Inactivation by ammonia of the photosynthetic reduction of nitrate in Nostoc muscorum particles.

Ammonia, as well as other uncouplers of photophosphorylation, strongly inhibit the photosynthetic reduction of nitrate by particles of the blue-green alga Nostoc muscorum. The enzyme responsible for nitrate reduction, nitrate reductase, can be reversibly inactivated by reduction in a ferredoxin-dependent reaction. Nitrate protects against this inactivation, and molecular oxygen restores the original activity.     

Ammonium (methylammonium) is the co-repressor of nitrate reductase in Chlamydomonas reinhardii

Methylammonium cannot be used as a nitrogen source by the green alga Chlamydomonas reinhardii and, like ammonia, caused the repression of nitrate reductase without affecting the photosynthetic activity. Glutamine synthetase catalyzed the conversion ofmethylammonium to a single product, identified as γ-N-methylglutamine, which accumulated in the cells. Derepression of nitrate reductase was accompanied by a decrease in the intracellular concentration of methylammonium and a concomitant accumulation of γ-N-methylglutamine inside the cells. These facts strongly suggest that ammonium (methylammonium) per se, and not a product of its metabolism, is the co-repressor of nitrate reductase in C. reinhardii     

Nitrate reductase in agrostemma githago comparison of the inductive effects of nitrate and cytokinin

The effect of nitrate and cytokinin on the induction of nitrate reductase (NADH-nitrate oxidoreductase, EC 1.6.6.1) in embryos of Agrostemma githago was compared. Increased enzyme levels in response to treatment with the cytokinin benzyladenine were not correlated with a general stimulation of protein synthesis or a general reduction of protein breakdown. Actinomycin D did not inhibit the formation of nitrate reductase in response to nitrate or the cytokinin. Cycloheximide and puromycin inhibited the induction by the hormone to the same extent as, or even more than, the incorporation of [¹⁴C]leucine into protein. Induction of nitrate reductase by nitrate was much less susceptible to inhibition by cycloheximide and puromycin than induction of the enzyme by benzyladenine. When induction of nitrate reductase was carried out in the presence of ²H₂O, isopycnic equilibrium centrifugation in CsCl showed that incorporation of ²H into the enzyme had occured. The increase in the buoyant density of nitrate reductase was the same whether the enzyme was induced by nitrate or by benzyladenine, indicating that at least part of the nitrate reductase molecule was newly synthesized in both instances.     

Iron acquisition by plants: the reduction of ferrisiderophores by higher plant NADH: Nitrate reductase

Siderophores are avid Fe³⁺-chelators of microbial origin. Plant roots are colonized by fungi and bacteria which synthesize siderophores, and plants have been shown to metabolize these substances to obtain iron. We have previously shown that nitrate reductase from squash catalyzed the reduction of the ferrisiderophore ferrioxamine B with the subsequent loss of Fe²⁺. Using a spectrophotometric assay which traps Fe²⁺ in a ferrozine complex, we have noted that the substrate diversity of nitrate reductase as a ferrisiderophore reductase includes ferrichrome A, ferrichrome, ferrirhodotorulic acid, ferrischizokinen, and the novel siderophore ferri-‘AAHS’. These reductions were inhibited by polyclonal antibodies against nitrate reductase, but ferrisiderophore reductase activity, as evidenced with ferrirhodotorulic acid, was unaffected by low concentrations of azide. In addition, maximal activity occurred between pH 4 and 5, and appaarent K_m values were approx. 100 μmolar. Thus, we suggest that plant nitrate reductases might be involved in iron assimilation as well as nitrate reduction.     

