomes catalyze the oxidation of NOHA to citrulline and NO2- by cumylhydroperoxide. Inhibitors of these hemeproteins (N3- and CN- for HRP and miconazole for P450) strongly inhibit both citrulline and NO2- formation. Rates of NOHA oxidation by these hemeproteins markedly decrease with time presumably because of their denaturation by nitrogen oxides and of the formation of hemeprotein-iron-NO complexes. These results suggest that NO (and other nitrogen oxides) could be formed from oxidation of NOHA by other enzymes than NO-synthases.     

NO and NOx interactions with group 8 metalloporphyrins

There has been an ongoing interest in the reactions of nitric oxide (NO) with heme model compounds, with the goal of interpreting related reactions occurring in biology. With recent evidence that higher oxides (NO2-, *NO2, N2O3, etc.) may also be formed under bioregulatory conditions, there is a need to understand the reactivities of these compounds with such models. This review discusses the mechanistic studies of the reactions of iron, ruthenium, and osmium metalloporphyrin complexes with NO and the higher nitrogen oxides.     

Radical mechanisms of the decomposition of peroxynitrite and the peroxynitrite-CO(2) adduct and of reactions with L-tyrosine and related compounds as studied by (15)N chemically induced dynamic nuclear polarization.

Enhanced absorption is observed in the (15)N NMR spectra of (15)NO(-)(3) during decomposition of peroxynitrite and the peroxynitrite-CO(2) adduct at pH 5.25, indicating the formation of (15)NO(-)(3) in radical pairs [(15)NO(*)(2), HO(*)] and [(15)NO(*)(2), CO(*-)(3)]. During the reaction of peroxynitrite and the peroxynitrite-CO(2) adduct with L-tyrosine, the (15)N NMR signal of the nitration product 3-nitrotyrosine exhibits emission showing a radical pathway of its formation. The nuclear polarization is built up in radical pairs [(15)NO(*)(2), tyr(*)] generated by free radical encounters of nitrogen dioxide and tyrosinyl radicals. The (15)N NMR signal of (15)NO(-)(2) formed during reaction of peroxynitrite with L-tyrosine appears in emission. It is concluded that tyrosinyl radicals are generated by reaction of nitrogen dioxide with L-tyrosine. In contrast to this, (15)NO(-)(2) does not show (15)N chemically induced dynamic nuclear polarization (CIDNP) during reaction of the peroxynitrite-CO(2) adduct with L-tyrosine, indicating a different reaction mechanism, which is assumed to be a hydrogen transfer between CO(*-)(3) and L-tyrosine. Emission is also observed in the (15)N NMR signals of 2-nitro-4-fluorophenol, 3-nitro-4-hydroxyphenylacetic acid, 2-nitrophenol, and 4-nitrophenol during reaction of 4-fluorophenol, 4-hydroxyphenylacetic acid, and phenol with peroxynitrite and the peroxynitrite-CO(2) adduct. 3-Nitro-4-hydroxyphenylacetic acid is also observed in emission during reaction of phenylacetic acid with peroxynitrite, but is not formed with the peroxynitrite-CO(2) adduct. The magnitude of the (15)N CIDNP effect during reaction of peroxynitrite with 4-fluorophenol and of the peroxynitrite-CO(2) adduct with 4-fluorophenol and phenol is determined. It excludes the occurrence of nonradical reactions. Only weak emission signals are observed during the reaction of peroxynitrite with phenol in (15)NO(-)(2), 2-nitrophenol, and 4-nitrophenol. 2-Nitrophenol is only formed in traces, and 4-nitrophenol is only formed in higher yields. The latter might be generated in part via a nonradical pathway.     

Reactions of PTIO and carboxy-PTIO with *NO, *NO2, and O2-*

Nitronyl nitroxides, such as derivatives of 2-phenyl-4,4,5,5,-tetramethylimidazoline-1-oxyl 3-oxide (PTIOs), react with *NO to form the corresponding imino nitroxides (PTIs) and *NO2. PTIOs are considered as monitors of *NO, stoichiometric sources of *NO2, biochemical and physiological effectors, specific tools for the elimination of *NO, and potential therapeutic agents. However, a better understanding of the chemical properties of PTIOs, especially following their reaction with *NO, is necessary to resolve many of the reported discrepancies surrounding the effects of PTIOs and to better characterize their potential therapeutic activity. We have generated electrochemically the oxidized and reduced forms of PTIO and carboxy-PTIO (C-PTIO), characterized their absorption spectra, and determined the reduction potentials for the oxoammonium/nitroxide and nitroxide/hydroxylamine couples. The rate constants for the reaction of *NO2 with PTIO and C-PTIO to form the corresponding oxoammonium cations (PTIO+s) and nitrite were determined to be (1.5 - 2) x 10(7) m-1 s-1. We have also shown that the reactions of PTIO+s with *NO form PTIOs and NO2-. The rate constants for these reactions are approximately 30-fold higher than those for the reactions of PTIOs with *NO or O2-*. The present results show that (i) the reaction of PTIOs with *NO forms solely PTIs and NO2- where [NO2-]/[PTI] varies between 1 and 2 depending on the steady-state concentrations of *NO. Consequently, quantitation of *NO is valid only at sufficiently low fluxes of *NO; (ii) the reaction of PTIOs with *NO can be used as a valid source of *NO2 only when the latter is effectively scavenged by an appropriate reductant; and (iii) the formation of peroxynitrite cannot be efficiently inhibited by PTIOs even under relatively low fluxes of *NO and O2-* and millimolar levels of PTIOs.     

