NADH-nitrate Reductase Research Articles

Composition and structure of assimilatory nitrate reductase from Ankistrodesmus braunii.

Assimilatory NAD(P)H-nitrate reductase (EC 1.6.6.2) from Ankistrodesmus braunii has been purified to homogeneity by affinity chromatography on blue Sepharose. The specific activity of the purified enzyme is in the range of 72 to 80 units/mg of protein. The electronic spectrum of the native enzyme shows absorption maxima at 278, 414 (Soret), 532 (beta), 562 (alpha), and 669 nm and shoulders at 455 and 484 nm, with an A278/A414 ratio of 2.56. The reduced enzyme shows absorption maxima at 424 (Soret), 528 (beta), 557 (alpha),and 669 n. The enzyme complex (Mr = 467,400) is composed of eight similar subunits (Mr = 58,750) and contains 4 molecules of FAD, 4 heme groups, and 2 atoms of molybdenum. Labile sulfide and nonheme iron were not detected. Electron micrographs show the eight subunits arranged alternately in two planes, and an 8-fold rotational symmetry was deduced from highly magnified images processed by optical superposition.

Spinach nitrate reductase. Kinetic studies of NADH-diaphorase

NADH-cytochrome c reductase (diaphorase), a functional moiety of the spinach NADH-nitrate reductase complex, uses one molecule of NADH to reduce two of ferricytochrome c. Initial velocity experiments in the absence of products produce patterns of parallel lines when plotting 1/ v vs. 1/(substrate) at different fixed concentrations of the other substrate. The results agree with a Ping Pong mechanism in which the first product (NAD +) is released, with formation of a reduced free form of the enzyme, prior to binding of the first molecule of the second substrate (ferricytochrome c). Product inhibition experiments indicate the production of an abortive complex between NAD + and the oxidized enzyme form that binds NADH. The data are consistent with a Hexa Uni Ping Pong mechanism in which an abortive complex is formed between NAD + and the enzyme form binding NADH.

In vitro complementation of assimilatory NAD(P)H-nitrate reductase from mutants of Chlamydomonas reinhardii

In vitro complementation of the soluble assimilatory NAD(P)H-nitrate reductase (NAD(P)H:nitrate oxidoreductase, EC 1.6.6.2) was attained by mixing cell-free preparations of Chlamydomonas reinhardii mutant 104, uniquely possessing nitrate-inducible NAD(P)H-cytochrome c reductase, and mutant 305 which possesses solely the nitrate-inducible FMNH 2- and reduced benzyl viologen-nitrate reductase activities. Full activity and integrity of NAD(P)H-cytochrome c reductase from mutant 104 and reduced benzyl viologen-nitrate reductase from mutant 305 are needed for the complementation to take place. A constitutive and heat-labile molybdenum-containing cofactor, that reconstitutes the NAD(P)H-nitrate reductase activity of nit-1 Neurospora crassa but is incapable of complementing with 104 from C. reinhardii, is present in the wild type and 305 algal strains. The complemented NAD(P)H-nitrate reductase has been purified 100-fold and was found to be similar to the wild enzyme in sucrose density sedimentation, molecular size, pH optimum, kinetic parameters, substrate affinity and sensitivity to inhibitors and temperature. From previous data and data presented in this article on 104 and 305 mutant activities, it is concluded that C. reinhardii NAD(P)H-nitrate reductase is a heteromultimeric complex consisting of, at least, two types of subunits separately responsible for the NAD(P)H-cytochrome c reductase and the reduced benzyl viologen-nitrate reductase activities.

