Purification and Properties of Nitrite Reductase from Spinach Leaves

Nitrite reductase (EC 1.6.6.4) has been purified 730-fold from spinach leaves. The enzyme catalyzes the reduction of nitrite to ammonia, with the use of reduced form of methyl viologen and ferredoxin. A stoichiometry of one molecule of nitrite reduced per molecule of ammonia formed has been found. KCN at 2.5×10^-4 m inhibited nitrite reductase activity almost completely. Purified enzyme was almost homogeneous by disk electrophoresis with polyacrylamide gel. The molecular weight of the enzyme was estimated to be 61,000 from gel filtration. Nitrite reductase, in the oxidized form, has absorption maxima at 276, 388 and 573 mμ. Both methyl viologen and ferredoxin linked nitrite reductase activities of the enzyme were inactivated on exposure to low ionic strength.     

Reduction of Nitrate and Nitrite by Subcellular Preparations of Anabaena cylindrica. I. Reduction of Nitrite to Ammonia

Reduction of nitrite by cell-free preparations of Anabaena cylindrica in the dark has been investigated. Nitrite-reducing activity was recovered in a supernatant fraction. The nitrite reductase system was partially purified by column chromatography on Sephadex G-75. NADPH could serve as an H-donor. NADH was completely inactive. The reduction required ferredoxin which mediated the transfer of electrons from NADPH to nitrite. Ferredoxin was successfully replaced with methyl viologen, benzyl viologen and diquat. The nitrite-reducing activity was inhibited by KCN, and by 2,4-dinitrophenol and arsenate at higher concentrations. The extent of nitrite reduction by NADPH was dependent on the oxidation-reduction states of NADP and ferredoxin.     

Nitrite,hydroxylamine and sulphite reductases in wheat leaves

The inclusion of cysteine and Na-EDTA in the extracting buffer lowered the activity of sulphite reductase extracted from wheat leaves while nitrite and hydroxylamine reductases were not so affected. Maximum activity for the three enzymes was achieved with reduced methyl viologen as the electron donor. The three enzyme activities were found in the chloroplasts. Nitrite reductase was detected in the leaves of the seedlings only when grown with nitrate and exposed to light. Sulphite and hydroxylamine reductases were not, however, influenced by either of these treatments. These results suggest that nitrite reductase is a distinct enzyme and is not associated with sulphite reductase and hydroxylamine reductase in wheat leaves.     

nir1, a conditional-lethal mutation in barley causing a defect in nitrite reduction

Summary Eleven green individuals were isolated when 95000 M₂ plants of barley (Hordeum vulgare L.), mutagenised with azide in the M₁, were screened for nitrite accumulation in their leaves after nitrate treatment in the light. The selected plants were maintained in aerated liquid culture solution containing glutamine as sole nitrogen source. Not all plants survived to flowering and some others that did were not fertile. One of the selected plants, STA3999, from the cultivar Tweed could be crossed to the wild-type cultivar and analysis of the F₂ progeny showed that leaf nitrite accumulation was due to a recessive mutation in a single nuclear gene, which has been designated Nir1. The homozygous nir1 mutant could be maintained to flowering in liquid culture with either glutamine or ammonium as sole nitrogen source, but died within 14 days after transfer to compost. The nitrite reductase cross-reacting material seen in nitrate-treated wild-type plants could not be detected in either the leaf or the root of the homozygous nir1 mutant. Nitrite reductase activity, measured with dithionite-reduced methyl viologen as electron donor, of the nitrate-treated homozygous nir1 mutant was much reduced but NADH-nitrate reductase activity was elevated compared to wild-type plants. We conclude that the Nir1 locus determines the formation of nitrite reductase apoprotein in both the leaf and root of barley and speculate that it represents either the nitrite reductase apoprotein gene locus or, less likely, a regulatory locus whose product is required for the synthesis of nitrite reductase, but not nitrate reductase. Elevation of NADH-nitrate reductase activity in the nir1 mutant suggests a regulatory perturbation in the expression of the Narl gene.     

