Reduced Methyl Viologen-nitrate Reductase Research Articles

Occurrence of Nitrate Reductase and Molybdopterin in Xanthomonas maltophilia

Fifteen of 23 ATCC strains and 2 of 9 clinical isolates of Xanthomonas maltophilia, all of which grew aerobically on ammonia, but not nitrate, as a sole nitrogen source, reduced nitrate to nitrite. X. maltophilia failed to grow anaerobically on complex medium with or without nitrate, so it is considered an obligate aerobe. Nitrate-reducing strains contained reduced methyl viologen nitrate reductase (MVH-NR) with specific activities ranging from 49.2 to 192 U mg of protein. Strain ATCC 17666 doubled its cell mass after 3 h of growth on nitrate broth under low aeration, possessed maximal MVH-NR activity, and converted the added nitrate to nitrite, which accumulated. Dissolved oxygen above 15% saturation greatly suppressed nitrite formation. All strains, except ATCC 14535, possessed between 0.25 and 5.05 pmol of molybdopterin mg of protein as measured by the Neurospora crassa nit-1 assay. The molybdopterin activity in the soluble fraction sedimented as a single symmetrical peak with an s(20,w) of 5.1. Studies identified MVH-NR in selected strains as a membrane-bound protein. The deoxycholate-solubilized MVH-NR sedimented as a single peak in sucrose density gradients with an s(20,w) of 8.8. The MVH-NR of X. maltophilia has the physical characteristics of a respiratory nitrate reductase and may enable cells to use nitrate as an electron sink under semiaerobic conditions.

Limited proteolysis of the nitrate reductase from spinach leaves.

The functional structure of assimilatory NADH-nitrate reductase from spinach leaves was studied by limited proteolysis experiments. After incubation of purified nitrate reductase with trypsin, two stable products of 59 and 45 kDa were observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The fragment of 45 kDa was purified by Blue Sepharose chromatography. NADH-ferricyanide reductase and NADH-cytochrome c reductase activities were associated with this 45-kDa fragment which contains FAD, heme, and NADH binding fragment. After incubation of purified nitrate reductase with Staphylococcus aureus V8 protease, two major peaks were observed by high performance liquid chromatography size exclusion gel filtration. FMNH2-nitrate reductase and reduced methyl viologen-nitrate reductase activities were associated with the first peak of 170 kDa which consists of two noncovalently associated (75-90-kDa) fragments. NADH-ferricyanide reductase activity, however, was associated with the second peak which consisted of FAD and NADH binding sites. Incubation of the 45-kDa fragment with S. aureus V8 protease produced two major fragments of 28 and 14 kDa which contained FAD and heme, respectively. These results indicate that the molybdenum, heme, and FAD components of spinach nitrate reductase are contained in distinct domains which are covalently linked by exposed hinge regions. The molybdenum domain appears to be important in the maintenance of subunit interactions in the enzyme complex.

NADH‐dependent nitrate reductase from two‐row barley roots: Purification, characteristics and comparison with leaf enzyme

NADH‐nitrate reductase (EC 1.6.6.1) was purified 800‐fold from roots of two‐row barley (Hordeum vulgare L. cv. Daisen‐gold) by a combination of Blue Sepharose and zinc‐chelate affinity chromatographies followed by gel filtration on TSK‐gel (G3000SW). The specific activity of the purified enzyme was 6.2 μmol nitrite produced (mg protein)−1 min−1 at 30°C.Besides the reduction of nitrate by NADH, the root enzyme, like leaf nitrate reductase, also catalyzed the partial activities NADH‐cytochrome c reductase, NADH‐ferricyanide reductase, reduced methyl viologen nitrate reductase and FMNH2‐nitrate reductase. Its molecular weight was estimated to be about 200 kDa, which is somewhat smaller than that for the leaf enzyme. A comparison of root and leaf nitrate reductases shows physiologically similar or identical properties with respect to pH optimum, requirements of electron donor, acceptor, and FAD, apparent Km for nitrate, NADH and FAD, pH tolerance, thermal stability and response to inorganic orthophosphate. Phosphate activated root nitrate reductase at high concentration of nitrate, but was inhibitory at low concentrations, resulting in increases in apparent Km for nitrate as well as Vmax whereas it did not alter the Km for NADH.

