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PubMed1992

Mutants of Pseudomonas fluorescens deficient in dissimilatory nitrite reduction are also altered in nitric oxide reduction.

Five Tn5 mutants of Pseudomonas fluorescens AK-15 deficient in dissimilatory reduction of nitrite were isolated and characterized. Two insertions occurred inside the nitrite reductase structural gene (nirS) and resulted in no detectable nitrite reductase protein on a Western immunoblot. One mutant had Tn5 inserted inside nirC, the third gene in the same operon, and produced a defective nitrite reductase protein. Two other mutants had insertions outside of this nir operon and also produced defective proteins. All of the Nir- mutants characterized showed not only loss of nitrite reductase activity but also a significant decrease in nitric oxide reductase activity. When cells were incubated with 15NO in H2(18)O, about 25% of the oxygen found in nitrous oxide exchanged with H2O. The extent of exchange remained constant throughout the reaction, indicating the incorporation of 18O from H2(18)O reached equilibrium rapidly. In all nitrite reduction-deficient mutants, less than 4% of the 18O exchange was found, suggesting that the hydration and dehydration step was altered. These results indicate that the factors involved in dissimilatory reduction of nitrite influenced the subsequent NO reduction in this organism.

Bacterial Proteins↗

PubMed1997

Characterization and regulation of the gene encoding nitrite reductase in Rhodobacter sphaeroides 2.4.3.

Nitrite reductase catalyzes the reduction of nitrite to nitric oxide, the first step in denitrification to produce a gaseous product. We have cloned the gene nirK, which encodes the copper-type nitrite reductase from a denitrifying variant of Rhodobacter sphaeroides, strain 2.4.3. The deduced open reading frame has significant identity with other copper-type nitrite reductases. Analysis of the promoter region shows that transcription initiates 31 bases upstream of the translation start codon. The transcription initiation site is 43.5 bases downstream of a putative binding site for a transcriptional activator. Maximal expression of a nirK-lacZ construct in 2.4.3 requires both a low level of oxygen and the presence of a nitrogen oxide. nirK-lacZ expression was severely impaired in a nitrite reductase-deficient strain of 2.4.3. This suggests that nirK expression is dependent on nitrite reduction. The inability of microaerobically grown nitrite reductase-deficient cells to induce nirK-lacZ expression above basal levels in medium unamended with nitrate demonstrates that changes in oxygen concentrations are not sufficient to modulate nirK expression.

Aerobiosis↗

PubMed1998

Nitrogen and oxygen regulation of Bacillus subtilis nasDEF encoding NADH-dependent nitrite reductase by TnrA and ResDE.

The nitrate and nitrite reductases of Bacillus subtilis have two different physiological functions. Under conditions of nitrogen limitation, these enzymes catalyze the reduction of nitrate via nitrite to ammonia for the anabolic incorporation of nitrogen into biomolecules. They also function catabolically in anaerobic respiration, which involves the use of nitrate and nitrite as terminal electron acceptors. Two distinct nitrate reductases, encoded by narGHI and nasBC, function in anabolic and catabolic nitrogen metabolism, respectively. However, as reported herein, a single NADH-dependent, soluble nitrite reductase encoded by the nasDE genes is required for both catabolic and anabolic processes. The nasDE genes, together with nasBC (encoding assimilatory nitrate reductase) and nasF (required for nitrite reductase siroheme cofactor formation), constitute the nas operon. Data presented show that transcription of nasDEF is driven not only by the previously characterized nas operon promoter but also from an internal promoter residing between the nasC and nasD genes. Transcription from both promoters is activated by nitrogen limitation during aerobic growth by the nitrogen regulator, TnrA. However, under conditions of oxygen limitation, nasDEF expression and nitrite reductase activity were significantly induced. Anaerobic induction of nasDEF required the ResDE two-component regulatory system and the presence of nitrite, indicating partial coregulation of NasDEF with the respiratory nitrate reductase NarGHI during nitrate respiration.

Aerobiosis↗

PubMed2005

Hypoxia, red blood cells, and nitrite regulate NO-dependent hypoxic vasodilation.