Nitrate transport system in Neurospora crassa

Nitrate uptake in Neurospora crassa has been investigated under various conditions of nitrogen nutrition by measuring the rate of disappearance of nitrate from the medium and by determining mycelial nitrate accumulation. The nitrate transport system is induced by either nitrate or nitrite, but is not present in mycelia grown on ammonia or Casamino Acids. The appearance of nitrate uptake activity is prevented by cycloheximide, puromycin, or 6-methyl purine. The induced nitrate transport system displays a K_m for nitrate of 0.25 mM. Nitrate uptake is inhibited by metabolic poisons such as 2,4-dinitrophenol, cyanide, and antimycin A. Furthermore, mycelia can concentrate nitrate 50-fold. Ammonia and nitrite are non-competitive inhibitors with respect to nitrate, with K_i values of 0.13 and 0.17 mM, respectively. Ammonia does not repress the formation of the nitrate transport system. In contrast, the nitrate uptake system is repressed by Casamino Acids. All amino acids individually prevent nitrate accumulation, with the exception of methionine, glutamine, and alanine. The influence of nitrate reduction and the nitrate reductase protein on nitrate transport was investigated in wild-type Neurospora lacking a functional nitrate reductase and in nitrate non-utilizing mutants, nit-1, nit-2, and nit-3. These mycelia contain an inducible nitrate transport system which displays the same characteristics as those found in the wild-type mycelia having the functional nitrate reductase. These findings suggest that nitrate transport is not dependent upon nitrate reduction and that these two processes are separate events in the assimilation of nitrate.     

Hydroxylamine inhibition of the nitrate reductase complex from Amaranthus

Nitrate reductase from Amaranthus viridis is similar to nitrate reductase from other plant sources. NH₂OH inhibits nitrate reduction from NADH by the nitrate reductase complex, but it does not inhibit either the NADH-dehydrogenase activity or nitrate reduction from reduced flavin mononucleotides. The inhibition observed was non-competitive with nitrate when the enzyme was pre-incubated with NH₂OH and NADH, and competitive with nitrate without pre-incubation. The K_i values for NH₂OH were 5 μM and 30 μM with or without pre-incubation respectively.     

Nitrate reductase from Spinacea oleracea. Reversible inactivation by NAD(P)H and by thiols

The enzymatic complex nitrate reductase from Spinacea oleracea is inactivated by NADH or NADPH and by simple thiols. The inactivation affects FNH₂-nitrate reductase but not NADH-diaphorase. Reactivation can be achieved by addition of ferricyanide. The extent of inactivation by dithioerythritol is increased by NAD⁺, but not by NADP⁺. Nitrate protects against inactivation by NADH or NADPH, and abolishes the effect of NAD⁺ on the inactivation by dithioerythritol. The NAD(P)H-inactivation of nitrate reductase requires that the diaphorase moiety of the complex be functional. However, there is no proportionality between NADH-diaphorase or NADH-nitrate reductase activities and the susceptibility of the enzymatic preparation to NADH or NADPH. It seems likely that the nitrate reductase complex contains a specific regulatory site, different from the catalytic site, the reduction of which is accompanied by the production of an inactive form of the complex.     

Nitrate reductase activity as a function of in situ nitrate uptake and environmental factors of euphotic zone profiles

The activity of nitrate reductase in euphotic zone profiles from several oceanic areas has been compared with in situ nitrate uptake rates estimated by the ¹⁵N technique. There were marked variations in the ratio of uptake to reduction and these have been related to the available light intensity, nitrate concentration, and the absolute value of nitrate uptake. It is shown that uptakereduction ratios greater than one are not due to dephasing between the two processes and that accumulation of nitrate inside the cells cannot account for the discrepancy. Nitrate uptake depends primarily on the external nitrate concentration, while nitrate reductase activity seems to be controlled by the intracellular nitrate level. The importance of those results as regards the significance of the nitrate reductase assay as an index of nitrate assimilation by marine phytoplankton is discussed.     

