Properties of Bromphenol Blue as an Electron Donor for Higher Plant NADH: Nitrate Reductase

Bromphenol blue, which was reduced with dithionite, was found to support nitrate reduction catalyzed by squash NADH:nitrate reductase at a rate about 5 times greater than NADH with freshly prepared enzyme and 10 times or more with enzyme having been frozen and thawed. Kinetic analysis of bromphenol blue as a substrate for squash nitrate reductase yielded apparent K_m values of 60 micromolar for bromphenol blue at 10 millimolar nitrate and 500 micromolar for nitrate at 0.2 millimolar bromphenol blue. With the same preparation of enzyme the apparent K_m values were 9 micromolar for NADH at 10 millimolar nitrate and 50 micromolar nitrate at 0.1 millimolar NADH. Bromphenol blue was found to be a noncompetitive inhibitor versus NADH with a K_i of 0.3 millimolar. When squash NADH:nitrate reductase activity was inactivated with p-hydroxymercuribenzoate or denatured by heating at 40°C, the bromphenol blue nitrate reductase activity was not lost. These results were taken to indicate that bromphenol blue and NADH donated electrons to nitrate reductase at different sites. When monoclonal antibodies prepared against corn and squash nitrate reductases were used to inhibit the nitrate reductase activities supported by NADH, bromphenol blue, and methyl viologen, differential inhibition was found which tended to indicate that the three electron donors were interacting with the enzyme at different sites. One monoclonal antibody prepared against squash nitrate reductase inhibited all three activities of both corn and squash nitrate reductase. It appears this antibody may bind to a highly conserved antigenic site in the nitrate binding region of the enzyme.     

Purification and Characterization of NAD(P)H:Nitrate Reductase and NADH:Nitrate Reductase from Corn Roots

The nitrate reductase activity of 5-day-old whole corn roots was isolated using phosphate buffer. The relatively stable nitrate reductase extract can be separated into three fractions using affinity chromatography on blue-Sepharose. The first fraction, eluted with NADPH, reduces nearly equal amounts of nitrate with either NADPH or NADH. A subsequent elution with NADH yields a nitrate reductase which is more active with NADH as electron donor. Further elution with salt gives a nitrate reductase fraction which is active with both NADH and NADPH, but is more active with NADH. All three nitrate reductase fractions have pH optima of 7.5 and Stokes radii of about 6.0 nanometers. The NADPH-eluted enzyme has a nitrate K_m of 0.3 millimolar in the presence of NADPH, whereas the NADH-eluted enzyme has a nitrate K_m of 0.07 millimolar in the presence of NADH. The NADPH-eluted fraction appears to be similar to the NAD(P)H:nitrate reductase isolated from corn scutellum and the NADH-eluted fraction is similar to the NADH:nitrate reductases isolated from corn leaf and scutellum. The salt-eluted fraction appears to be a mixture of NAD(P)H: and NADH:nitrate reductases.     

Some characteristics of nitrate reductase from higher plants

With respect to cofactor requirements, NADH, and FMNH₂ were equally effective as electron donors for nitrate reductase obtained from leaves of maize, marrow, and spinach, when the cofactors were supplied in optimal concentrations. The concentration of FMNH₂ required to obtain half-maximal activity was from 40- to 100-fold higher than for NADH. For maximal activity with the corn enzyme, 0.8 millimolar FMNH₂ was required. In contrast, NADPH was functional only when supplied with NADP:reductase and exogenous FMN (enzymatic generation of FMNH₂).

All attempts to separate the NADH₂- and FMNH₂-dependent nitrate reductase activities were unsuccessful and regardless of cofactor used equal activities were obtained, if cofactor concentration was optimal. Unity of NADH to FMNH₂ activities were obtained during: A) purification procedures (4 step, 30-fold); B) induction of nitrate reductase in corn seedlings with nitrate; and C) inactivation of nitrate reductase in intact or excised corn seedlings. The NADH- and FMNH₂-dependent activities were not additive.

A half-life for nitrate reductase of approximately 4 hours was estimated from the inactivation studies with excised corn seedlings. Similar half-life values were obtained when seedlings were incubated at 35° in a medium containing nitrate and cycloheximide (to inhibit protein synthesis), or when both nitrate and cycloheximide were omitted.

In those instances where NADH activity but not FMNH₂ activity was lost due to treatment (temperature, removal of sulfhydryl agents, addition of p-chloromercuribenzoate), the loss could be explained by inactivation of the sulfhydryl group (s) required for NADH activity. This was verified by reactivation with exogenous cysteine.