NADH-nitrate reductase in barley leaves: Identification and amino acid composition of subunit protein

NADH-nitrate reductase (NADH:nitrate oxidoreductase, EC 1.6.6.1) isolated from barley ( Hordeum vulgate L. cv. Steptoe) leaves was identified on a native slab gel after electrophoresis by staining for reduced methyl viologen nitrate reductase activity, NO 2 − production and diaphorase activity. The nitrate reductase protein, when subjected to a second-dimensional SDS-polyacrylamide gel electrophoresis, migrated as a single protein band of M r 110000. Thus, barley nitrate reductase (native molecular weight 221000) consists of two identical subunits. Preparative SDS-polyacrylamide gel electrophoresis was used to isolated highly homogeneous nitrate reductase subunit protein. Fluorograms of peptide maps indicated no homology among the nitrate reductase subunit and other polypeptides (60 kDa diaphorase, 41 kDa, 35 kDa and 25 kDa species) which are common contaminants of the affinity-purified nitrate reductase. Thus, the 60 kDa diaphorase and 41 kDa polypeptide observed under our experimental conditions are not breakdown products of the 110 kDa nitrate reductase subunit. The amino acid composition of the barley nitrate reductase subunit was similar to that reported for the nitrate reductase of Chlorella vulgaris and Ankistrodesmus braunii.

Isoelectric focusing and two-dimensional analysis of purified nitrate reductase from Aspergillus nidulans

The assimilatory NADPH-nitrate oxidoreductase (EC 1.6.6.3) from Aspergillus nidulans was purified by means of affinity chromatography and analyzed by agarose isoelectric focusing and two-dimensional electrophoresis. NADPH-nitrate reductase activity was not activated by oxidation with potassium ferricyanide and was irreversibly inhibited by acrylamide. Electrophoresis of nitrate reductase in 7% polyacrylamide gels resulted in rapid loss of enzyme activity. Isoelectric focusing of purified enzyme in agarose gels resulted in one homogeneous band that exhibited NADPH-nitrate reductase, NADPH-cytochrome c reductase and reduced methyl viologen-nitrate reductase activities, which corresponded to an isoelectric point of 6.12 ± 0.05 at 22°C. Two-dimensional electrophoresis of focused nitrate reductase on SDS-polyacrylanide gel slabs yielded a single subunit of 54000 molecular weight. Acid treatment of the enzyme and subsequent isoelectric focusing resulted in a protein with a strongly acidic isoelectric point and reduced methyl viologen-nitrate reductase activity. It released another protein with a strongly basic isoelectric point which was inactive. It is postulated that the overall association of fiavoprotein protomers with both heme and cytochrome b 1 components confers a small net negative charge upon the native heteromultimer and accounts for its slightly acidic isoelectric point.

Purification and characterization of the assimilatory NADPH-nitrate reductase of Aspergillus nidulans.

NADPH-nitrate reductase [NADPH : nitrate oxidoreductase, EC 1.6.6.3] was purified 500-fold from Aspergillus nidulans with an overall yield of about 20%. The purified enzyme catalyzed NADPH-nitrate, NADPH-cytochrome c, FADH2-nitrate and reduced methyl viologen-nitrate reductase activities. Its molecular weight was estimated to be 180,000 from the Stokes radius and sedimentation coefficient. The oxidized enzyme exhibited an absorption spectrum having a peak at 412 nm and a broad shoulder at about 450 nm. When reduced with NADPH, absorption peaks appeared at 423 (Soret), 527 (beta) and 557 (alpha) nm, and absorption in the 450 nm region decreased. Upon treatment of the reduced enzyme with KNO3, the spectrum returned to that of the oxidized enzyme. The presence of protoheme in the enzyme was confirmed by the absorption spectrum of reduced pyridine hemochromogen. It was concluded that a b-type cytochrome ("cytochrome b-557") is present in the enzyme and is involved in the intramolecular electron transport from NADPH to nitrate. The NADPH-nitrate and NADPH-cytochrome c reductase activities, but not the other two activities, were significantly decreased by incubation of the enzyme at 37 degrees C in the absence of FAD. Analysis by SDS slab gel electrophoresis suggested that the nitrate reductase consists of two each of two subunits of 59,000 and 38,000 daltons and that a dissociation of 38,000 subunits from the native enzyme occurs during heat treatment, resulting in alteration of the catalytic activity.

Repression of nitrate reductase activity and loss of antigenically detectable protein in Neurospora crassa.