Activation of Nitrate Reductase by Extracts from Corn Scutella

NADH-nitrate reductase (NR) from the primary leaves and root tips of corn seedlings (var. W64A x W182E) were activated by extracts from corn scutella. The activator extracted in potassium phosphate buffer (pH 7.5) or 80% (v/v) ethanol and fractionated by Dowex 1 (acetate) and Dowex 50 (H(+)) resins was recovered in the cationic fraction. The activator was not detected in extracts from shoots, roots, or endosperm of the seedlings. It activated the nitrate-induced cytochrome c reductase of NR complex but had slight inhibitory effects on the activities of FMNH(2)-NR and reduced methylviologen-NR. In addition the activator inhibited the activities of purified NR-inactivating proteins from corn roots (var. Wf9 x 38-11) and rice cell cultures.

Ferrocyanide-activated NADH cytochrome c reductase from barley

A single ferrocyanide-activated NADH cytochrome c reductase species was present in primary leaf extracts from both nitrate-treated and untreated barley plants. The enzyme had a sedimentation coefficient of 3.9 S, a Stokes radius of 2.8 nm, a calculated mol. wt of 45 000, a frictional ratio of 1.19 and an axial ratio of 3 : 1. The enzyme was not detected under conditions which led to the partial breakdown of NADH nitrate reductase to 3.1 S and 3.8 S NADH cytochrome c reductase species. We conclude that the ferrocyanide-activated NADH cytochrome c reductase species is not related to NADH nitrate reductase.

Reduction of Nitrate and Nitrite in Lambsquarters (Chenopodium album) Biotypes Resistant and Susceptible to Atrazine Toxicity

The nitrite-reducing activity of the normal susceptible biotype of lambsquarters (Chenopodium album L.) was strongly inhibited by atrazine in the assay medium, both in the case of the in vivo assays of leaf discs in light, and in vitro photoreduction assays of crude extracts. In vitro assays of crude extracts with methylviologen or ferredoxin supplying the reducing potential were not inhibited by atrazine. In the resistant biotype, inhibition of nitrite reduction did not occur with any of the above assays. Thus, it appears that atrazine does not inhibit nitrite reductase itself, but rather the availability of photosynthetically supplied electrons for the reduction. Atrazine had no effect when added to the media for either in vivo or in vitro assays of nitrate reduction by either the susceptible or resistant biotype.Young lambsquarters plants were treated with atrazine by spraying the leaves at a rate which was lethal for susceptible plants after 5 or 6 days, but had little effect on the resistant biotype. Nitrite did not accumulate in either biotype, but remained present at the level of about 0.1 microgram nitrite N per gram fresh weight. The nitrate content of susceptible-type leaves did increase to two or three times the initial level, during the first four days after spraying. Usually the only visible effect on the plants during this time was a decreased growth rate. Twenty-four hours after spraying the following activities had fallen to 25% or less of the activities of solvent-sprayed control plants: in vivo nitrite reductase, in vivo nitrate reductase, in vitro NADH-nitrate reductase, in vitro reduced flavin mononucleotidenitrate reductase, and in vitro NADH-diaphorase. In these atrazine-treated plants, in vitro nitrite reductase activity with reducing potential supplied by methylviologen was not affected, nor were any of the above activities in leaves of atrazine-treated resistant plants. The abrupt fall in nitrate reductase represents an effect of atrazine not directly related to inhibition of photosynthesis.

Catalytic properties of Ankistrodesmus braunii nitrate reductase

Woolf plots of the initial reaction velocities at varying concentrations of substrates have been performed to determine the substrates affinity for the purified A. branuii NAD(P)H-nitrate reductase complex. p-Hydroxymercuribenzoate, cyanide or azide inhibits the reduction of nitrate by NAD(P)H catalysed by this enzyme. The inhibition of p-hydroxymercuribenzoate is localised in the first activity of the complex, i.e. NAD(P)H-cytochrome c reductase, and this inhibition is specifically protected by NADH, but not by NADPH. Cyanide or azide inhibits the terminal activity of the complex, i.e. reduced methyl viologen-nitrate reductase. Lineweaver-Burk plots of the initial velocities, at different concentrations of NADH and nitrate, and product inhibition patterns show an iso ping pong bi bi steady state kinetic mechanism, with isomeration of the enzymatic form which binds NADH, for the NADH-nitrate reductase activity.