Based on these current findings, and previous work, it is concluded that nitrate reductase is a single moiety with the ability to utilize either NADH or FMNH₂ as cofactor. However the high concentration of FMNH₂ required for optimal activity suggests that in vivo NADH is the electron donor and that nitrate reductase in higher plants should be designated NADH:nitrate reductase (E.C. 1.6.6.1).

     

Characteristics of a Nitrate Reductase in a Barley Mutant Deficient in NADH Nitrate Reductase

A barley (Hordeum vulgare L.) mutant, nar1a (formerly Az12), deficient in NADH nitrate reductase activity is, nevertheless, capable of growth with nitrate as the sole nitrogen source. In an attempt to identify the mechanism(s) of nitrate reduction in the mutant, nitrate reductase from nar1a was characterized to determine whether the residual activity is due to a leaky mutation or to the presence of a second nitrate reductase. The results obtained indicate that the nitrate reductase in nar1a differs from the wild-type enzyme in several important aspects. The pH optima for both the NADH and the NADPH nitrate reductase activities from nar1a were approximately pH 7.7, which is slightly greater than the pH 7.5 optimum for the NADH activity and considerably greater than the pH 6.0 to 6.5 optimum for the NADPH activity of the wild-type enzyme. The nitrate reductase from nar1a exhibits greater NADPH than NADH activity and has apparent K_m values for nitrate and NADH that are approximately 10 times greater than those of the wild-type enzyme. The nar1a nitrate reductase has apparent K_m values of 170 micromolar for NADPH and 110 micromolar for NADH. NADPH, but not NADH, inhibited the enzyme at concentrations greater than 50 micromolar.     

Different effects of 2-n-heptyl-4-hydroxyquinoline-N-oxide on oxygen and nitrate respiration in Klebsiella aerogenes

The inhibition of the oxidase and respiratory nitrate reductase activity in membrane preparations from Klebsiella aerogenes by 2-n-heptyl-4-hydroxyquinoline-N-oxide (HQNO) has been investigated. Addition of HQNO only slightly affected the aerobic steady-state reduction of cytochrome b₅₅₉ with NADH, but caused a significantly lower nitrate reducing steady-state of this cytochrome. The changes in the redox states of the cytochromes during a slow transition from anaerobic to aerobic conditions in the presence and absence of HQNO showed that the inhibition site of HQNO is located before cytochrome d. Inhibition patterns obtained upon titration of the NADH oxidase and NADH nitrate reductase activity with HQNO indicated one site of inhibitor interaction in the NADH nitrate reductase pathway and suggested a multilocated inhibition of the NADH oxidase pathway. Difference spectra with ascorbate-dichlorophenolindophenol as electron donor indicated the presence of a cytochrome b₅₆₃ component which was not oxidized by nitrate, but was rapidly oxidized by oxygen. The latter oxidation was prevented by HQNO. A scheme for the electron transport to oxygen and nitrate is presented. In the pathway to oxygen, HQNO inhibits both at the electron-accepting side of cytochrome b₅₅₉ and at the electron-donating side of cytochrome b₅₆₃, whereas in the pathway to nitrate, inhibition occurs only at the electron-accepting side of cytochrome b₅₅₉.     

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 Reductases from Wild-Type and nr(1)-Mutant Soybean (Glycine max [L.] Merr.) Leaves : I. Purification, Kinetics, and Physical Properties

NADH:nitrate reductase (EC 1.6.6.1) and NAD(P)H:nitrate reductase (EC 1.6.6.2) were purified from wild-type soybean (Glycine max [L.] Merr., cv Williams) and nr₁-mutant soybean plants. Purification included Blue Sepharose- and hydroxylapatite-column chromatography using acetone powders from fully expanded unifoliolate leaves as the enzyme source.

Two forms of constitutive nitrate reductase were sequentially eluted with NADPH and NADH from Blue Sepharose loaded with extract from wild-type plants grown on urea as sole nitrogen source. The form eluted with NADPH was designated c₁NR, and the form eluted with NADH was designated c₂NR. Nitrate-grown nr₁ mutant soybean plants yielded a NADH:nitrate reductase (designated iNR) when Blue Sepharose columns were eluted with NADH; NADPH failed to elute any NR form from Blue Sepharose loaded with this extract. Both c₁NR and c₂NR had similar pH optima of 6.5, sedimentation behavior (s_20,w of 5.5-6.0), and electrophoretic mobility. However, c₁NR was more active with NADPH than with NADH, while c₂NR preferred NADH as electron donor. Apparent Michaelis constants for nitrate were 5 millimolar (c₁NR) and 0.19 millimolar (c₂NR). The iNR from the mutant had a pH optimum of 7.5, s_20,w of 7.6, and was less mobile on polyacrylamide gels than c₁NR and c₂NR. The iNR preferred NADH over NADPH and had an apparent Michaelis constant of 0.13 millimolar for nitrate.