Effects of electron donors on nitrate removal by nitrate and nitrite reductases

Effects of artificial electron donors to deliver reducing power on enzymic denitrification were investigated using nitrate reductase and nitrite reductase obtained fromOchrobactrum antropi. The activity of nitrite reductase in the soluble portion was almost the same as that in the precipitated portion of the cell extract. Nitrate removal efficiency was higher with benzyl viologen than with methyl viologen or NADH as an artificial electron donor. The turn-over numbers of nitrate and nitrite reductase were 14.1 and 1.9 μmol of nitrogen reduced/min·mg cell extracts, respectively when benzyl viologen was used as an electron donor.     

Some properties of two forms of nitrite reductase from corn (Zea mays L.) scutellum

Summary Nitrite reductase from corn scutellum-a non-chlorophyllous tissue-can use methyl viologen, benzyl viologen or ferredoxin as electron donor. Little or no reduction occurs with nicotinamide or flavin nucleotides. Activity is inhibited by p-chloromercuribenzoate and by cyanide. Organic chelates, with the exception of bathocuproine disulphonate and bathophenanthroline disulphonate, are not inhibitory. Ammonia is the reaction product. Ion exchange chromatography resolves the nitrite reductase activity into two peaks with apparently represent two forms of the enzyme. Both have a molecular weight of 61–63000 as determined by molecular exclusion chromatography, and a pH optimum of 6.7–6.8. Although their properties are generally similar, they show a marked difference in thermal stability, ionic charge and behaviour during isoelectric focusing. Nitrite reductase is found largely in the soluble fraction although some particulate activity is also obtained. Both forms of the enzyme are present in the soluble and particulate fractions.     

Nitrite and hydroxylamine reduction in higher plants. Fractionation, electron donor and substrate specificity of leaf enzymes, principally from vegetable marrow (Cucurbita pepo L.)

Nitrite reductase was purified between 760- and 1300-fold from vegetable marrow (Cucurbita pepo L.) and residual hydroxylamine reductase activity was low or negligible by comparison. With ferredoxin as electron donor, nitrite loss and ammonia formation at pH7.5 were stoicheiometrically equivalent. Crude nitrite reductase preparations showed negligible activity with NADPH as electron donor maintained in the reduced state by glucose 6-phosphate, whereas by comparison, activity was high when either ferredoxin or benzyl viologen were also present and reduced by the NADPH-glucose 6-phosphate system, whereas FMNH(2) produced variable and relatively low activity under the same conditions. At pH values below 7, non-enzymic reactions occurred between reduced benzyl viologen and nitrite, and intermediate reduction products were inferred to be produced instead of ammonia. Activity with ferredoxin (0.1mm), reduced by chloroplast grana in the light, was 25 times that produced with ferredoxin (40mum) reduced with NADPH and glucose 6-phosphate. For an approximate molecular weight 61000-63000 derived by chromatography on Sephadex G-100 and G-200, and a specific activity of 46mumol of nitrite reduced/min per mg of protein with light and chloroplast grana, a minimum turnover number of 3x10(3)mol of nitrite reduced/min per mol of enzyme was found. Two hydroxylamine reductases were separated on Sephadex gels. One (HR1) was initially associated with nitrite reductase during gel filtration but disappeared during later fractionation. This HR1 fraction showed nearly comparable activity with reduced benzyl viologen, ferredoxin or FMNH(2). The other (HR2), of molecular weight approx. 35000, reacted with reduced benzyl viologen but showed negligible activity with ferredoxin or NADPH. Activity with FMNH(2) was associated with an irregular trailing boundary during gel filtration, with much diminished activity in the HR2 region. Activity with NADPH was about 30% of that with FMNH(2), reduced benzyl viologen or ferredoxin and was considered to reside in fraction HR1. Hydroxylamine yielded ammonia under all assay conditions. No activity with hyponitrite or sulphite was observed with reduced benzyl viologen as electron donor in either the nitrite reductase or the hydroxylamine reductase systems, but pyruvic oxime produced about 4% of the activity of hydroxylamine.     