Purification and partial characterization of nitrate reductase from barley leaves

NADH-nitrate reductase from barley leaves ( Hordeum vulgare L. cv. Steptoe) was purified 570-fold with over 50% yield by a procedure using ammonium sulfate fractionation and blue dextran-agarose affinity chromatography. The purified enzyme had NADH-, FMNH 2-, and reduced methyl viologen-nitrate reductase and NADH-cytochrome c reductase activities, but not NADPH nitrate reductase activity. The NADH-nitrate reductase has a pH optimum at 7.5 and a Michaelis constant ( K m) for nitrate of 2.4 × 10 −4 M. The specific activity of the purified enzyme was 8 μ mol NO 2 −/min/mg protein. A single protein band corresponding to reduced methyl viologen-nitrate reductase activity was observed by disc gel electrophoresis. A S 20,w of 7.9 S was found by a sucrose density centrifugation and a Stokes radius of 68 Å was determined by gel filtration. From these values, a molecular weight of 221 000 was estimated for the native enzyme. SDS-gel electrophoresis of the native enzyme indicated a subunit molecular weight of approximately 100 000. Thus, the barley nitrate reductase contains two subunits of the same size.

Characterization of nitrate reductase-deficient barley mutants

Ten nitrate reductase-deficient Hordeum vulgare mutants were characterized for NADH and FMNH2 nitrate reductase (NR), cytochrome C reductase (CR) and nitrite reductase (NiR) activities. The mutants sort into four major groups. Group I represented by mutants Az 12, Az 23, Az 29 and Az 30 have low Nr and Cr activities. Group II represented by mutants Az 13, Az 31, Az 33 and Az 34 have low NR activities but intermediate CR activities. Group III represented by mutant Az 28 has low NR activity, but above normal CR activity. Group IV represented by Az 32 has low NADH-NR, low CR, but above normal FMNH2-NR activity. All ten mutants have elevated NiR activities. None of the ten mutants were constitutive for nitrite reductase activity. Only Az 34 showed a definite high temperature sensitivity when the NADH nitrate reductase activity was compared in the 12 to 26° C range. The mutants Az 12, Az 13, Az 23, Az 28, Az 29, Az 30, Az 31, Az 32 and Az 33 are allelic and were assigned the locus designation nar1. Mutant Az 34 represents a different genetic locus designated nar2. The nar1 gene is codominant and the nar2 gene is recessive.

NADH nitrate reductase and related NADH cytochrome c reductase species in barley

Nitrate and nitrate-less barley ( Hordeum vulgare cv Golden Promise) shoot extracts were examined by Sephadex G200 gel filtration and sucrose density gradient analysis and the MWs of NR and CR species present were determined from their Stokes radii and sedimentation coefficients by the method of Siegel and Monty. Nitrate-less plant extracts possessed a CR species of MW 27 800 whilst nitrate-plant extracts possessed CR species of MW 203 000, 61 000, 40 000 and 27 800. The MW 203 000 CR species was associated with NADH-NR, FMNH-NR and MV°-NR activities and represents the NR complex. The MW 40 000 and 61 000 CR species were shown to be derived from the NR complex. We suggest that the MW 40 000 and 61 000 CR species represent either subunits of the NR complex or domains cleaved from the intact NR complex by endogenous proteinases.