Experiments were performed to determine whether conditions which cause the rapid loss of nitrate reductase activity in Neurospora crassa mycelia were accompanied by the loss of antigenically detectable nitrate reductase protein. When mycelia with nitrate reductase activity were transferred to ammonia media, there was a rapid loss in the reduced nicotinamide adenine dinucleotide-nitrate reductase activity plus the parallel loss of the reduced nicotinamide adenine dinucleotide-diaphorase and the reduced methyl viologen-nitrate reductase activities associated with the nitrate reductase. In addition, there was the loss of cross-reacting material to anti-nitrate reductase antisera that was concomitant with the loss of nitrate reductase activity. When mycelia were exposed to either ammonia plus cycloheximide, nitrate plus cycloheximide, or nitrogen-free media, or to media which lacked an assimilable carbon source, the amount of cross-reacting material declined in concert with the nitrate reductase activity. The mutant nit-6, which lacks nitrite reductase activity, was exposed to ammonia or nitrate plus cycloheximide media. The nitrate reductase and the amount of cross-reacting material declined together as in the wild-type mycelia. We conclude that the loss of nitrate reductase activity was accompanied by the specific loss of this protein and that no pool of inactivated nitrate reductase molecules existed.

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.

Action of Corn and Rice-inactivating Proteins on a Purified Nitrate Reductase from Chlorella vulgaris

When nitrate reductase (NR) purified from Chlorella was incubated with NR-inactivating proteins purified from corn roots and rice cell suspension cultures or with trypsin there was a loss in NADH-NR and NADH cytochrome c reductase (NADH-CR) activities with time whereas the reduced methylviologen NR (MV-NR) remained active. When NADH-NR and NADH-CR activities were inactivated completely by the incubation with corn protein, the major protein band obtained by polyacrylamide gel electrophoresis shifted from an R(F) value of 0.12 to an R(F) of 0.25 and reduced MV-NR activity moved to the new position on the gel. When NADH-NR and NADH-CR activities were partially inactivated by the corn protein, NADH-NR activity was detected in an intermediate position (R(F) value of 0.18). Incubation with trypsin also caused a change in the NR protein migration pattern (R(F) value of 0.20). This protein band also had reduced MV-NR activity. Thus, the corn inactivator degrades NR in a fashion similar to but not identical with trypsin. The incubation of NR with rice inactivating protein resulted in a loss of NADH-NR but had no effect on the migration of NR protein or on the reduced MV-NR activity or mobility suggesting that the rice protein binds to Chlorella NR.

Further characterization of the reduced nicotinamide adenine dinucleotide phosphate: nitrate oxidoreductase in Aspergillus nidulans.

The reduced nicotinamide adenine dinucleotide phosphate (NADPH):nitrate oxidoreductase (EC 1.6.6.2) from Aspergillus nidulans wild-type bi-1 was purified by means of salt fractionation, gel filtration, affinity chromatography, and polyacrylamide gel electrophoresis. Enzyme which was adsorbed on Cibacron blue agarose could be eluted with 2 mM NADPH or 2 mM oxidized NADP (NADP(+)), the former being about three times more effective than the latter. About half the total NADPH:nitrate reductase activity adsorbed on agarose required elution with 1 M NaCl. This salt-elutable form remained active with NADPH and was not converted to the NADPH-elutable form after readsorption on Cibacron blue agarose. The NADPH-eluted enzyme exhibited a markedly different electrophoretic mobility than the enzyme eluted with NADP(+) or NaCl. After electrophoresis on polyacrylamide gels, the NADPH-eluted NADPH:nitrate reductase was separated into four proteins, two of which contained nonheme iron and exhibited reduced methyl viologen-nitrate reductase activity. None of these proteins, singly or in combination, reduced nitrate with NADPH as substrate. Difference spectra analyses and specific heme iron stains revealed the presence of cytochrome b(557) in the largest of the proteins. The molecular weights of the four proteins, which were determined from the relationship of their mobilities on varied concentrations of acrylamide gel, were 360,000, 300,000, 240,000, and 118,000. The subunit molecular weights of these, which are determined via sodium dodecyl sulfate slab gel electrophoresis, were 49,000, 50,000, and 75,000. The key role of NADPH in maintenance of the active form of the heteromultimer is further substantiated.