Local vasodilation in response to hypoxia is a fundamental physiologic response ensuring oxygen delivery to tissues under metabolic stress. Recent studies identify a role for the red blood cell (RBC), with hemoglobin the hypoxic sensor. Herein, we investigate the mechanisms regulating this process and explore the relative roles of adenosine triphosphate, S-nitrosohemoglobin, and nitrite as effectors. We provide evidence that hypoxic RBCs mediate vasodilation by reducing nitrite to nitric oxide (NO) and ATP release. NO dependence for nitrite-mediated vasodilation was evidenced by NO gas formation, stimulation of cGMP production, and inhibition of mitochondrial respiration in a process sensitive to the NO scavenger C-PTIO. The nitrite reductase activity of hemoglobin is modulated by heme deoxygenation and heme redox potential, with maximal activity observed at 50% hemoglobin oxygenation (P(50)). Concomitantly, vasodilation is initiated at the P(50), suggesting that oxygen sensing by hemoglobin is mechanistically linked to nitrite reduction and stimulation of vasodilation. Mutation of the conserved beta93cys residue decreases the heme redox potential (ie, decreases E(1/2)), an effect that increases nitrite reductase activity and vasodilation at any given hemoglobin saturation. These data support a function for RBC hemoglobin as an allosterically and redox-regulated nitrite reductase whose "enzyme activity" couples hypoxia to increased NO-dependent blood flow.

Adenosine Triphosphate↗

PubMed2003

Need for cytochrome bc1 complex for dissimilatory nitrite reduction of Pseudomonas aeruginosa.

Pseudomonas aeruginosa strains deficient in the genes for cytochrome c1, a subunit of the cytochrome bc1 complex, or the tetraheme membrane protein NapC, which is similar to NirT of Pseudomonas stutzeri, were constructed and their growth was investigated. The cytochrome c1 mutant could not grow under anaerobic conditions with nitrite as an electron acceptor and did not reduce nitrite in spite of its producing active nitrite reductase. NirM (cytochrome c551) and azurin, which are the direct electron donors for nitrite reductase, were reduced by succinate in the presence of the membrane fraction from the wild-type strain as a mediator but not in the presence of that from the cytochrome c1 mutant. These results indicated that cytochrome bc1 complex was necessary for electron transfer from the membrane quinone pool to nitrite reductase. The NapC mutant grew anaerobically at the expense of nitrite, indicating that NapC was not necessary for nitrite reduction.

Aerobiosis↗

PubMed1984

Oxidation of deoxyhemerythrin to semi-methemerythrin by nitrite.

In anaerobic phosphate buffer, pH 6.3-7.5, deoxyhemerythrin is oxidized to semi-methemerythrin (semi-met) by excess sodium nitrite. This oxidation is quantitative as judged by EPR spectroscopy. Further oxidation to methemerythrin is not detected. The absorbance changes of hemerythrin during the oxidation are biphasic. The rate of the faster first phase is linearly dependent on [H+] and [NO2-] suggesting that the oxidant is nitrous acid rather than nitrite. During the slower second phase, the characteristic EPR spectrum of semi-methemerythrin appears. The first phase can be interpreted by a scheme in which nitrous acid transforms deoxyhemerythrin (FeIIFeII) to the semi-met nitrosyl adduct (FeIIFeIIINO) and hydroxide. Independent experiments confirm that the combination of semi-met plus NO produces an EPR-silent adduct. The rates of the absorbance changes for the second phase are nearly independent of nitrite concentration and pH in the range 6.3-7.5. This slower phase involves the transformation of the EPR-silent intermediate to the semi-met nitrite adduct (FeIIFeIIINO2-) and is consistent with rate-limiting dissociation of nitric oxide followed by rapid attachment of nitrite. Nitrite appears to be a unique oxidant of deoxyhemerythrin in that when employed in excess, the final, stable product is semi-met- rather than methemerythrin. The lack of reactivity of ethyl nitrite with deoxyhemerythrin suggests that HONO oxidizes deoxyhemerythrin via an "inner-sphere" process in contrast to oxidants such as Fe(CN)6(3-). A proposed generalization is that excesses of "inner-sphere" oxidants convert deoxy to (semi-met)R, which is stabilized with respect to (semi-met)R, which is stabilized with respect to (semi-met)0 and met because the oxidant and/or a product of the oxidant can bind to the iron site.

