Product Of Nitrite Reduction Research Articles

Kinetics of Nitrite Reduction by Zinc Metal: Influence of Metal Shape on the Determination of Kinetic Parameters

Heterogeneous chemical reactions are complicated, especially in the case of competitive reactions. The aim of this study was to investigate the elimination of nitrite (NO2(-)) by applying a metallic reduction using zero-valent zinc (Zn0). The effect of pH, stirring, and metal shape (powder and chips) on the rate and products of nitrite reduction were studied in a batch-stirred reactor. The obtained data have been used to optimize the conditions for metallic reduction of NO2(-) and for kinetic parameters identification. It was found that the dissolution of zinc involves a pseudo-first-order reaction independent of the shape of the metal. Further, the influence of operating conditions on nitrogen (N2) and ammonium (NH4(+)) formation has been determined. It was found that a decrease in pH and in the Zn0 content enhances NH4(+) production. If kinetic parameters can be approximated easily for constant surface area, it was demonstrated that surface evolution had to be integrated for metal powder. Finally, a numerical simulation has been used to determine the kinetic parameters for NO2(-) reduction with zinc powder.

The kinetics of nitrite reduction by thiourea dioxide in the presence of cobalt octasulfophenyltetrapyrazinoporphyrazine

Cobalt octasulfophenyltetrapyrazinoporphyrazine was shown to be an effective catalyst of nitrite reduction by thiourea dioxide. The intermediate product of nitrite reduction was nitrogen(II) oxide, and the final product was ammonia. The kinetic and activation parameters of catalytic process stages were determined.

Electroreduction of nitrite catalyzed by a dinuclear ruthenium phthalocyanine modified graphite electrode

The electrocatalytic reduction of nitrite on a dinuclear ruthenium phthalocyanine ( RuPc )2modified electrode is studied, using cyclic voltammetry (cyclic voltammogram), rotating disc electrode (RDE) and ring-disc electrode (RRDE) techniques. The products of nitrite reduction are ammonia, nitrous oxide, and hydroxylamine. Depending on the experimental conditions, ammonia or nitrous oxide is formed as the main product. The initial step in the electroreduction of nitrite catalyzed by ( RuPc )2is a one electron RuII/ RuIprocess, followed by coordination of NO2−to the metal center. Formation of a nitrosyl complex prior to the electroreduction does not occur. A suitable mechanism is proposed by analyzing the rate equation and the Tafel slope.

The nrfI gene is essential for the attachment of the active site haem group of Wolinella succinogenes cytochrome c nitrite reductase.

The cytochrome c nitrite reductase complex (NrfHA) is the terminal enzyme in the electron transport chain catalysing nitrite respiration of Wolinella succinogenes. The catalytic subunit NrfA is a pentahaem cytochrome c containing an active site haem group which is covalently bound via the cysteine residues of a unique CWTCK motif. The lysine residue serves as the axial ligand of the haem iron. The other four haem groups of NrfA are bound by conventional haem-binding motifs (CXXCH). The nrfHAIJ locus was restored on the genome of the W. succinogenes DeltanrfAIJ deletion mutant by integration of a plasmid, thus enabling the expression of modified alleles of nrfA and nrfI. A mutant (K134H) was constructed which contained a nrfA gene encoding a CWTCH motif instead of CWTCK. NrfA of strain K134H was found to be synthesized with five bound haem groups, as judged by matrix-assisted laser-desorption/ionization (MALDI) mass spectrometry. Its nitrite reduction activity with reduced benzyl viologen was 40% of the wild-type activity. Ammonia was formed as the only product of nitrite reduction. The mutant did not grow by nitrite respiration and its electron transport activity from formate to nitrite was 5% of that of the wild-type strain. The predicted nrfI gene product is similar to proteins involved in system II cytochrome c biogenesis. A mutant of W. succinogenes (stopI) was constructed that contained a nrfHAIJ gene cluster with the nrfI codons 47 and 48 altered to stop codons. The NrfA protein of this mutant did not catalyse nitrite reduction and lacked the active site haem group, whereas the other four haem groups were present. Mutant (K134H/stopI) which contained the K134H modification in NrfA in addition to the inactivated nrfI gene had essentially the same properties as strain K134H. NrfA from strain K134H/stopI contained five haem groups. It is concluded that NrfI is involved in haem attachment to the CWTCK motif in NrfA but not to any of the CXXCH motifs. The nrfI gene is obviously dispensable for maturation of a modified NrfA protein containing a CWTCH motif instead of CWTCK. Therefore, NrfI might function as a specific haem lyase that recognizes the active site lysine residue of NrfA. A similar role has been proposed for NrfE, F and G of Escherichia coli, although these proteins share no overall sequence similarity to NrfI and belong to system I cytochrome c biogenesis, which differs fundamentally from system II.