Chloride inhibition of spinach nitrate reductase

Initial rate studies of spinach (Spinacia oleracea L.) nitrate reductase showed that NADH:nitrate reductase activity was ionic strength dependent with elevated ionic concentration resulting in inhibition. In contrast, NADH:ferricyanide reductase was markedly less ionic strength dependent. At pH 7.0, NADH:nitrate reductase activity exhibited changes in the V_max and K_m for NO₃⁻ yielding V_max values of 6.1 and 4.1 micromoles NADH per minute per nanomoles heme and K_m values of 13 and 18 micromolar at ionic strengths of 50 and 200 millimolar, respectively. Control experiments in phosphate buffer (5 millimolar) yielded a single K_m of 93 micromolar. Chloride ions decreased both NADH:nitrate reductase and reduced methyl viologen:nitrate reductase activities, suggesting involvement of the Mo center. Chloride was determined to act as a linear, mixed-type inhibitor with a K_i of 15 millimolar for binding to the native enzyme and 176 millimolar for binding to the enzyme-NO₃⁻ complex. Binding of Cl⁻ to the enzyme-NO₃⁻ complex resulted in an inactive E-S-I complex. Electron paramagnetic resonance spectra showed that chloride altered the observed Mo(V) lineshape, confirming Mo as the site of interaction of chloride with nitrate reductase.     

Metabolite control of squash nitrate reductase

Nitrate reductase (EC 1.6.6.1) catalyzes the pyridine nucleotide-linked reduction of nitrate to nitrite in higher plants. We have shown that in squash (Cucurbita maxima Duchesne var. Buttercup), exogenous nitrate increases nitrate reductase activity by increasing steady-state levels of nitrate reductase protein, while glutamine diminishes nitrate reductase activity both by decreasing steady-state levels of nitrate reductase protein and by decreasing cellular nitrate concentrations in plant cells. Other amino acids affect nitrate reductase similarly to glutamine; other metabolites tested including nitrate did not cause major perturbations in the synthesis of other cellular proteins. Thus, it appears that the effects of nitrate and reduced nitrogen compounds on enzymes of the nitrate assimilatory pathway are highly specific for these enzymes, and have little effect on other cellular proteins.     

The roles of nitrate,photosynthesis and protein turnover in the formation of nitrate reductase in the nit A mutant of Chlamydomonas

The appearance of nitrate reductase activity in derepressed cultures of the Nit A mutant of Chlamydomonas reinhardtii required concomitant photosynthetic CO₂ fixation and was inhibited when protein turnover was prevented. Provided leupeptin was included in the extraction buffer, a single species of nitrate reductase (molecular mass, m = 390 kDa) was extracted from Nit A cultures incubated in nitrate medium for 4 h. Cultures of the mutant incubated in nitrate-free medium contained a number of nitrate reductase species (m = 52–500 kDa). This evidence suggests that nitrate plays a role in the stabilisation of the structure of the mutant nitrate reductase. Only one species of nitrate reductase (m = 188 kDa) was extracted from wild type cultures grown with nitrate.     

Stability of nitrate reductase and source of its reductant in Sorghum seedlings

Pre-incubation of nitrate reductase from Sorghum seedlings with NADH increased enzyme activity by 25%. Ferricyanide had no effect. NADH protected the enzyme from inactivation during storage. Malonate inhibited in vivo nitrate reduction in Sorghum leaves by 95%. The inhibitory effect of malonate was reversed by fumarate. Sodium fluoride in the presence of phosphate also inhibited in vivo nitrate reduction by 60%. It is suggested that NADH generated via the citric acid cycle is utilized for nitrate reduction in Sorghum seedlings.     

In vivo nitrate reduction in relation to nitrate uptake, nitrate content, and in vitro nitrate reductase activity in intact barley seedlings

A study was done to relate the in vivo reduction of nitrate to nitrate uptake, nitrate accumulation, and induction of nitrate reductase activity in intact barley seedlings (Hordeum vulgare L. var. `Numar'). The characteristics of nitrate uptake in response to both time and ambient concentration of nitrate regulated reduction and accumulation. Uptake, accumulation, and in vivo reduction achieved steady state rates in 3 to 4 hours, whereas extractable (in vitro) nitrate reductase activity was still increasing at 12 hours. In vivo reduction of nitrate was better correlated exponentially than linearly over time with in vitro activity of nitrate reductase. A similar relationship occurred over increasing concentration of nitrate in the ambient solution. The results suggest that the rate of in vivo reduction of nitrate in barley seedlings may be regulated by the rate of uptake at the ambient concentrations of nitrate employed in the study.     