Animals↗

PubMed2001

Nitrite and nitrate measurement in human urine by capillary electrophoresis.

Nitrite and nitrate have been widely used as markers for nitric oxide (NO) formation in vivo and represent the major NO oxidation products in biological fluids. In the present study, the use of capillary electrophoresis (CE) in the measurement of nitrite and nitrate in human urine is described. Urine samples were electrophoresed in an extended light path fused-silica capillary (104 cm; 75 microm ID) at an applied negative potential of 30 kV, and UV detection at 214 nm. Using electrokinetic sample injection (-6 kV x 20 s), we found that urine concentration, pH, sodium and chloride interfered with nitrite and nitrate detection. The detection of nitrite and nitrate was decreased when hydrodynamic sample injection was used (30 mbar x 60 s). However, basal levels of urinary nitrite (0.25 +/- 0.05 microM) and nitrate (591 +/- 115 microM) were detected and no interference by variations in urine concentration and pH was noted when hydrodynamic sample injection was used. Thus, hydrodynamic sample injection is convenient for the measurement of urinary nitrite and nitrate and avoids the effect of variations in urine matrices and pH on nitrite and nitrate detection.

Electrophoresis, Capillary↗

PubMed1994

Lactate-dependent killing of Escherichia coli by nitrite plus hydrogen peroxide: a possible role of nitrogen dioxide.

The killing of Escherichia coli by nitrite plus hydrogen peroxide was observed in lactate, but not in phosphate or acetate. Although nitrite or hydrogen peroxide alone caused a slight decrease in bacterial survival, nitrite plus hydrogen peroxide killed bacteria synergistically in time-, dose-, and pH-dependent manners. The killing was increased with decreasing pH. The plot of viable cells versus [nitrous acid] was linear. Among the hydroxyl radical scavengers used, only benzoate and formate at concentrations higher than that of lactate inhibited the killing by nitrite plus hydrogen peroxide, whereas dimethyl sulfoxide enhanced it. The generation of peroxynitrous acid during the reaction of nitrite and hydrogen peroxide was confirmed by the formation of malondialdehyde using deoxyribose as a hydroxyl radical-like oxidant detector. The nitration of glycyl-tryosine was observed only in lactate buffer, but not in phosphate and acetate buffers. Benzoate and formate inhibited the nitration, whereas dimethyl sulfoxide and ethanol enhanced it. No evidence for the formation of nitric oxide and superoxide during the reaction of nitrite and hydrogen peroxide was found. These data suggest that nitrogen dioxide from the decomposition of peroxynitrous acid or secondary oxidants formed from the reaction of peroxynitrous acid with lactate is responsible for the lactate-dependent killing of E. coli induced by the reaction of protonated nitrite and hydrogen peroxide.

Animals↗

PubMed1998

Myeloperoxidase and horseradish peroxidase catalyze tyrosine nitration in proteins from nitrite and hydrogen peroxide.

Nitration of tyrosine residues in proteins occurs in a wide range of inflammatory diseases involving neutrophil and macrophage activation. We report that both myeloperoxidase (MPO) and horseradish peroxidase (HRP) utilize nitrite (NO2-) and hydrogen peroxide (H2O2) as substrates to catalyze tyrosine nitration in proteins. MPO was approximately 10 times more effective than HRP as a nitration catalyst of bovine serum albumin (BSA). Nitration of BSA by MPO did not require chloride as a cofactor. Physiologic levels of chloride did not significantly inhibit nitration by MPO. Oxidation of chloride to hypochlorous acid (HOCl) is catalyzed by MPO but not by HRP, yet HRP also catalyzed nitration from hydrogen peroxide plus nitrite. Therefore, HOCl formation was not obligatory for tyrosine nitration. Although HOCl plus nitrite can nitrate the amino acid tyrosine in simple solutions, protein nitration by HOCl plus nitrite was not observed in heart homogenates, probably due to the presence of multiple alternative targets of both HOCl and HOCl plus nitrite. In contrast, MPO catalyzed nitration of many proteins in rat heart homogenates using NO2- plus H2O2, suggesting that peroxidase-catalyzed nitration of tyrosine could occur in the presence of competing substrates in vivo. HOCl could substitute for H2O2 as the oxidizing substrate for nitration of either BSA or tissue homogenates catalyzed by either peroxidase. Activated neutrophils may generate nitrotyrosine by several mechanisms, including peroxynitrite, HOCl plus nitrite, and a chloride-independent mechanism involving MPO, nitrite, and hydrogen peroxide.