Nitrate reductase-deficient mutant cell lines of Nicotiana tabacum

The wild-type line and 14 nitrate reductase-deficient mutant cell lines of Nicotiana tabacum were tested for the presence of nitrate reductase partial activities, and for nitrite reductase and xanthine dehydrogenase activity. Data characterizing the electron donor specificity of nitrate reductase (EC 1.6.6.1., NADH:nitrate oxidoreductase) and nitrite reductase (EC 1.7.7.1., ferredoxin:nitrite oxidoreductase) of the wild-type line are presented. Three lines (designated cnx) simultaneously lack NADH-, FADH2-, red. benzyl viologen-nitrate reductase, and xanthine dehydrogenase activities, but retain the nitrate reductase-associated NADH-cytochrome c reductase activity. These mutants are, therefore, interpreted to be impaired in gene functions essential for the synthesis of an active molybdenum-containing cofactor. For cnx-68 and cnx-101, the sedimentation coefficient of the defective nitrate reductase molecules does not differ from that of the wild-type enzyme (7.6S). In 11 lines (designated nia) xanthine dehydrogenase activity is unaffected, and the loss of NADH-nitrate reductase is accompanied by a loss of all partial activities, including NADH-cytochrome c reductase. However, one line (nia-95) was found to possess a partially active nitrate reductase molecule, retaining its FADH2- and red. benzyl viologen nitrate reductase activity. It is likely that nia-95 is a mutation in the structural gene for the apoprotein. Both, the nia and cnx mutant lines exhibit nitrite reductase activity, being either nitrate-inducible or constitutive. Evidence is presented that, in Nicotiana tabacum, nitrate, without being reduced to nitrite, is an inducer of the nitrate assimilation pathway.

Involvement of Oxygen in Chlorella fusca Nitrate Reductase Inactivation by Reduced Nicotinamide Adenine Dinucleotide

Summary In vitro inactivation of Chlorella fusca NADH-nitrate reductase can be obtained by preincubation of the enzyme with reduced nicotinamide adenine dinucleotide. ADP cooperates with NADH in this inactivation. Anaerobic experiments reveal that oxygen is required in order to obtain inactivation of nitrate reductase by preincubation with NADH, alone or in the presence of ADP. Moreover, inactivation is prevented if superoxide dismutase and/or catalase are included in the preincubation mixture. The presence of hydrogen peroxide in the preincubation mixture increases the sensitivity of nitrate reductase to its inactivation by NADH. In vivo inactivation of Chlorella fusca NADH-nitrate reductase by addition of methylamine to the cells appears to depend on the intracellular catalase activity levels.

In Vitro Stability of Nitrate Reductase from Wheat Leaves

A nitrate reductase (EC 1.6.6.1)-inactivating factor has been isolated from 8-day-old wheat leaves. The purification schedule involved ammonium sulfate precipitation, Sephadex G-100 filtration, DEAE-cellulose chromatography, and Sephadex G-150 filtration. No accurate assessment could be made as to the degree of purification relative to crude extract as the inactivating factor could not be detected in crude extract. However a 2,446-fold purification was achieved from the ammonium sulfate fraction to the pooled enzyme from the Sephadex G-150 step.The inactivating factor was heat-labile and had a molecular weight of 37,500. The inactivating factor was particularly sensitive to the divalent metal chelators, 1,10-phenanthroline and bathophenanthroline. Evidence indicated that Fe(2+) may be the functional metal. The trypsin inhibitors N-alpha-p-tosyl-l-lysine chloromethyl ketone and alpha-N-benzoyl-l-arginine were inhibitory. However, phenylmethyl sulfonyl fluoride, an inhibitor of serine peptide hydrolases, was not inhibitory. Neither casein nor hemoglobin nor a range of artificial substrates were hydrolyzed by the inactivating factor. Highly purified wheat leaf nitrite reductase (EC 1.7.99.3) and ribulose 1,5-bisphosphate carboxylase:oxygenase (EC 4.1.1.39) were not affected by the nitrate reductase-inactivating factor.The inactivating factor was more active toward the NADH-nitrate reductase compared to either of the component enzymic activities flavin adenine mononucleotide-nitrate reductase and methyl viologen-nitrate reductase. The NADH-ferricyanide reductase (diaphorase) component was the least sensitive.

Anaerobic respiration and energy conservation in Paracoccus denitrificans. Functioning of iron-sulfur centers and the uncoupling effect of nitrite.