Iron assimilation in plants: reduction of a ferriphytosiderophore by NADH:nitrate reductase from squash

NADH:nitrate reductase (EC 1.6.6.1) from squash (Cucurbita maxima Duch., cv. Buttercup) can catalyze the reduction of a ferriphytosiderophore from barley (Hordeum vulgare L. cv. Europa). Maximal activity occurs at pH 6, with an apparentK _m andV _max of 76 M and 21 nmol·min^-1·(mg protein)^-1, respectively. The ferriphytosiderophore strongly inhibits nitrate reduction catalyzed by nitrate reductase at the optimal pH for nitrate reduction, i.e. 7.5. On the contrary, nitrate is a poor inhibitor of ferriphytosiderophore reduction catalyzed by nitrate reductase at the optimal pH for this reaction, pH 6.0. Thus, squash has the potential to assimilate the iron from a ferriphytosiderophore synthesized by another plant.     

Effects of NADH and thiol compounds on wheat leaf nitrate reductase

The initial activity of wheat leaf nitrate reductase was depressed on inclusion of the following thiol compounds; dithiothreitol, dithioerythreitol or mercaptoethanol, but not cysteine and glutathione. This thiol effect simply resulted from an interference with the chemical determination of nitrite. Preincubation of the enzyme with NAD⁺ and these thiols enhanced the inhibition of nitrate reductase activity. This effect was mediated by NADH production by the thiol reduction of NAD⁺. The inactivation by NAD⁺ in the presence of thiol compounds which was enhanced by cyanide ions could be reversed by ferricyanide, as has been observed previously for NADH-mediated inactivation of nitrate reductase.     

On the regulation of spinach nitrate reductase

A coupled assay has been worked out to study spinach (Spinacea oleracea L.) nitrate reductase under low, more physiological concentrations of NADH. In this assay the reduction of nitrate is coupled to the oxidation of malate catalyzed by spinach NAD-malate dehydrogenase. The use of this coupled system allows the assay of nitrate reductase activity at steady-state concentrations of NADH below micromolar. We have used this coupled assay to study the kinetic parameters of spinach nitrate reductase and to reinvestigate the putative regulatory role of adenine nucleotides, inorganic phosphate, amino acids, and calcium and calmodulin.     

Genetic and Biochemical Evidence for the Involvement of a Molybdenum-Dependent Enzyme in One of the Selenite Reduction Pathways of Rhodobacter sphaeroides f. sp. denitrificans IL106

Selenite reduction in Rhodobacter sphaeroides f. sp. denitrificans was observed under photosynthetic conditions, following a 100-h lag period. This adaptation period was suppressed if the medium was inoculated with a culture previously grown in the presence of selenite, suggesting that selenite reduction involves an inducible enzymatic pathway. A transposon library was screened to isolate mutants affected in selenite reduction. Of the eight mutants isolated, two were affected in molybdenum cofactor synthesis. These moaA and mogA mutants showed an increased duration of the lag phase and a decreased rate of selenite reduction. When grown in the presence of tungstate, a well-known molybdenum-dependent enzyme (molybdoenzyme) inhibitor, the wild-type strain displayed the same phenotype. The addition of tungstate in the medium or the inactivation of the molybdocofactor synthesis induced a decrease of 40% in the rate of selenite reduction. These results suggest that several pathways are involved and that one of them involves a molybdoenzyme. Although addition of nitrate or dimethyl sulfoxide (DMSO) to the medium increased the selenite reduction activity of the culture, neither the periplasmic nitrate reductase NAP nor the DMSO reductase is the implicated molybdoenzyme, since the napA and dmsA mutants, with expression of nitrate reductase and DMSO reductase, respectively, eliminated, were not affected by selenite reduction. A role for the biotine sulfoxide reductase, another characterized molybdoenzyme, is unlikely, since its overexpression in a defective strain did not restore the selenite reduction activity.     

Inactivation of nitrate reductase by NADH in Nitrobacter agilis

Nitrate reductase from Nitrobacter agilis was inactivated by NADH (but not by NADPH) in the absence of nitrate.The inactivation of the enzyme by over-reduction with NADH was overcome by oxidizing the reduced enzyme with nitrate, ferricyanide, NAD⁺ or NADP⁺.     