Animals↗

PubMed1998

Evaluation of the magnitude and rate of nitric oxide production from nitrite in biological systems.

Recently it was shown that the formation of nitric oxide (NO) is increased in biological tissues during ischemia, and that there is an enzyme-independent pathway of NO generation due to reduction of tissue nitrite under the acidic conditions which occur. To investigate the quantitative importance of this mechanism of NO generation in biological systems where pH and nitrite concentrations vary, electron paramagnetic resonance and chemiluminescence studies were performed to characterize the kinetics and magnitude of the nitrite disproportionation process. The reaction process and the corresponding rate law of NO formation from nitrite were derived. The generation and accumulation of NO from typical nitrite concentrations found in biological tissues increased 100-fold when pH decreased from normal values of 7.4 to the acidic values found in ischemic tissues, such as the heart, where pH falls to 5.5. It was also observed that ischemic heart tissue contains reducing equivalents which reduce nitrite to NO, further increasing the rate of NO formation more than 40-fold. Under these conditions the magnitude of this enzyme-independent NO generation may exceed that which can be generated by tissue concentrations of nitric oxide synthase. Thus, in ischemic tissues nitrite can be primarily a source rather than a product of NO.

Animals↗

PubMed1995

Simultaneous determination of nitrate and nitrite in biological samples by multichannel flow injection analysis.

An automated method for the simultaneous determination of nitrite and nitrate in biological samples by using a multichannel flow injection analyzer has been developed. The method was based on the reaction of nitrite with Greiss reagent. The sample solution was injected and equally divided into two channels; channel one (1) represented total nitrite obtained by cadmium reduction of nitrate to nitrite while channel two (2) represented only nitrite. The absorbance of the color product was measured by photometric detectors with 540-nm filters. This method combines high reproducibility of sample introduction via flow injection and sensitivity of spectrophotometric detection. The detection limit is 25 nM for both nitrite and nitrate. The chemistry manifolds are constructed of Teflon tubing which, along with a low-pressure Flowfit connector system, provides for low maintenance, ease of use, and high sample throughput. We demonstrated that the system can be used for the determination of both nitrate and nitrite in a variety of biological samples as well as a comparison of the results from this system and the HPLC system.

Animals↗

PubMed2002

Crystal structure of the soluble domain of the major anaerobically induced outer membrane protein (AniA) from pathogenic Neisseria: a new class of copper-containing nitrite reductases.

The major anaerobically induced outer membrane protein (AniA) from pathogenic Neisseria gonorrhoeae is essential for cell growth under oxygen limiting conditions in the presence of nitrite and is protective against killing by human sera. A phylogenic analysis indicates that AniA is a member of a new class of copper-containing nitrite reductases. Expression of the soluble domain of AniA yields a protein capable of reducing nitrite with specific activity of 160 units/mg, approximately 50 % of that measured for the nitrite reductase from the strong soil denitrifier Alcaligenes faecalis S-6. The crystal structure of the soluble domain of AniA was solved by molecular replacement and sixfold averaging to a resolution of 2.4 A. The nitrite soaked AniA crystal structure refined to 1.95 A reveals a bidentate mode of substrate binding to the type II copper. Despite low sequence identity (approximately 30 %), the core cupredoxin fold of AniA is similar to that found in copper-containing nitrite reductases from soil bacteria. The main structural differences are localized to two attenuated surface loops that map to deletions in the sequence alignment. In soil nitrite reductases, one of these surface loops is positioned near the type I copper site and contributes residues to the docking surface for proteaceous electron donors. In AniA, the attenuation of this loop results in a restructured hydrophobic binding surface that may be required to interact with a lipid anchored azurin. The second attenuated loop is positioned on the opposite side of AniA and may facilitate a more intimate interaction with the lipid membrane. A unique combination of structural effectors surrounding the type I copper site of sAnia contribute to a unusual visible absorption spectra with components observed previously in either green or blue type I copper sites.