Purification and properties of nitrite reductase from the blue-green alga Anabaena cylindrical

Nitrite reductase was purified about 40-fold from the blue-greenalga Anabaena cylindrica by acetone precipitation and chromatographyon DEAE-cellulose columns. The nitrite reductase had its pHoptima at about 7.6 with Tris-HCl and at about 7.4 with phosphatewhen reduced methyl viologen was used as an electron donor.The Km's for nitrite, methyl viologen and ferredoxin were 510^–55,210^–4 and 510^–6M, respectively. A stoichiometryof one molecule of ammonia formation per one molecule of nitritedisappearance was confirmed. Ferredoxin which had been reducedeither chemically with dithionite or enzymatically with NADPHin the presence of diaphorase was active as an electron donor.Dithionite-reduced FAD and FMN were inactive. NADPH could notgive electrons directly to nitrite reductase. Hydroxylaminereductase was segregated from nitrite reductase by DEAE-cellulosecolumn chromatography. Purified nitrite reductase showed noactivity for sulfite reduction. A molecular weight of 68,000was estimated for nitrite reductase using a calibrated SephadexG-200 column. ¹This work was supported by grants 4090 and 955008 from theMinistry of Education. ²This work was supported by grants 4090 and 955008 from theMinistry of Education. 2 Present address: Department of Botany,Faculty of Science, University of Tokyo, Tokyo.     

Utilization of nitrate by bacteroids of Bradyrhizobium japonicum in the soybean root nodule

Bacteroids of Bradyrhizobium japonicum strain CB1809, unlike CC705, do not have a high level of constitutive nitrate reductase (NR; EC 1.7.99.4) in the soybean (Glycine max. Merr.) nodule. Ex planta both strains have a high activity of NR when cultured on 5 mM nitrate at 2% O₂ (v/v). Nitrite reductase (NiR) was active in cultured cells of bradyrhizobia, but activity with succinate as electron donor was not detected in freshly-isolated bacteroids. A low activity was measured with reduced methyl viologen. When bacteroids of CC705 were incubated with nitrate there was a rapid production of nitrite which resulted in repression of NR. Subsequently when NiR was induced, nitrite was utilized and NR activity recovered. Nitrate reductase was induced in bacteroids of strain CB1809 when they were incubated in-vitro with nitrate or nitrite. Increase in NR activity was prevented by rifampicin (10 g· ml^-1) or chloramphenicol (50 g·ml^-1). Nitrite-reductase activity in bacteroids of strain CB1809 was induced in parallel with NR. When nitrate was supplied to soybeans nodulated with strain CC705, nitrite was detected in nodule extracts prepared in aqueous media and it accumulated during storage (1°C) and on further incubation at 25°C. Nitrite was not detected in nodule extracts prepared in ethanol. Thus nitrite accumulation in nodule tissue appears to occur only after maceration and although bacteroids of some strains of B. japonicum have a high level of a constitutive NR, they do not appear to reduce nitrate in the nodule because this anion does not gain access to the bacteroid zone. Soybeans nodulated with strains CC705 and CB1809 were equally sensitive to nitrate inhibition of N₂ fixation.Abbreviations NR nitrate reductase - NiR nitrite reductase - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

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.     

Effect of xenobiotics on inorganic nitrogen assimilation by the phototrophic bacteriumRhodobacter capsulatus E1F1