Purification and characterization of homogeneous assimilatory reduced nicotinamide adenine dinucleotide phosphate-nitrate reductase from Neurospora crassa

Neurospora crassa wild type STA4 NADPH-nitrate reductase (NADPH : nitrate oxidoreductase, EC 1.6.6.3) has been purified 5000-fold with an overall yield of 25–50%. The final purified enzyme contained 4 associated enzymatic activities: NADPH-nitrate reductase, FADH 2-nitrate reductase, reduced methyl viologen-nitrate reductase and NADPH-cytochrome c reductase. Polyacrylamide gel electrophoresis yielded 1 major and 1 minor protein band and both bands exhibited NADPH-nitrate and reduced methyl viologen-nitrate reductase activities. SDS gel electrophoresis yielded 2 protein bands corresponding to molecular weights of 115 000 and 130 000. A single N- terminal amino acid (glutamic acid) was found and proteolytic mapping for the two separated subunits appeared similar. Purified NADPH-nitrate reductase contained 1 mol of molybdenum and 2 mol of cytochrome b 557 per mol protein. Non-heme iron, zinc and copper were not detectable. It is proposed that the Neurospora assimilatory NADPH-nitrate reductase consists of 2 similar cytochrome b 557- containing 4.5-S subunits linked together by one molybdenum cofactor. A revised electron flow scheme is presented. p- Hydroxymercuribenzoate inhibition was reversed by sulfhydryl reagents. Inhibitory pattern of p- hydroxymercuribenzoate and phenylglyoxal revealed accessible sulfhydryl and arginyl residue(s) as functional group(s) in the earlier part of electron transport chain as possibly the binding site of NADPH or FAD.

Nitrate reductase-deficient mutants in barley

MUTANTS have long been used to study genetic and biochemical regulation of metabolic pathways in microorganisms. Often it is a relatively simple task to screen large bacterial populations for an individual lacking a specific enzyme or enzyme product. Defined enzyme mutants would also be valuable research tools for plant geneticists and biochemists. Unfortunately, very few biochemically defined mutants have been selected in higher plants, principally because of the genetic and biochemical complexity, and the long generation time of higher plants1. But, these obstacles could be overcome in plants by inducing high mutation frequencies and developing effective techniques for selecting biochemically defined mutants. High mutation frequencies can be readily induced in some higher plant species by sodium azide, a potent base substitution mutagen in barley2,3. Nitrate reductase, which has an important function in higher plants4, has been extensively studied in bacteria and fungi5, is highly regulated5,6, and has several catalytic activities including NAD(P)H, FMNH2 and reduced methyl viologen nitrate reductase activities, and NAD(P)H cytochrome c reductase activity6. Nitrate reductase is therefore an excellent enzyme in which defined mutants in higher plants can be selected, studied, and compared with microorganisms. We describe here a procedure for the induction and selection of nitrate reductase-deficient mutants in higher plants, and the partial characterisation of three nitrate reductase-deficient mutants in barley.

Localization and solubilization of the respiratory nitrate reductase of Bacillus stearothermophilus.

Membranes were isolated from Bacillus stearothermophilus 2184D by lysozyme digestion of the cell wall and subsequent differential centrifugation. Observations with the electron microscope indicate that such membranes are relatively intact and have a typical membrane appearance. Nitrate will preferentially oxidize the cytochrome b of such membranes. Approximately 80% of the total respiratory nitrate reductase activity of whole cells can be localized in the washed membrane fraction and the process of membrane isolation results in a sixfold purification of this enzyme. Of several detergents tested, sodium dodecyl sulfate, Triton 114, and Triton X-100 are most effective in converting reduced methyl viologen-nitrate reductase to a form which will not pellet at 130,000 x g. Density gradient analysis reveals that such detergent-mediated solubilization converts virtually all membrane protein to a form of lighter density.

Invitro formation of assimilatory reduced nicotinamide adenine dinucleotide phosphate: nitrate reductase from a Neurospora mutant and a component of molybdenum-enzymes.

An active Neurospora-like assimilatory NADPH-nitrate reductase (EC 1.6.6.2), which can be formed in vitro by incubation of extracts of nitrate-induced Neurospora crassa mutant nit-1 with extracts of (a) certain other nonallelic nitrate reductase mutants, (b) uninduced wild type, or (c) xanthine oxidizing and liver aldehyde-oxidase systems was also formed by combination of the nit-1 extract with other acid-treated enzymes known to contain molybdenum. These molybdenum enzymes included (a) nitrogenase, or its molybdenum-iron protein, from Clostridium, Azotobacter, and soybeannodule bacteroids, (b) bovine liver sulfite oxidase, (c) respiratory formate-nitrate reductase from Escherichia coli, (d) NADH-nitrate reductase from foxtail grass (Setaria faberii), and (e) FADH(2)- and reduced methyl viologennitrate reductase preparations from certain Neurospora mutants. Several molybdenum-amino-acid complexes, as possible catalytic models of nitrogenase, were inactive (as were some previously tested 20 nonmolybdenum enzymes) in place of the acid-treated molybdenum-containing enzymes. The results imply the existence of a molybdenum-containing component shared by the known molybdenum-enzymes.