Amino Acid Sequence↗

PubMed2002

Electron donation between copper containing nitrite reductases and cupredoxins: the nature of protein-protein interaction in complex formation.

In denitrifying organisms with copper containing dissimilatory nitrite reductases, electron donation from a reduced cupredoxin is an essential step in the reduction of nitrite to nitric oxide. Copper nitrite reductases are categorised into two subgroups based on their colour, green and blue, which are found in organisms where the cupredoxins are pseudoazurins and azurins, respectively. In view of this and some in vitro electron donation experiments, it has been suggested that copper nitrite reductases have specific electron donors and that electron transfer takes place in a specific complex of the two proteins. We report results from the first comprehensive electron donation experiments using three copper nitrite reductases, one green and two blue, and five cupredoxins, one pseudoazurin and four azurins. Our data show that pseudoazurin can readily donate electrons to both blue and green copper nitrite reductases. In contrast, all of the azurins react very sluggishly as electron donors to the green nitrite reductase. These results are discussed in terms of surface compatibility of the component proteins, complex formation, overall charges, charge distribution, hydrophobic patches and redox potentials. A docking model for the complexes is proposed.

Azurin↗

PubMed1984

Depression of murine natural killer cell cytotoxicity by isobutyl nitrite.

We have investigated the effect of isobutyl nitrite on murine NK-cell antitumor-directed cytotoxicity. This agent has been suggested as one of the factors underlying immunodeficiency syndrome (AIDS) in man. We demonstrated that two injections, each of 0.25 ml isobutyl nitrite, resulted in significant depression of endogenous splenic and peripheral blood natural killer (NK) cell cytotoxicity against T-cell lymphoma, YAC-1. In addition to endogenous NK cells, activity of pyrimidinol-activated NK cells was also substantially depressed by this agent. The latter observation is of the utmost importance, since it suggests that the attempt to augment NK-cell activity (to promote resistance to infections and malignancies) could fail in patients with AIDS who are isobutyl nitrite users. Isobutyl nitrite was NK-cell-suppressive not only after in vivo administration but, most importantly, also after inhalation. This indicates that isobutyl nitrite, via its NK-cell suppressive effect, could contribute to immunodeficiency in AIDS. Studies on the mechanism of NK-cell depression by isobutyl nitrite demonstrated that the NK-cell tumor-binding properties as well as NK-cell cytotoxic potential were substantially depressed. Mixing experiments failed to reveal any regulation by suppressor cell activities. The results of these studies clearly indicate that isobutyl nitrite is an immunosuppressive agent and that its use should be avoided.

Acquired Immunodeficiency Syndrome↗

PubMed1976

A reduced pyridine nucleotides-diaphorase activity associated to the assimilatory nitrite reductase complex from Neurospora crassa.

The Neurospora crassa assimilatory NAD(P)H-nitrite reductase complex has associated a NAD(P)H-diaphorase activity. 1. This NAD(P)H-diaphorase activity can use either mammalian cytochrome c, 2,6--dichlorophenol-indophenol, ferricyanide, or menadione as electron acceptor from the reduced pyridine nucleotides, and requires flavin adenine dinucleotide for maximal activity. 2. It is inhibited by p-hydroxymercuribenzoate, 1 muM, and it is unaffected by cyanide, sulfite, or arsenite at concentrations which completely inhibit the NAD(P)H-nitrite reductase activity. 3. Flavin adenine dinucleotide specifically protects the NAD(P)H-diaphorase activities, but not the NAD(P)H-nitrite reductase activities, against thermal inactivation. 4. In vitro preincubation of the Neurospora crassa nitrite reductase complex with reduced pyridine nucleotides plus flavin adenine dinucleotide inactivates the NAD(P)H-nitrite reductase activities, but does not affect the NAD(P)H-diaphorase activities, indicating that this nitrite reductase inactivation occurs in the part of the enzyme that contain the nitrite reducing center.