In the presence of nitroaromatic and haloaromatic derivatives,Rhodobacter capsulatus E1F1 growth was affected in different degrees depending on the nitrogen source used. Phototrophic growth on glutamate or ammonium was not inhibited by 2,4-dinitrophenol (2,4-DNP), 4-nitrophenol (4-NP), 2-amino-4-nitrophenol (2,4-ANP), 4-aminophenol (4-AP), or 4-chlorophenol (4-CIP), whereas 2,4-dinitrophenol and 4-chlorophenol partially inhibited bacterial growth in nitrate, nitrite, and dinitrogen. On the other hand, although photosynthetic nitrate uptake was significantly inhibited by 2,4-dinitrophenol, 4-chlorophenol inhibited it to a lesser extent. Nitrogen fixation was severely inactivated in vivo by 2,4-dinitrophenol, but nitrate reductase activity was activated in vivo by 2,4-dinitrophenol, 4-nitrophenol, and 4-chlorophenol. Similar effects were found in cells growing with nitrate and 2,4-dinitrophenol under dark and aerobiosis conditions. None of the enzymatic activities related to inorganic nitrogen assimilation were affected by xenobiotics in vitro.     

Further Characterization of Ferredoxin Nitrite Reductase and the Relationship between the Enzyme and Methyl Viologen-dependent Nitrite Reductase

Ferredoxin-dependent nitrite reductase of spinach has been further characterized and the relationship between this enzyme and methyl viologen-dependent nitrite reductase studied.

Purified ferredoxin nitrite reductase, having a molecular weight of 86,000, showed 2.5 times higher ferredoxin-dependent activity than methyl viologen-linked activity. Besides 4 mol of labile sulfide the enzyme contained about 2 mol of siroheme per mol. When dithionite, methyl viologen and nitrite were added, ESR signals of a heme nitrosyl complex at g = 2.14, 2.07 and 2.02 were observed. Moreover, hyperfine splitting of the signal due to ¹⁴N nuclear spin was also observed at 2.033, 2.023 and 2.013. The sole addition of hydroxylamine to the ferric enzyme also caused the same but much less intense signals with the hyperfine splitting.

On treatment of the ferredoxin nitrite reductase (native enzyme) with DEAE-Sephadex A-50 chromatography, a modified nitrite reductase having a molecular weight of 61,000 and a protein fraction having an apparent molecular weight of 24,000 were separated. The modified enzyme contained about one mol of siroheme and 4 mol of labile sulfide per mol and showed essentially the same heme ESR signals as the native enzyme. Contrary to the native enzyme, this modified enzyme accepted electrons more efficiently from methyl viologen than ferredoxin and the reduction of nitrite to ammonia catalyzed by the modified enzyme was not stoichiometric. The observed nitrite to ammonia ratio was 1 to less than 0.6. Cyanide at concentrations between 0.02 to 0.2 mm inhibited the activity of the native enzyme almost completely but the modified enzyme was inhibited only partially.

From the results obtained, it is suggested that the native ferredoxin-linked nitrite reductase consists of two components (or subunits) and removal of the light component results in formation of a modified enzyme with increased relative affinity to methyl viologen.     

Evaluation of Nitrate Reductase Activity in Rhizobium japonicum

Nitrate reductase activity was evaluated by four approaches, using four strains of Rhizobium japonicum and 11 chlorate-resistant mutants of the four strains. It was concluded that in vitro assays with bacteria or bacteroids provide the most simple and reliable assessment of the presence or absence of nitrate reductase. Nitrite reductase activity with methyl viologen and dithionite was found, but the enzyme activity does not confound the assay of nitrate reductase.     

Nitrite reduction in barley-root plastids: Dependence on NADPH coupled with glucose-6-phosphate and 6-phosphogluconate dehydrogenases,and possible involvement of an electron carrier and a diaphorase