Thus, wild-type soybean contains two forms of constitutive nitrate reductase, both differing in their physical properties from nitrate reductases common in higher plants. The inducible nitrate reductase form present in soybeans, however, appears to be similar to most substrateinduced nitrate reductases found in higher plants.

     

In vivo assay of nitrate reductase in cotton leaf discs: effect of oxygen and ammonium

Factors affecting nitrate reduction by leaf discs of cotton (Gossypium hirsutum L.) were investigated. When incubated in 30 mm nitrate, discs reduced nitrate much more slowly under air or O₂ than under N₂. Inhibition by O₂ did not occur at nitrate levels of 100 mm or greater. Treatment with arsenate had little effect under N₂ but stimulated nitrate reduction under air. Similarly, ammonium inhibited nitrate reduction, with the inhibition being partially relieved by arsenate. Uptake of nitrate was unaffected by ammonium. The NAD/NADH ratio increased in response to both oxygen and ammonium. The effects of these treatments on nitrate reduction can be explained by competition with nitrate for NADH generated by glycolysis.     

Specificity for nicotinamide adenine dinucleotide by nitrate reductase from leaves

Preliminary work revealed that nitrate reductase in crude extracts prepared from leaves of certain corn genotypes as well as soybeans could utilize NADPH as well as NADH as the electron donor. Isoelectric focusing and diethylaminoethyl cellulose chromatography confirmed previous findings that NADH and NADPH activities could not be separated, which suggests the involvement of a single enzyme. Nitrate reduction with both cofactors varies with plant species, plant age, and assay conditions. The ability of the nitrate reductase from a given genotype to utilize NADPH was associated with the amount of NADPH-phosphatase in the extract. While diethylaminoethyl cellulose chromatography of plant extracts separated nitrate reductase from the bulk (90%) of the phosphatase and caused a decrease in the NADPH activity, the residual level of phosphatase was sufficient to account for the apparent NADPH nitrate reductase activity. Addition of KH₂PO₄ and KF, inhibitors of NADPH-phosphatase activity in in vitro assays, caused a drastic reduction or abolishment of NADPH-mediated nitrate reductase activity but were without effect on NADH nitrate reductase activity. It is concluded that NADPH-nitrate reduction, in soybean and certain corn genotypes, is an artifact resulting from the conversion of NADPH to NADH by a phosphatase and that the enzyme in leaf tissue is NADH-dependent (E.C.1.6.6.1).     

Mode of action of natural inactivator proteins from corn and rice on a purified assimilatory nitrate reductase

The molecular basis for the action of two natural inactivator proteins, isolated from rice and corn, on a purified assimilatory nitrate reductase has been examined by several physical techniques. Incubation of purified Chlorella nitrate reductase with either rice inactivator protein or corn inactivator protein results in a loss of NADH:nitrate reductase and the associated partial activity, NADH:cytochrome c reductase, but no loss in nitrate-reducing activity with reduced methyl viologen as the electron donor. The molecular weight of the reduced methyl viologen:nitrate reductase species, determined by sedimentation equilibrium in the Beckman airfuge after complete inactivation with rice inactivator protein or with corn inactivator protein, was 595,000 and 283,000, respectively, compared to a molecular weight of 376,000 for the untreated control determined under the same conditions. Two protein peaks were observed after molecular-sieve chromatography on Sephacryl S-300 of nitrate reductase inactivated by corn inactivator protein. The Stokes radii of these fragments were 68 and 24 Å, compared to a value of 81 Å for untreated nitrate reductase. The large fragment contained molybdenum and heme but no flavin, and had nitrate-reducing activity with reduced methyl viologen as electron donor. The small fragment contained FAD but had no NADH:cytochrome c reductase or nitrate-reducing activities. Molecular weights determined by sodium dodecyl sulfate-gel electrophoresis were 67,000 and 28,000 for the large and small fragments, respectively, compared to a subunit molecular weight of 99,000 determined for the untreated control. No change in subunit molecular weight of nitrate reductase after inactivation by rice inactivator protein was observed. These results indicate that rice inactivator protein acts by binding to nitrate reductase. The stoichiometry of binding is 1–2 molecules of rice inactivator protein to one tetrameric molecule of nitrate reductase. Corn inactivator protein, in contrast, acts by cleavage of a M_r 30,000 fragment from nitrate reductase which is associated with FAD. The remaining fragment is a tetramer of M_r 70,000 subunits which retains nitrate-reducing activity and contains molybdenum and heme but has no NADH:dehydrogenase activity. The action of rice inactivator protein was partially prevented by NADH and completely prevented by a combination of NADH and cyanide, while the action of corn inactivator protein was not significantly affected by these effectors.     