Cytochrome Reductases↗

PubMed1985

Effect of sulfite or nitrite on the ATP content and the carbohydrate metabolism in yeast.

Low concentrations of sulfite or nitrite (about 0.5 mmol) when applied at pH 3.6, caused a rapid and drastic decrease of the concentration of ATP in yeast cells. Under these conditions, alcoholic fermentation was inhibited by sulfite and to a lesser extent by nitrite. Ethanol consumption under aerobic conditions was shown to be more sensitive to nitrite than to sulfite. This indicates a higher sensitivity of respiratory processes to nitrite than to sulfite. Among 15 enzyme activities assayed in extracts from yeast cells after incubation with sulfite or nitrite, glyceraldehyde-3-phosphate dehydrogenase was shown to be the most sensitive. Analysis of the steady-state concentrations of intermediates of alcoholic fermentation in intact yeast cells also implies inhibition by sulfite or nitrite of the glyceraldehyde-3-phosphate dehydrogenase step of fermentation. In contrast to nitrite, sulfite had an additional effect by accumulating the intracellular steady state concentration of glyceraldehyde-3-phosphate 10 to 100-fold over the concentration in the absence of sulfite. In vitro studies on the equilibrium catalyzed by triosephosphate isomerase or aldolase confirmed the postulated shift of equilibrium concentrations by a formation of complex of glyceraldehyde-3-phosphate with sulfite.

Adenosine Triphosphate↗

PubMed1993

Effect of nitrite on ureagenesis and carbohydrate metabolism in isolated rat hepatocytes.

The effect of three NaNO2 concentrations (0.5, 2.0, and 5.0 mM) on 15N-ammonia utilization, ureagenesis, glucose, pyruvate and lactate formation and glycogen breakdown were studied in isolated rat hepatocytes. Nitrite failed to affect the rate of glycogenolysis as well as the lactate and pyruvate formation, but at the same time it markedly increased the glucose formation. It is concluded that the increase in the glucose formation results from the nitrite stimulation of the rate of gluconeogenesis. An increased sodium nitrite concentration caused a significant decrease in the ammonia utilization and urea synthesis; there are strong linear correlations between the nitrite concentration and the amount of utilized ammonia (r = -0.93) and the formed urea (r = -0.96). The observed lower rate of ureagenesis in the presence of nitrite resulted from the diminished incorporation of the added 15N-ammonia into urea, as well as from the diminished urea formation from endogenous nitrogen. It is concluded that the disturbances in carbohydrate and nitrogen metabolism observed in the nitrite-poisoned animals are attributed to the direct effect of nitrite on metabolism.

Animals↗

PubMed1996

Solvent-free sample preparation by headspace solid-phase microextraction applied to the tracing of n-butyl nitrite abuse.

The most common alkyl nitrites encountered in forensic toxicology are iso-butyl, n-butyl and iso-pentyl(amyl) nitrites. All have become popular as an aphrodisiac, especially among the homosexual population. Alkyl nitrites are a volatile and unstable group of compounds, which hydrolyse in aqueous matrices to the alcohol and nitrite ion. Here we describe a fast, clean and sensitive procedure for the detection of hydrolysed n-butyl nitrite in whole human blood using a new, solvent-free sampling technique, the headspace solid-phase micro-extraction (HSPME), combined with GC/FID analysis. Sample preparation was investigated using two different stationary phases (100 microns polydimethylsiloxane and 85 microns polyacrylate), coating a fused silica fibre. The effect of different sampling times at fixed temperatures was also studied. Our results demonstrate that the HSPME/GC/FID procedure allows tracing of n-butyl nitrite abuse and detects hydrolysed n-butyl nitrite, i.e., released n-butanol, in whole blood at the 1 ng/mL level.

Autopsy↗