Is N2O3 the main nitrosating intermediate in aerated nitric oxide (NO) solutions in vivo? If so,where, when,and which one?

The widespread opinion that N(2)O(3) as a product of NO oxidation is the only nitros(yl)ating agent under aerobic conditions is based on experiments in homogeneous buffered water solutions. In vivo NO is oxidized in heterogeneous media and this opinion is not correct. The equilibrium in the system being dependent on temperature and DeltaG((sol)) for NO, NO(2), isomers of both N(2)O(3), and N(2)O(4). For polar solvents including water, DeltaG((sol)) for N(2)O(3) is high enough, and a stationary concentration of N(2)O(3) in the mixture with other oxides is sufficient to guarantee the hydrolysis of N(2)O(3) to nitrite. In heterogeneous media, the mixture contains solvates NO(2(sol)), N(2)O(3(sol)), and N(2)O(4(sol)) at stationary nonequilibrium concentrations. As far as DeltaG((sol)) is decreased in heterogeneous mixtures with low polar solvents and/or at increased temperatures, the equilibrium in such a system shifts to NO(2). Although NO(2) is a reactive free radical, it almost does not react with water. In contrast, the reaction with most functional protein groups efficiently proceeds by a radical type with the formation of nitrite and new radicals (X) further stabilized in various forms. Therefore, the ratio of the nitrosylated and nitrated products yields depends on actual concentrations of all NO(x).     

Oxidation of active center cysteine of bovine 1-Cys peroxiredoxin to the cysteine sulfenic acid form by peroxide and peroxynitrite.

Peroxiredoxins are antioxidant enzymes whose peroxidase activity depends on a redox-sensitive cysteine residue at the active center. In this study we investigated properties of the active center cysteine of bovine 1-Cys peroxiredoxin using a recombinant protein (BRPrx). The only cysteine residue of the BRPrx molecule was oxidized rapidly by an equimolar peroxide or peroxynitrite to the cysteine sulfenic acid. Approximate rates of oxidation of BRPrx by different peroxides were estimated using selenium glutathione peroxidase as a competitor. Oxidation of the active center cysteine of BRPrx by H2O2 proceeded only several times slowly than that of the selenocysteine of glutathione peroxidase. The rate of oxidation varied depending on peroxides tested, with H2O2 being about 7 and 80 times faster than tert-butyl hydroperoxide and cumene hydroperoxide, respectively. Peroxynitrite oxidized BRPrx slower than H2O2 but faster than tert-butyl hydroperoxide. Further oxidation of the cysteine sulfenic acid of BRPrx to higher oxidation states proceeded slowly. Oxidized BRPrx was reduced by dithiothreitol, dihydrolipoic acid, and hydrogen sulfide, and demonstrated peroxidase activity (about 30 nmol/mg/min) with these reductants as electron donors. beta-Mercaptoethanol formed a mixed disulfide and did not support peroxidase activity. Oxidized BRPrx did not react with glutathione, cysteine, homocysteine, N-acetyl-cysteine, and mercaptosuccinic acid.     

Chemical and biological interactions during nitrate and goethite reduction by Shewanella putrefaciens 200

Although previous research has demonstrated that NO(3)(-) inhibits microbial Fe(III) reduction in laboratory cultures and natural sediments, the mechanisms of this inhibition have not been fully studied in an environmentally relevant medium that utilizes solid-phase, iron oxide minerals as a Fe(III) source. To study the dynamics of Fe and NO(3)(-) biogeochemistry when ferric (hydr)oxides are used as the Fe(III) source, Shewanella putrefaciens 200 was incubated under anoxic conditions in a low-ionic-strength, artificial groundwater medium with various amounts of NO(3)(-) and synthetic, high-surface-area goethite. Results showed that the presence of NO(3)(-) inhibited microbial goethite reduction more severely than it inhibited microbial reduction of the aqueous or microcrystalline sources of Fe(III) used in other studies. More interestingly, the presence of goethite also resulted in a twofold decrease in the rate of NO(3)(-) reduction, a 10-fold decrease in the rate of NO(2)(-) reduction, and a 20-fold increase in the amounts of N(2)O produced. Nitrogen stable isotope experiments that utilized delta(15)N values of N(2)O to distinguish between chemical and biological reduction of NO(2)(-) revealed that the N(2)O produced during NO(2)(-) or NO(3)(-) reduction in the presence of goethite was primarily of abiotic origin. These results indicate that concomitant microbial Fe(III) and NO(3)(-) reduction produces NO(2)(-) and Fe(II), which then abiotically react to reduce NO(2)(-) to N(2)O with the subsequent oxidation of Fe(II) to Fe(III).     