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.     

The participation of cytochromes in the process of nitrate respiration in Klebsiella (Aerobacter) aerogenes

1. The participation of cytochromes in the membrane-bound, nitrate and oxygen respiratory systems of Klebsiella (Aerobacter) aerogenes has been investigated. The membrane preparations contained the NADH, succinate, lactate and formate oxidase systems, and in addition a high respiratory nitrate reductase activity.2. Difference spectra indicated the presence of cytochromes b, a₁, d, and o. Cytochromes of the c-type could not be detected in these membranes. Both cytochrome b content and respiratory nitrate reductase activity were the highest in bacteria grown anaerobically in the presence of nitrate.3. Cytochrome b was the only cytochrome which, after being reduced by NADH, could be partially reoxidized anaerobically in the presence of nitrate. Furthermore, nitrate caused a lower aerobic steady state reduction only of cytochrome b.4. NADH oxidase and NADH-linked respiratory nitrate reductase activities were both inhibited by antimycin A, 2-n-heptyl-4-hydroxyquinoline-N-oxide and KCN. NADH oxidase activity was selectively inhibited by CO, while azide was found to inhibit only the respiratory nitrate reductase. In the presence of azide, nitrate did not affect the level of reduction of cytochrome b.5. The evidence presented suggests that cytochrome b is a carrier in the electron transport systems to both nitrate and oxygen; from cytochrome b branching occurs, with one branch linked to the respiratory nitrate reductase and one branch linked to oxidase systems, containing the cytochromes a₁, d and o.     

Determination of the Apparent Km of Nitrate Reductase from Spinach Leaves for NADH by a Coupled Assay

Using a novel coupled enzyme activity assay, with a partially purified preparation of spinach leaf nitrate reductase, the apparent K_m for NADH was determined as 1.4 μM. These measurements were carried out in the presence of 0.5 mM NAD, which is within the physiological range found in the cytosol of a leaf cell. The results show that an NADH/NAD ratio of 3 × 10^?3 is sufficient for a half maximal rate of nitrate reductase.     

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.     

NADH-Linked oxidative phosphorylation inNitrobacter agilis

A particulate cell-free fraction (144,000-X-g pellet) fromNitrobacter agilis catalyzes the acrobic or anaerobic oxidation of NADH. Phosphorylation coupled to the aerobic oxidation of NADH yields P/O ratios of 1.1. The net formation of ATP coupled to the anaerobic oxidation of NADH by nitrate yields P/NO₃ ratios of 0.7. Phosphate esterification is uncoupled by carbonylcyanide-m-chlorophenyl-hydrozone and is sensitive to inhibitors of the electron transport system.     

UV-B Radiation Mediated Alterations in the Nitrate Assimilation Pathway of Crop Plants 1. Kinetic Characteristics of Nitrate Reductase

The kinetics and other characteristics of nitrate reductase (NR, EC 1.6.6.1) in cowpea [Vigna unguiculata (L.) Walp.] seedlings irradiated with biologically effective UV-B radiation (280-320 nm, 3.2 W m^-2 s^-1) were recorded. The in vivo and in vitro NR activities were inhibited by 34 and 41 % under UV-B treatment, respectively. Both V_max and K_m for the substrate were enhanced by UV-B radiation. The K_m for nitrate increased from 1.2 to 1.7 mM after the UV-B irradiation. The change in K_m for NADH was from 0.12 to 0.17 mM. The increases in K_m indicate that UV-B radiation seriously changes the topology of NR, particularly with respect to the nitrate and NADH binding sites. The rate of NR turnover indicates the extent of damage inflicted by UV-B radiation on the nitrate metabolism. The half-life (t_1/2) of NR was reduced from 7 to 4 h in the UV-B treated seedlings. UV-B also inhibited the kinetics of nitrate uptake by plants: its K_m increased from 0.08 to 0.12 mM. This revised version was published online in June 2006 with corrections to the Cover Date.     