Formation of assimilatory nitrate reductase by in vitro inter-cistronic complementation in Neurospora crassa.

In vitro complementation of the soluble assimilatory nicotinamide adenine dinucleotide phosphate, reduced form (NADPH)-nitrate reductase was attained by mixing cell-free preparations of certain Neurospora nitrate reductase mutants: induced nit-1 (uniquely possessing inducible NADPH-cytochrome c reductase) with (a) uninduced or induced nit-2 or nit-3, or (b) uninduced wild type. The complementing activity of induced nit-1 is soluble while that of nit-2, nit-3, and wild type is particulate but not of mitochondrial origin. All fractions are inactivated by heat or trypsin. The NADPH-nitrate reductase enzymes formed in the above three complementing mixtures are similar to the wild-type enzyme in sucrose density gradient profiles, molecular weight, substrate affinity, sensitivity to inhibitors and temperature, but show different ratios of associated enzyme activities. The data suggest that nitrate reductase consists of at least two protein subunits: a nitrate-inductible subunit as reflected by inductible NADPH-cytochrome c reductase, and a constitutive protein which is activated (as indicated by the appearance of flavine adenine dinucleotide, reduced form (FADH(2))- and reduced methyl viologen-nitrate reductase activities) when it combines with the inductible subunit.

Further Purification and Properties of Neurospora Nitrate Reductase

Abstract Neurospora crassa (5297a) NADPH-nitrate reductase (NADPH:nitrate oxidoreductase, EC 1.6.6.2), a soluble, sulfhydryl-containing protein with FAD, a cytochrome b designated cytochrome b557 (N. crassa), and molybdenum as prosthetic groups, has been purified 500-fold by procedures including pH adjustment, ammonium sulfate fractionation of the resultant supernatant solution, phase separation to remove nucleic acids, diethylaminoethyl cellulose chromatography, hydroxylapatite chromatography, and, finally, Sephadex G-200 gel filtration. Other enzymatic activities associated with NADPH-nitrate reductase throughout the purification and maintaining a proportional relationship with it are NADPH-cytochrome c reductase, FADH2-nitrate reductase, and reduced methyl viologen-nitrate reductase. Polyacrylamide gel electrophoresis of the most highly purified enzyme preparations yielded a major buffalo black-staining zone containing the above enzymatic activities, a somewhat smaller zone, and several faint zones. In unstained gels, a red zone, which was apparently due to the presence of cytochrome b557, was visible at the position corresponding to the major buffalo black-staining zone. By the use of sucrose density gradient centrifugation, a relative s20, w0.725 value of 8.0 for the nitrate reductase was found, and with Sephadex G-200 gel filtration techniques, a Stokes radius of 70 A was determined. From the relationship, mol wt = 6πeNas/(1 - vp), a molecular weight of 228,000 was calculated, assuming v = 0.725 cc per g. All enzymatic activities associated with nitrate reductase are heat-labile but to varying degrees, with the NADPH-nitrate and -cytochrome c reductases being most sensitive, and FADH2- and reduced methyl viologen-nitrate reductase activities being progressively less so, in that order. The reduced methyl viologen-nitrate reductase activity showed a marked increase in activity as the NADPH activities declined. The cytochrome b557 associated with nitrate reductase activity shows a typical cytochrome b type visible absorption spectrum at liquid nitrogen temperature and is a protoporphyrin IX heme as judged from the spectrum of its pyridine hemochromogen derivative. Studies with the inhibitor, p-hydroxymercuribenzoate, reveal the presence of one or more readily accessible sulfhydryl groups functioning between NADPH and FAD. FADH2-nitrate reductase and reduced methyl viologen-nitrate reductase are considerably less sensitive to this —SH reagent. Inhibition by metal-binding agents has suggested the possible involvement of a second metal moiety (in addition to the molybdenum), functioning earlier in the electron transfer scheme. No negative feedback phenomena at the enzymatic level could be found with the variety of nitrogen compounds tested.