Protein tyrosine nitration in cytokine-activated murine macrophages. Involvement of a peroxidase/nitrite pathway rather than peroxynitrite.

Peroxynitrite, formed in a rapid reaction of nitric oxide (NO) and superoxide anion radical (O(2)), is thought to mediate protein tyrosine nitration in various inflammatory and infectious diseases. However, a recent in vitro study indicated that peroxynitrite exhibits poor nitrating efficiency at biologically relevant steady-state concentrations (Pfeiffer, S., Schmidt, K., and Mayer, B. (2000) J. Biol. Chem. 275, 6346-6352). To investigate the molecular mechanism of protein tyrosine nitration in intact cells, murine RAW 264.7 macrophages were activated with immunological stimuli, causing inducible NO synthase expression (interferon-gamma in combination with either lipopolysaccharide or zymosan A), followed by the determination of protein-bound 3-nitrotyrosine levels and release of potential triggers of nitration (NO, O(2)*, H(2)O(2), peroxynitrite, and nitrite). Levels of 3-nitrotyrosine started to increase at 16-18 h and exhibited a maximum at 20-24 h post-stimulation. Formation of O(2) was maximal at 1-5 h and decreased to base line 5 h after stimulation. Release of NO peaked at approximately 6 and approximately 9 h after stimulation with interferon-gamma/lipopolysaccharide and interferon-gamma/zymosan A, respectively, followed by a rapid decline to base line within the next 4 h. NO formation resulted in accumulation of nitrite, which leveled off at about 50 microm 15 h post-stimulation. Significant release of peroxynitrite was detectable only upon treatment of cytokine-activated cells with phorbol 12-myristate-13-acetate, which led to a 2.2-fold increase in dihydrorhodamine oxidation without significantly increasing the levels of 3-nitrotyrosine. Tyrosine nitration was inhibited by azide and catalase and mimicked by incubation of unstimulated cells with nitrite. Together with the striking discrepancy in the time course of NO/O(2) release versus 3-nitrotyrosine formation, these results suggest that protein tyrosine nitration in activated macrophages is caused by a nitrite-dependent peroxidase reaction rather than peroxynitrite.     

Intervention by nitric oxide, NO, and its oxide derivatives particularly in mammals

Nitric oxide (NO) is a natural and stable free radical produced in soil and water by the bacteriological reduction of nitrites and nitrates and in animals by the enzyme oxidation of L-arginine. NO is biosynthesised by finely regulated enzymatic systems called NO-synthases and readily diffuses through tissues. It reacts rapidly with hemoproteins and iron-sulphur centers to form nitrosylated compounds. It oxidises more slowly to form nitrogen oxides that nitrosate thiols into thionitrite. NO is transported in these various forms and released spontaneously or through yet unclear mechanisms into most cells; it also regulates oxygen consumption at the mitochondrial respiratory chain level through interaction with cytochrome oxidase. In the cardiovascular system, NO lowers blood pressure by activating a hemoprotein, the guanylate cyclase present in muscle cells; through such interaction it acts also as a neuromediator and neuromodulator in the nervous system. However, many of NO's roles result from rapid coupling to other radicals; for example, it reacts with the superoxide anion (O2-) to form oxoperoxinitrate (ONOO-, also known as peroxynitrite). This strong oxidant of metallic centers, thiols, and antioxidants is also able to convert tyrosine to 3-nitrotyrosine and to act upon tyrosine residues contained in proteins. The biological aspects of the roles of NO are presented with particular respect to the rapid interactions of NO with hemoproteins' iron and other radicals. Concurrently, NO oxidation enables nitrosation reactions primarily of thiols but ultimately of nucleic bases. The thionitrite function (R-S-NO) thus formed and the dimerisation and nitration of tyrosine residues are protein post-translational modifications that are being investigated in animals.     