Plastids from roots of barley (Hordeum vulgare L.) seedlings were isolated by discontinuous Percoll-gradient centrifugation. Coinciding with the peak of nitrite reductase (NiR; EC 1.7.7.1, a marker enzyme for plastids) in the gradients was a peak of a glucose-6-phosphate (Glc6P) and NADP⁺-linked nitrite-reductase system. High activities of phosphohexose isomerase (EC 5.3.1.9) and phosphoglucomutase (EC 2.7.5.1) as well as glucose-6-phosphate dehydrogenase (Glc6PDH; EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (6PGDH; EC 1.1.1.44) were also present in the isolated plastids. Thus, the plastids contained an overall electron-transport system from NADPH coupled with Glc6PDH and 6PGDH to nitrite, from which ammonium is formed stoichiometrically. However, NADPH alone did not serve as an electron donor for nitrite reduction, although NADPH with Glc6P added was effective. Benzyl and methyl viologens were enzymatically reduced by plastid extract in the presence of Glc6P+ NADP⁺. When the plastids were incubated with dithionite, nitrite reduction took place, and ammonium was formed stoichiometrically. The results indicate that both an electron carrier and a diaphorase having ferredoxin-NADP⁺ reductase activity are involved in the electron-transport system of root plastids from NADPH, coupled with Glc6PDH and 6PGDH, to nitrite.Abbreviations Cyt cytochrome - Glc6P glucose-6-phosphate - Glc6PDH glucose-6-phosphate dehydrogenase - MVH reduced methyl viologen - NiR nitrite reductase - 6PG 6-phosphogluconate - 6PGDH 6-phosphogluconate dehydrogenase     

Assimilatory nitrate reductase from Acinetobacter calcoaceticus

A soluble nitrate reductase from the bacterium Acinetobacter calcoaceticus grown on nitrate has been characterized. The reduction of nitrate to nitrite is mediated by an enzyme of 96000 molecular weight that can use as electron donors either viologen dyes chemically reduced with dithionite or enzymatically reduced with NAD(P)H, through specific diaphorases which utilize viologens as electron acceptors. Nitrate reductase activity is molybdenum-dependent as shown by tungstate antagonistic experiments and is sensitive to -SH reagents and metal chelators such as KCN.The enzyme synthesis is repressed by ammonia. Moreover, nitrate reductase activity undergoes a quick inactivation either by dithionite and temperature or by dithionite in the presence of small amounts of nitrate. Cyanate prevents this inactivating process and can restore the activity once the inactivation had occurred, thus suggesting that an interconversion mechanism may participate in the regulation of Acinetobacter nitrate reductase.Abbreviations EDTA ethylenediaminetetraacetate - BV benzyl viologen - MV methyl viologen - MW molecular weight - NEM N-ethylmaleimide - p-HMB p-hydroxymercuribenzoate - DCPIP 2,6-dichlorophenol-indophenol - FMN flavin mononucleotide - FAD flavin adenine dinucleotide - KCNO potassium cyanate     

Immunological studies of ferredoxin-nitrite reductases and ferredoxin-glutamate synthases from photosynthetic organisms

Polyclonal antiserum specific for ferredoxin-nitrite reductase (EC 1.7.7.1) from the green alga Chlamydomonas reinhardii recognized the nitrite reductase from other green algae, but did not cross-react with the corresponding enzyme from different cyanobacteria or higher plant leaves. An analogous situation was also found for ferredoxin-glutamate synthase (EC 1.4.7.1), using its specific antiserum. Besides, the antibodies raised against C. reinhardii ferredoxin-glutamate synthase were able to inactivate the ferredoxin-dependent activity of nitrite reductase from green algae.These results suggest that there exist similar domains in ferredoxin-nitrite reductases and ferredoxin-glutamate synthases from green algae. In addition, both types of enzymes share common antigenic determinants, probably located at the ferredoxin-binding domain. In spite of their physicochemical resemblances, no apparent antigenic correlation exists between the corresponding enzymes from green algae and those from higher plant leaves or cyanobacteria.Abbreviations Fd ferredoxin - GOGAT glutamate synthase - MV⁺ reduced methyl viologen (radical cation) - NiR nitrite reductase - PMSF phenylmethylsulphonyl fluoride - SDS sodium dodecyl sulfate     

Expression and characterization of the assimilatory NADH-nitrite reductase from the phototrophic bacterium Rhodobacter capsulatus E1F1