Effect of ph on the reduction of nitrite in water by metallic iron

Chemical reduction is an alternative technique to remove nitrogen oxides from contaminated groundwater and closed-surface water body. Metallic iron was employed as a reductant for the reduction of nitrite in water in this study. The effect of pH on the rate and products of nitrite reduction was investigated with a fixed dosage of metallic iron powder (12 mol-Fe/mol-N, size of the powder: 80 mesh). The reduction of nitrite by metallic iron was a pseudo-zero-order reaction under the experimental conditions. The reduction rate of nitrite was increased with decreasing the pH of reaction solution, and the pseudo-zero-order reaction rate constants were 180, 130, 60, 15, 10, and 1 mM/h at pH=2, 3, 4, 5, 6, and 7, respectively. The reduction products of nitrite were nitrogen gas and ammonium. The yields of nitrogen gas from nitrite reduction were 0.63, 0.74, 0.81, 0.87, 0.92, and 0.98 as molar ratios of nitrogen atom at pH=2, 3, 4, 5, 6, and 7, respectively. Neutral condition enhanced the formation of nitrogen gas from nitrite reduction.

Gonococcal nitric oxide reductase is encoded by a single gene, norB, which is required for anaerobic growth and is induced by nitric oxide.

The gene encoding a nitric oxide reductase has been identified in Neisseria gonorrhoeae. The norB gene product shares significant identity with the nitric oxide reductases in Ralstonia eutropha and Synechocystis sp. and, like those organisms, the gonococcus lacks a norC homolog. The gonococcal norB gene was found to be required for anaerobic growth, but the absence of norB did not dramatically decrease anaerobic survival. In a wild-type background, induction of norB expression was seen anaerobically in the presence of nitrite but not anaerobically without nitrite or aerobically. norB expression is not regulated by FNR or NarP, but a functional aniA gene (which encodes an anaerobically induced outer membrane nitrite reductase) is necessary for expression. When aniA is constitutively expressed, norB expression can be induced both anaerobically and aerobically, but only in the presence of nitrite, suggesting that nitric oxide, which is likely to be produced by AniA as a product of nitrite reduction, is the inducing agent. This was confirmed with the use of the nitric oxide donor, spermine-nitric oxide complex, in an aniA null background both anaerobically and aerobically. NorB is important for gonococcal adaptation to an anaerobic environment, a physiologically relevant state during gonococcal infection. The presence of this enzyme, which is induced by nitric oxide, may also have implications in immune evasion and immunomodulation in the human host.

Identification of transcription activators that regulate gonococcal adaptation from aerobic to anaerobic or oxygen-limited growth.

Analysis of the Neisseria gonorrhoeae DNA sequence database revealed the presence of two genes, one encoding a protein predicted to be 37. 5% identical (50% similar) in amino acid sequence to the Escherichia coli FNR protein and the other encoding a protein 41% and 42% identical (54 and 51% sequence similarity) to the E. coli NarL and NarP proteins respectively. Both genes have been cloned into E. coli and insertionally inactivated in vitro. The mutated genes have been transformed into gonococci and recombined into the chromosome. The fnr mutation totally abolished and the narP mutation severely diminished the ability of gonococci to: (i) grow anaerobically; (ii) adapt to oxygen-limited growth; (iii) initiate transcription from the aniA promoter (which directs the expression of a copper-containing nitrite reductase, AniA, in response to the presence of nitrite); and (iv) reduce nitrite during growth in oxygen-limited media. The product of nitrite reduction was identified to be nitrous oxide. Immediately upstream of the narL/narP gene is an open reading frame that, if translated, would encode a homologue of the E. coli nitrate- and nitrite-sensing proteins NarX and NarQ. As transcription from the aniA promoter was not activated during oxygen-limited growth in the presence of nitrate, the gonococcal two-component regulatory system is designated NarQ-NarP rather than NarX-NarL. As far as we are aware, this is the first well-documented example of a two-component regulatory system working in partnership with a transcription activator in pathogenic neisseria. A 45 kDa c-type cytochrome that was synthesized during oxygen-limited, but not during oxygen sufficient, growth was identified as a homologue of cytochrome c peroxidases (CCP) of other bacteria. The gene for this cytochrome, designated ccp, was located, and its regulatory region was cloned into the promoter probe vector pLES94. Transcription from the ccp promoter was repressed during aerobic growth and induced during oxygen-limited growth and was totally FNR dependent, suggesting that the gonococcal FNR protein is a transcription activator of at least two genes. However, unlike AniA, synthesis of the CCP homologue was insensitive to the presence of nitrite during oxygen-limited growth.