1. Electron paramagnetic resonance spectra at 8-60 K of NADH-reduced membrane particles prepared from Paracoccus denitrificans grown anaerobically with nitrate as terminal electron acceptor show the presence of iron-sulfur centers 1-4 in the NADH-ubiquinone segment of the respiratory chain. In addition resonance lines at g = 2.058, g = 1.953 and g = 1.88 are detectable in the spectra of succinate-reduced membranes at 15 K, which are attributed to the iron-sulfur-containing nitrate reductase. 2. Sulphate-limited growth under anaerobic conditions does not affect the iron-sulfur pattern of NADH dehydrogenase or nitrate reductase. Furthermore respiratory chain-linked electron transport and its inhibition by rotenone are not influenced. These results contrast those observed for sulphate-limited growth of P. denitrificans under aerobic conditions [Eur. J. Biochem. (1977) 81, 267-275]. 3. Proton translocation studies of whole cells indicate that nitrite increases the proton conductance of the cytoplasmic membrane, resulting in a collapse of the proton gradient across the membrane. Nitrite accumulates under anaerobic growth conditions with nitrate as terminal electron acceptor; the extent of accumulation depends on the specific growth conditions. Thus the low efficiencies of respiratory chain-linked energy conservation observed during nitrate respiration [Arch. Microbiol. (1977) 112, 17-23] can be explained by the uncoupling action of nitrite.

Inhibition of NADH-nitrate reductase activity in cucumber leaves due to NADH oxidation

Inhibition of NADH-nitrate reductase activity in cucumber leaves due to NADH oxidation

In Vitro Stability of Nitrate Reductase from Wheat Leaves

NADH-nitrate reductase has been highly purified from leaves of 8-day-old wheat (Triticum aestivum L. cv. Olympic) seedlings by affinity chromatography, using blue dextran-Sepharose 4B. Purification was assessed by polyacrylamide gel electrophoresis. The enzyme was isolated with a specific activity of 23 micromoles nitrite produced per minute per milligram protein at 25 C. At pH 7.5, the optimum pH for stability of NADH-nitrate reductase, this enzyme, and a component enzyme reduced flavin adenine mononucleotide (FMNH(2))-nitrate reductase has a similar stability at both 10 and 25 C. Two other component enzymes-methylviologen-nitrate reductase and NADH-ferricyanide reductase-also have a similar but higher stability. At this pH the Arrhenius plot for decay of NADH-nitrate reductase and methylviologen-nitrate reductase indicates a transition temperature at approximately 30 C above which the energy of activation for denaturation increases. FMNH(2)-nitrate reductase and NADH-ferricyanide reductase do now show this transition. The energy of activation for denaturation (approximately 9 kcal per mole) of each enzyme is similar between 15 and 30 C. The optimum pH for stability of the component enzymes was: NADH-ferricyanide reductase, 6.6; FMNH(2)-nitrate reductase and methylviologen-nitrate reductase, 8.9. All of our studies indicate that the NADH-ferricyanide reductase was the most stable component of the purified nitrate reductase (at pH 6.6, t((1/2)) [25 C] = 704 minutes). Data are presented which suggest that methylviologen and FMNH(2) do not donate electrons to the same site of the nitrate reductase protein.

The Occurrence and Role of Ubiquinone in Electron Transport to Oxygen and Nitrate in Aerobically, Anaerobically and Symbiotically Grown Rhizobium japonicum

SUMMARY: Ubiquinone was extracted from free-living Rhizobium japonicum, grown aerobically or anaerobically, and from the symbiotic bacteroid form; it was tentatively identified as the Q-10 homologue. The ubiquinone concentration was highest in symbiotically grown R. japonicum but the ratio ubiquinone:total cytochrome was about 1·5:1 in membrane particles from organisms grown under all three conditions. The ubiquinone was reduced 75% by NADH, completely oxidized by oxygen but not oxidized by nitrate. NADH oxidase activity and nitrate reductase activity in membrane particles from organisms grown under the different conditions were similar except that nitrate reductase activity was low in aerobically grown organisms. It is concluded that ubiquinone functions in electron transport to oxygen but not to nitrate.