A nas gene region from Rhodobacter capsulatus E1F1 containing the putative nasB gene for nitrite reductase was previously cloned. The recombinant His₆-NasB protein overproduced in E. coli showed nitrite reductase activity in vitro with both reduced methyl viologen and NADH as electron donors. The apparent K _m values for nitrite and NADH were 0.5 mM and 20 μM, respectively, at the pH and temperature optima (pH 9 and 30°C). The optical spectrum showed features that indicate the presence of FAD, iron-sulfur cluster and siroheme as prosthetic groups, and nitrite reductase activity was inhibited by sulfide and iron reagents. These results indicate that the phototrophic bacterium R. capsulatus E1F1 possesses an assimilatory NADH-nitrite reductase similar to that described in non-phototrophic organisms.     

ATP activation and properties of the methyl coenzyme M reductase system in Methanobacterium thermoautotrophicum.

The requirement of ATP for the methyl coenzyme M methylreductase in extracts of Methanobacterium thermoautotrophicum was found to be catalytic; for each mol of ATP added, 15 mol of methane was produced from methyl coenzyme M [2-(methylthio)ethanesulfonic acid]. Other nucleotide triphosphates partially replaced ATP in activation of the reductase. All components of the reaction were found in the supernatant fraction of cell extracts after centrifugation at 100,000 X g for 1 h; optimal reaction rates occurred at 65 degrees C, at a pH range of 5.6 to 6.0, and at concentrations of ATP and MgCl2 of 1 mM and 40 mM, respectively. Chloral hydrate, chloroform, nitrite, 2,4-dinitrophenol, and viologen dyes (compounds known to inhibit methanogenesis from a variety of substrates) were found to inhibit the conversion of methyl coenzyme M to methane. Methyl coenzyme M methylreductase was shown to be present in a variety of methanogens.     

Nitrate reductase from bacteroides of Rhizobium japonicum: enzyme characteristics and possible interaction with nitrogen fixation.

The soluble nitrate reductase of Rhizobium japonicum bacteroids has been purified and its properties compared to those of aerobically grown cells. The enzymes from both sources are similar with molecular weights of about 70 000 suggesting no close relationship with the molybdo-protein component of nitrogenase. Nitrite, the product of nitrate reductase, strongly inhibited the nitrogenase activity from bacteroids, at concentrations less than 100 muM. Thus, an interference in the rate of nitrogen fixation is possible as a result of nitrate reductase activity. A study of the distribution of nitrate reductase in bacteroids indicates that a proportion of the total activity is membrane-bound but that this activity is similar to that in the soluble fraction. Purified nitrate reductase required reduced viologen dyes for activity. Neither NADPH or NADH or FAD could substitute as electron donors. Dithionite is a strong inhibitor and inactivated nitrate reductase from all sources examined. This inactivation is prevented by methyl viologen. Purified nitrate reductase from bacteroids and bacteria Rhizobium japonicum is practically unaffected by exposure to oxygen.     

Nitrate and Nitrite Reduction in Wheat Leaves as Affected by Different Types of Water Stress

The effect of different types of water stress on nitrate and nitrite reductases of wheat (Triticum vulgare L. cv. Mivhor) leaves was investigated. Water stress was applied either to leaf tissue by its incubation in mannitol or various salt solutions, or to intact plants by exposure of the root system to low temperatures or to salinity. Nitrite reductase was much less sensitive to water stress than nitrate reductase, and was not sensitive to salinity up to osmotic potentials of about — 13 bars. The decrease in nitrite reductase activity by water stress was attributed to a direct inhibition of the enzyme rather than to a repression of enzyme synthesis. This was based on the fast response of the enzyme after exposure of leaf tissue to reduced osmotic potential, on the lack of a continuous decrease in enzyme activity during a prolonged stress, and on the fact that light activation of reductase was unaffected by water stress. The inhibition of nitrate reductase under water stress was attributed to both a direct inhibition and a reduced rate in enzyme synthesis. This is concluded from the fact that a decrease in its activity was obtained already within 1 h after stress application and from the fact that light induction of the enzyme was inhibited by stress.