The effect of some ammonium salts on nitrate reductase level,onin vivo nitrate reduction and on nitrate content in excisedpisum sativum roots

The effect of some ammonium salts on nitrate reductase (NR) level, onin vivo nitrate reduction and on nitrate content was followed in the presence of nitrate in the medium, under changing experimental conditions, in excisedPisum sativum roots, and their effect was compared with that of KNO₃, Ca(NO₃)₂ and NaNO₃ at 15 mM NO₃ ^- concentration, i.e. at a concentration which considerably exceeded the level of saturation with nitrate with respect to nitrate reductase. The effect of ammonium salts on NR level is indirect and changes from a positive one to a strongly negative one which is dependent on the time of action of the salt, on the presence of other cations, on pH of the solution of the ammonium salt and on the nature of the anion of the ammonium salt. A positive effect on the enzyme level can be observed in the presence of other cations than NH₄ ⁺ at suitable concentrations of those ammonium salts, the solutions of which have their pH values in the acid region (i.e. NH₄H₂PO₄, (NH₄)₂SO₄ and NH₄NO₃). However their positive effect is independent of the presence of NH₄ ⁺ ions, and it is obviously the result of an increased concentration of H⁺ ions. A clear-cut negative effect on NR level can be observed after 24 h in one-salt NH₄NO₃ solution where NH₄ ⁺ is not balanced with other cations and thus certainly can adversely influence many metabolic processes, and in the solutions containing neutral (pH 6.2) and dibasic ammonium phosphates in which dissolved undissociated ammonia [(NH₃). (H₂O) which can also affect many metabolic processes incl. proteosynthesis] probably has a toxic influence. Thein vivo nitrate reduction is always depressed in excised pea roots in the presence of ammonium salts in the medium, regardless of the level of nitrate reductase. Under the described conditions, no relationship could be established between the enzyme level and the so-called metabolic NO₃ ^- pool (i.e. NO₂ ^- production under anaerobic conditions), nor between NR level and the total nitrate content in the roots. One-salt solutions of NaNO₃, Ca(NO₃)₂ and KNO₃ exert different effects on the level of nitrate reductase and on the content of NO₃ ^- in the roots, but the in vivo NO₃ ^- reduction shows the same trend as NR level in the roots influenced by these salts. Cl^- ions, supplied in NH₄C1, depress both NR level and NO₃ ^- content in the roots at higher concentrations, but they do not significantly affect the in vivo nitrate reduction in comparison with other ammonium salts. These results indicate that NR level,in vivo nitrate reduction, and nitrate uptake can be regulated in pea roots independently of each other.     

The crystal structure of Cupriavidus necator nitrate reductase in oxidized and partially reduced states

The periplasmic nitrate reductase (NapAB) from Cupriavidus necator is a heterodimeric protein that belongs to the dimethyl sulfoxide reductase family of mononuclear Mo-containing enzymes and catalyzes the reduction of nitrate to nitrite. The protein comprises a large catalytic subunit (NapA, 91 kDa) containing the molybdenum active site plus one [4Fe-4S] cluster, as well as a small subunit (NapB, 17 kDa), which is a diheme c-type cytochrome involved in electron transfer. Crystals of the oxidized form of the enzyme diffracted beyond 1.5 Å at the European Synchrotron Radiation Facility. This is the highest resolution reported to date for a nitrate reductase, providing true atomic details of the protein active center, and this showed further evidence on the molybdenum coordination sphere, corroborating previous data on the related Desulfovibrio desulfuricans NapA. The molybdenum atom is bound to a total of six sulfur atoms, with no oxygen ligands or water molecules in the vicinity. In the present work, we were also able to prepare partially reduced crystals that revealed two alternate conformations of the Mo-coordinating cysteine. This crystal form was obtained by soaking dithionite into crystals grown in the presence of the ionic liquid [C₄mim]Cl⁻. In addition, UV-Vis and EPR spectroscopy studies showed that the periplasmic nitrate reductase from C. necator might work at unexpectedly high redox potentials when compared to all periplasmic nitrate reductases studied to date.