Inhibitory effects of ferrocytochrome c on NADH-nitratereductase activity of spinach ( Spinacea oleracea L.)

Spinach NADH-nitrate oxido-reductase (EC 1.6.6.1) is inactivated progressively during several min by NADH, or less rapidly, by ferrocytochrome c in a biphasic reaction. Inactivation is synergistically increased by the presence of both during the first phase of the reaction. The enzyme is immune to inactivation by either compound during turnover with nitrate. When the enzyme is incubated with ferrocytochrome c without NADH, inactivation does not affect binding of NADH or nitrate, but when incubated together with NADH, ferrocytochrome c appears to impede binding by nitrate in addition to causing inactivation. The ping-pong kinetics of NADH nitrate reductase are thought to protect the enzyme against inactivation by NADH or ferrocytochrome c in the presence of nitrate.

Nitrate Reductase Deficient Mutants of Chlamydomonas reinhardii. Biochemical Characteristics

Two mutants of Chlamydomonas reinhardii that cannot grow with nitrate as nitrogen source have been examined biochemically. Each mutant differs from the wild-type strain by a single mutation in a Mendelian gene. One mutant (14/15) carries a mutation in a gene designated nitB. This organism took up nitrate and converted nitrate-nitrogen to insoluble-nitrogen; nevertheless no enzymic activities associated with nitrate reductase (NADH-nitrate reductase, reduced benzyl viologen nitrate reductase or inducible NADH-cytochrome c reductase) could be demonstrated in frozen/thawed cells or cell-free extracts. At high nitrate concentrations (5 mM) the rate of nitrate uptake by this mutant [V max 59 nmol (mg dry wt)–1 h–1] was considerably lower than that of wild-type [V max 920 nmol (mg dry wt)–1 h–1]. It is concluded that nitB is probably a regulatory gene for nitrate reductase. The other mutant (17/4) has a mutation in a gene designated nitA. This organism did not take up nitrate and had no NADH-nitrate reductase or inducible NADH-cytochrome c reductase activities. Frozen/thawed cells and cell-free extracts had reduced benzyl viologen nitrate reductase activity. The enzyme from the nitA mutant eluted from a Sepharose 4B column later than wild-type enzyme; it was therefore probably of lower molecular weight. The nitA mutation of C. reinhardii closely resembles the nit-3 mutation of Neurospora crassa and, like this mutation, it probably results in loss of the NADH combining sub-unit of nitrate reductase. Strain 137c of C. reinhardii had no enzymic activities associated with nitrate reductase and did not take up nitrate. This organism may have mutations in both the nitA and nitB loci.

Preparation and some properties of homogeneous Neurospora crassa assimilatory NADPH-nitrite reductase

The Neurospora crassa assimilatory NADPH-nitrite reductase (NAD(P)H: nitrite oxidoreductase, EC 1.6.6.4), which catalyzes the NADPH-dependent formation of ammonia from nitrite, has been purified to homogeneity as judged by polyacrylamide gel electrophoresis. The specific activity of the purified enzyme is 26.9 μmol nitrite reduced/min per mg protein, which corresponds to a turnover number of 7800 min −1. The enzyme also has associated NADH-nitrate reductase, NADPH-hydroxylamine reductase activities. The stoichiometry of 3 mol NADPH oxidized per mol nitrite reduced and ammonia formed has been confirmed. The visible absorption spectrum of the nitrite reductase reveals maxima at 280, 390 (Soret) and 580 (α) nm. The latter bands are indicative of the occurrence of siroheme as a prosthetic group. The A 280nm/ A 390nm ratio of 7.0 and the Soretα ratio of 3.8 are compatible with values reported for other purified siroheme-containing enzymes. These results are discussed in terms of the comparative biochemistry of various enzymes involved in nitrite, hydroxylamine and sulfite metabolism in Neurospora crassa and other organisms.