Regulation of Nitrite Reductase Activity under CO2 Limitation in the Cyanobacterium Synechococcus sp. PCC7942

During photoautotrophic growth under CO2-limited conditions, cells of Synechococcus sp. PCC7942 excreted into the medium about 30% of the nitrite produced by reduction of nitrate. No nitrite was excreted under CO2-sufficient conditions. After transfer of high-CO2-grown cells to CO2-limited conditions, nitrite reductase activity started to decline within 0.5 h and decreased to 50% of the initial level in 3 h, whereas nitrate reductase activity was virtually unchanged. Nitrite started to accumulate in the medium about 3 h after the transfer of the cells to CO2-limited conditions and reached a concentration of >0.4 mM at 17 h. These findings suggested that the nitrite excretion was due to an imbalance of the activities of nitrite reductase and nitrate reductase. Since ammonium, the product of nitrite reduction, was not detected in the medium, it was concluded that the step of nitrite reduction limits the rate of nitrate assimilation under CO2-limited conditions. The extent of decrease in nitrite reductase activity under CO2-limited conditions was much larger than that caused by rifampicin (an inhibitor of RNA synthesis) treatment under high-CO2 conditions. Addition of CO2, in the form of sodium bicarbonate, to the CO2-limited culture increased the nitrite reductase activity, but rifampicin inhibited this increase. These findings suggested the presence of a mechanism that irreversibly inactivates nitrite reductase under CO2-limited conditions.

Type 1, blue copper proteins constitute a respiratory nitrite-reducing system in Pseudomonas aureofaciens.

Pseudomonas aureofaciens truncates the respiratory reduction of nitrate (denitrification) at the level of N2O. The nitrite reductase from this organism was purified to apparent electrophoretic homogeneity and found to be a blue copper protein. The enzyme contained 2 atoms of copper/85 kDa, both detectable by electron paramagnetic resonance (EPR) spectroscopy. The protein was dimeric, with subunits of identical size (40 +/- 3 kDa). Its pI was 6.05. The EPR spectrum showed an axial signal g at 2.21(8) and g at 2.04(5). The magnitude of the hyperfine splitting (A parallel = 6.36 mT) indicated the presence of type 1 copper only. The electronic spectrum had maxima at 280 nm, 474 nm and 595 nm (epsilon = 7.0 mM-1 cm-1), and a broad shoulder around 780 nm. A copper protein of low molecular mass (15 kDa), with properties similar to azurin, was also isolated from P. aureofaciens. The electronic spectrum of this protein showed a maximum at 624 nm in the visible range (epsilon = 2.5 mM-1 cm-1) and pronounced structures in the ultraviolet region. The EPR parameters were g parallel = 2.26(6) and g perpendicular = 2.05(6), with A parallel = 5.8 mT. The reduced azurin transferred electrons efficiently to nitrite reductase; the product of nitrite reduction was nitric oxide. The specific nitrite-reducing activity with ascorbate-reduced phenazine methosulfate as electron donor was 1 mumol substrate min-1 mg protein-1. The reaction product again was nitric oxide. Nitrous oxide was the reaction product from hydroxylamine and nitrite and from dithionite-reduced methyl viologen and nitrite. No 'oxidase' activity could be demonstrated for the enzyme. Our data disprove the presumed exclusiveness of cytochrome cd1 as nitrite reductase within the genus Pseudomonas.

The purification and properties of a cd-cytochrome nitrite reductase from Paracoccus halodenitrificans.