Contributions of Autotrophic and Heterotrophic Nitrifiers to Soil NO and N(2)O Emissions

Soil emission of gaseous N oxides during nitrification of ammonium represents loss of an available plant nutrient and has an important impact on the chemistry of the atmosphere. We used selective inhibitors and a glucose amendment in a factorial design to determine the relative contributions of autotrophic ammonium oxidizers, autotrophic nitrite oxidizers, and heterotrophic nitrifiers to nitric oxide (NO) and nitrous oxide (N(2)O) emissions from aerobically incubated soil following the addition of 160 mg of N as ammonium sulfate kg. Without added C, peak NO emissions of 4 mug of N kg h were increased to 15 mug of N kg h by the addition of sodium chlorate, a nitrite oxidation inhibitor, but were reduced to 0.01 mug of N kg h in the presence of nitrapyrin [2-chloro-6-(trichloromethyl)-pyridine], an inhibitor of autotrophic ammonium oxidation. Carbon-amended soils had somewhat higher NO emission rates from these three treatments (6, 18, and 0.1 mug of N kg h after treatment with glucose, sodium chlorate, or nitrapyrin, respectively) until the glucose was exhausted but lower rates during the remainder of the incubation. Nitrous oxide emission levels exhibited trends similar to those observed for NO but were about 20 times lower. Periodic soil chemical analyses showed no increase in the nitrate concentration of soil treated with sodium chlorate until after the period of peak NO and N(2)O emissions; the nitrate concentration of soil treated with nitrapyrin remained unchanged throughout the incubation. These results suggest that chemoautotrophic ammonium-oxidizing bacteria are the predominant source of NO and N(2)O produced during nitrification in soil.     

Production of NO, N2O and N2 by extracted soil bacteria, regulation by NO2(-) and O2 concentrations.

The oxygen control of denitrification and its emission of NO/N2O/N2 was investigated by incubation of Nycodenz-extracted soil bacteria in an incubation robot which monitors O2, NO, N2O and N2 concentrations (in He+O2 atmosphere). Two consecutive incubations were undertaken to determine (1) the regulation of denitrification by O2 and NO2(-) during respiratory O2 depletion and (2) the effects of re-exposure to O2 of cultures with fully expressed denitrification proteome. Early denitrification was only detected (as NO and N2O) at 相似文献   

Bacterium-based NO2- biosensor for environmental applications

A sensitive NO2- biosensor that is based on bacterial reduction of NO2- to N2O and subsequent detection of the N2O by a built-in electrochemical N2O sensor was developed. Four different denitrifying organisms lacking NO3- reductase activity were assessed for use in the biosensor. The relevant physiological aspects examined included denitrifying characteristics, growth rate, NO2- tolerance, and temperature and salinity effects on the growth rate. Two organisms were successfully used in the biosensor. The preferred organism was Stenotrophomonas nitritireducens, which is an organism with a denitrifying pathway deficient in both NO3- and N2O reductases. Alternatively Alcaligenes faecalis could be used when acetylene was added to inhibit its N2O reductase. The macroscale biosensors constructed exhibited a linear NO2- response at concentrations up to 1 to 2 mM. The detection limit was around 1 microM NO2-, and the 90% response time was 0.5 to 3 min. The sensor signal was specific for NO2-, and interference was observed only with NH2OH, NO, N2O, and H2S. The sensor signal was affected by changes in temperature and salinity, and calibration had to be performed in a system with a temperature and an ionic strength comparable to those of the medium analyzed. A broad range of water bodies could be analyzed with the biosensor, including freshwater systems, marine systems, and oxic-anoxic wastewaters. The NO2- biosensor was successfully used for long-term online monitoring in wastewater. Microscale versions of the NO2- biosensor were constructed and used to measure NO2- profiles in marine sediment.     

Heme-hemopexin: a 'chronosteric' heme-protein

Hemopexin (HPX) serves as scavenger and transporter of toxic plasma heme to the liver. HPX is formed by two four-bladed beta-propeller domains, resembling two thick disks that lock together at a 90 degrees angle. The heme is bound between the two beta-propeller domains in a pocket formed by the interdomain linker peptide. Residues His213 and His266 coordinate the heme iron atom giving a stable bis-histidyl complex. The HPX-heme geometry is reminiscent of heme-proteins endowed with ligand binding and (pseudo-)enzymatic properties. HPX-heme binds reversibly CO, (*)NO, and cyanide by detaching His213; however, O(2) induces HPX-heme(II) oxidation. Furthermore, HPX-heme(II) facilitates (*)NO/O(2) and (*)NO/peroxynitrite scavenging. Heme sequestering by HPX prevents heme-mediated activation of oxidants which induce the low-density lipoprotein oxidation. Here, ligand binding and (pseudo-)enzymatic properties of HPX-heme are reviewed. HPX, acting not only as a heme carrier but also displaying transient heme-based ligand binding and (pseudo-)enzymatic properties, could be considered a 'chronosteric' heme-protein.