Oxygen Inhibition of Nitrate Reductase Biosynthesis in Detached Corn Leaves via Inhibition of Total Soluble Protein Synthesis

Detached first leaves of 3-day-old corn seedlings (Zea mays L. W64AxW183E) were incubated with nitrate in air or 100% O₂ in the light. Nitrate accumulation in the leaves was not depressed by O₂. NADH:nitrate reductase activity and enzyme protein, as measured with an enzyme-linked immunosorbent assay, increased in parallel during the 8 h nitrate treatment in air, but in O₂ the levels of enzyme activity and protein were depressed. NADH:nitrate reductase mRNA levels were the same in the air-and O₂-treated leaves. Total soluble protein levels in leaves were slightly depressed by O₂ and shifting from O₂ to an air environment increased the protein level. Incorporation of [³⁵S]methionine during nitrate treatment revealed that total soluble protein and nitrate reductase protein synthesis were both depressed by the O₂ environment relative to air, but both recovered when leaves were shifted from O₂ to air. Although O₂ accelerated inactivation of nitrate reductase in vitro, the in vivo inactivation rate appeared to be too low to account for the depressed level of nitrate reductase activity in O₂-treated leaves. We concluded that O₂ inhibition of nitrate reductase biosynthesis in detached corn leaves was largely due to inhibition of total soluble protein synthesis at the level of translation.     

Optimized nitrate reductase assay predicts the rate of nitrate utilization in the halotolerant microalga Dunaliella viridis

An in situ method for measuring nitrate reductase (NR) activity in Dunaliella viridis was optimized in terms of incubation time, concentration of KNO₃, permeabilisers (1-propanol and toluene), pH, salinity, and reducing power (glucose and NADH). NR activity was measured by following nitrite production and was best assayed with 50 mM KNO₃, 1.2 mM NADH, 5% 1-propanol (v/v), at pH 8.5. The estimated half-saturation constant (K_s) for KNO₃ was 5 mM. Glucose had no effect as external reducing power source, and NADH concentrations >1.2 mM inhibited NR activity. Nitrite production was linear up to 20 min; longer incubation did not lead to higher nitrate reduction. The use of the optimized assay predicted the rate of NO ₃ ⁻ removal from the external medium by D. viridis with high degree of precision. This revised version was published online in September 2006 with corrections to the Cover Date.     

Spinach Nitrate Reductase : Effects of Ionic Strength and pH on the Full and Partial Enzyme Activities

Initial velocity studies of immunopurified spinach nitrate reductase have been performed under conditions of controlled ionic strength and pH and in the absence of chloride ions. Increased ionic strength stimulated NADH:ferricyanide reductase and reduced flavin:nitrate reductase activities and inhibited NADH:nitrate reductase, NADH:cytochrome c reductase and reduced methyl viologen:nitrate reductase activities. NADH:dichlorophenolindophenol reductase activity was unaffected by changes in ionic strength. All of the partial activities, expressed in terms of micromole 2 electron transferred per minute per nanomole heme, were faster than the overall full, NADH:nitrate reductase activity indicating that none of the partial activities included the rate limiting step in electron transfer from NADH to nitrate. The pH optimum for NADH:nitrate reductase activity was determined to be 7 while values for the various partial activities ranged from 6.5 to 7.5. Chlorate, bromate, and iodate were determined to be alternate electron acceptors for the reduced enzyme. These results indicate that unlike the enzyme from Chlorella vulgaris, intramolecular electron transfer between reduced heme and Mo is not rate limiting for spinach nitrate reductase.     

Regulation of nitrate reduction in wheat leaves

Wheat leaves exposed to 710 nm monochromatic light, when only photosystem 1 operates, reduced small but significant amount of nitrate to nitrite. This could be due to partial inhibition of mitochondrial oxidation of NADH, brought about by cyclic photo-phosphorylation. Under dark aerobic conditions, citric acid cycle intermediates only slightly stimulated nitrate reduction. Under dark anaerobic conditions, when maximum reduction of nitrate occurred, the time course showed a 1:1 stoichiometry between nitrite and CO₂. It is suggested that for maximum reduction of nitrate under physiological conditions, CO₂ fixation and export of ATP via triose phosphate shuttle is essential.