Paracoccus halodenitrificans, grown anaerobically in the presence of nitrite, contained membrane and cytoplasmic nitrite reductases. When assayed in the presence of phenazine methosulfate and ascorbate, the membrane-bound enzyme produced nitrous oxide whereas the cytoplasmic enzyme produced nitric oxide. When both enzymes were assayed in the presence of methyl viologen and dithionite, the cytoplasmic enzyme produced ammonia. Following solubilization, the membrane-bound enzyme behaved like the cytoplasmic enzyme, producing nitric oxide in the presence of phenazine methosulfate and ascorbate, and ammonia when assayed in the presence of methyl viologen and dithionite. The cytoplasmic and membrane-bound enzymes were purified to essentially the same specific activity. Only a single nitrite-reductase activity was detected on electrophoretic gels and the electrophoretic behavior of both enzymes suggested they were identical. The spectral properties of both enzymes suggested they were cd-type cytochromes. These data suggest that the products of nitrite reduction by the cd-cytochrome nitrite reductase are determined by the location of the enzyme and the redox potential of the electron donor.

Patterns of product inhibition for bacterial nitrite reductase

Product inhibition has been examined in the turnover kinetics of cytochrome cd1 from Pseudomonas aeruginosa (ATCC 19429) and from Paracoccus denitrificans1 (ATCC 13456). A common characteristic was a decrease in rate during the time course of assays that was not due to substrate depletion or irreversible inactivation. The product of nitrite reduction, nitric oxide (NO), acted as a product inhibitor in anaerobic assays with an apparent Ki of 0.2 microM, but only if the enzyme was first preincubated with NO for 15 min. The enzyme was inhibited by the oxidized form of electron donors and this could account for the decrease in rate during an assay. For the donors hydroquinone, ascorbate, TMPD, and azurin, measured values of the inhibition constant were at least ten fold lower than measured Km's. Cytochromes c as donors demonstrated a complex pattern of product inhibition by the ferric form. Although numerical values of Ki in these cases were not obtained, trends indicated that apparent values would be less than Km.

Purification and Characterization of a Dissimilatory Nitrite Reductase from the Phototrophic Bacterium Rhodopseudomonas palustris

A dissimilatory nitrite reductase from the facultatively phototrophic bacterium, Rhodopseudomonas palustris strain 1a1 was studied. A basic level of the enzyme (10-50 mU/mg protein) was measured in dark, aerated and anaerobic, photosynthetic cultures. A marked derepression of enzyme synthesis occurred under conditions of oxygen limitation (200-300 mU/mg protein). The addition of nitrite (or nitrate) to the culture medium had only a slight effect on the maximal nitrite reductase titer of cells. The enzyme was purified from photosynthetically grown cells by precipitation with ammonium sulfate, gel filtration through Sepharose 6B and repeated chromatography on DE 52-cellulose. As estimated by gel filtration, the nitrite reductase had a molecular weight of about 120 000 +/- 12 000 and yielded only one band (mol. wt. of about 68 000 +/- 7000) in SDS-gel electrophoresis. The isoelectric point of the enzyme was at pH 5.1. Nitric oxide (NO) was identified as the reaction product of nitrite reduction. The enzyme also exhibited cytochrome c-oxidase activity and was active with chemically reduced viologen dyes, FMN and cytochrome c as electron donors. Highly purified nitrite reductase preparations contained 10 mol% of a c-type cytochrome. Trace metal analyses indicated the presence of Cu in the enzyme. Consistent with the detection of Cu was the finding that the Cu-chelator, diethyldithiocarbamate, strongly inhibited the nitrite reductase.

Nitrite reduction in Veillonella alcalescens

Nitrite reduction was examined in Veillonella alcalescens C-1, and obligate anaerobe with an ATP-yielding nitrate-reducing system. Hydrogen donors for nitrite reduction included hydrosulfite, hydrogen gas, and pyruvate, but not pyridine nucleotides, in the presnece or absence of flavins. Pyruvate-linked nitrite reduction was not inhibited by 4,4,4-trifluoro-1-(2-thienyl) 1,3-butanedione, dicoumarol, or 2-heptyl-4-hydroxy-quinoline-N-oxide. The noninvolvement of membrane-bound factors was supported by the fact that 100% of pyruvate-linked activity remained in the soluble fraction after fractionation of crude extracts by ultracentrifugation. Using DEAE-cellulose column chromatography, however, the participation of ferredoxin in nitrite reduction was demonstrated. The product of nitrite reduction appeared to be ammonia, as determined from H2-to-NO2- ratios. Nitrite reductase was induced by nitrate or nitrite and was repressed by increased levels of reduced nitrogenous compounds.

Sulphide-linked Nitrite Reductase from Thiobacillus denitrificans with Cytochrome Oxidase Activity: Purification and Properties

A membrane-bound, sulphide-linked nitrite reductase from Thiobacillus denitrificans was solubilized and after further purification its properties were examined. The purified enzyme, mol. wt 120 000, contained cytochromes c and d in the ratio of 1:1. Both cytochromes were reduced by sulphide and re-oxidized with nitrite or air. Oxidation by nitrite resulted in the appearance of an absorption peak at 572 nm. The kinetics of the reduction of the enzyme with sulphide indicated that cytochrome c was reduced before cytochrome d. The redox potential of cytochrome d was 22 mV more positive than that of cytochrome c. Cytochromes c and d were dissociated from the purified enzyme by treatment with sodium dodecyl sulphate. The purified nitrite reductase also had cytochrome oxidase activity and both the activities were stimulated by cytochrome c-551 isolated from T. denitrificans. Reduced cytochrome c-551 was an effective electron donor for the purified enzyme with either nitrite or air as the terminal electron acceptor. Neither cytochrome c-554 (also isolated from T. denitrificans) nor mammalian cytochrome c was effective as reductant for the enzyme. NO and N2O were identified as the products of nitrite reduction by the purified sulphide-linked nitrite reductase.

A nitrite-reducing enzyme from Nitrosomonas europaea Preliminary characterization with hydroxylamine as electron donor

1. A soluble enzyme which catalyzes the reduction of nitrite in the presence of hydroxylamine has been extracted from Nitrosomonas cells and partially purified. 2. The hydroxylamine-nitrite reductase was also able to utilize leuco-pyocyanine as an electron donor. 3. The enzyme had a pH optimum for HNO 2 reduction of 5.75 and approximate K m values for HNO 2 and NH 2OH of 1.6 mM and 0.24 M, respectively. 4. The net evolution of approx. 1 mole of gas and disappearance of 1.8 moles of hydroxylamine occurred per mole of nitrite utilized during the reaction. The compounds N 2O and NO were identified as the products of nitrite reduction. 5. Nitrite reduction by hydroxylamine was inhibited 50% by 2 μM diethyl-dithiocarbamate, 8 μM α,α′-dipyridyl, 2×10 −4 M KCN or 1.5 mM NaN 3. 6. The partially purified enzyme fraction contained mammalian cytochrome c oxidase, hydroxylamine oxidase, hydroquinone oxidase and NH 2OH-cytochrome c reductase activity and catalyzed the production of HNO 2 from NH 2OH. 7. The rate of O 2 utilization coupled to hydroxylamine oxidation was inhibited in the presence of nitrite, whereas the rate of aerobic hydroxylamine disappearance was stimulated approx. 100% in the presence of nitrite. 8. Hydroxylamine-nitrite reductase (hydroxylamine:nitrite oxidoreductase) appears to involve the combined action of Nitrosomonas hydroxylamine dehydrogenase and nitrite reductase.

The purification and properties of nitrite reductase from higher plants, and its dependence on ferredoxin.

1. NADPH-dependent nitrite reductase from the leaves of higher plants was purified at least 70-fold and separated into two enzyme fractions. The first enzyme, a diaphorase with ferredoxin-NADP-reductase activity, is required only to transfer electrons from NADPH to a suitable electron acceptor, which then donates electrons to nitrite reductase proper. 2. Purified nitrite reductase accepted electrons from ferredoxin (the natural donor) or from reduced dyes. Ferredoxin was reduced by illuminated chloroplasts or dithionite, or by NADPH when diaphorase was present. The purified enzyme did not accept electrons directly from NADPH. 3. Ferredoxins purified from maize, spinach or Clostridium were interchangeable in the nitrite-reductase system. 4. Nitrite reductase had K(m) 0.15mm for nitrite. The pH optimum varied with plant and method of assay. The preparation had low sulphite-reductase activity. Ammonia was the product of nitrite reduction. 5. For some plants, the assay of crude preparations with NADPH was limited by diaphorase and the addition of diaphorase gave a better estimate of nitrite-reductase activity. A simple method of assay is described that uses dithionite with benzyl viologen as electron donor.