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PubMed1985

Inactivation of the NADH-dependent activities of nitrate reductase by ferrate.

Nitrate reductase (EC 1.6.6.1) from Chlorella vulgaris, a flavin-cytochrome-molybdenum enzyme, catalyses two types of partial reactions: reduction of exogenous cytochrome c by NADH and reduction of nitrate to nitrite by reduced methyl viologen (reduced 1,1'-dimethyl-4,4'-dipyridine dichloride). Ferrate, an analogue of orthophosphate acting on the phosphate-binding region of the enzymes, abolishes the NADH-nitrate reductase as well as the NADH-cytochrome c activities. In addition, the ability of NADH to reduce the endogenous cytochrome b component of the enzyme is also impaired. The reduction of nitrate by reduced methyl viologen is only partially affected. The results indicate that the ferrate primarily disrupts the NADH site.

Bacterial Proteins↗
PubMed1979

In Vitro Stability of Nitrate Reductase from Wheat Leaves: III. Isolation and Partial Characterization of a Nitrate Reductase-inactivating Factor.

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.

Journal Article↗
PubMed1993

Molecular characterization of conventional and new repeat-induced mutants of nit-3, the structural gene that encodes nitrate reductase in Neurospora crassa.

Nitrate reductase of Neurospora crassa is a dimeric protein composed of two identical subunits, each possessing three separate domains, with flavin, heme, and molybdenum-containing cofactors. A number of mutants of nit-3, the structural gene that encodes Neurospora nitrate reductase, have been characterized at the molecular level. Amber nonsense mutants of nit-3 were found to possess a truncated protein detected by a specific antibody, whereas Ssu-1-suppressed nonsense mutants showed restoration of the wild-type, full-length nitrate reductase monomer. The mutants show constitutive expression of the truncated nitrate reductase protein; however normal control, which requires nitrate induction, was restored in the suppressed mutant strains. Three conventional nit-3 mutants were isolated by the polymerase chain reaction and sequenced; two of these mutants were due to the deletion of a single base in the coding region for the flavin domain, the third mutant was a nonsense mutation within the amino-terminal molybdenum-containing domain. Homologous recombination was shown to occur when a deleted nit-3 gene was introduced by transformation into a host strain with a single point mutation in the resident nit-3 gene. New, severely damaged, null nit-3 mutants were created by repeat-induced point mutation and demonstrated to be useful as host strains for transformation experiments.

Alleles↗
PubMed1979

Nitrate reductase activities of rhizobia and the correlation between nitrate reduction and nitrogen fixation.

All species of Rhizobium except R. lupini had nitrate reductase activity. Only R. lupini was incapable of growth with nitrate as the sole source of nitrogen. However, the conditions necessary for the induction of nitrate reductase varied among species of Rhizobium. Rhizobium japonicum and some Rhizobium species of the cowpea strains expressed nitrate reductase activities both in the root nodules of appropriate leguminous hosts and when grown in the presence of nitrate. Rhizobium trifolii, R. phaseoli, and R. leguminosarum did not express nitrate reductase activities in the root nodules, but they did express them when grown in the presence of nitrate. In bacteroids of R. japonicum and some strains of cowpea Rhizobium, high N2 fixation activities were accompanied by high nitrate reductase activities. In bacteroids of R. trifolii, R. leguminosarum, and R. phaseoli, high N2 fixation activities were not accompanied by high nitrate reductase activities.

Culture Media↗
PubMed2003

The role of nitrate reductase in the regulation of the nitrate assimilation pathway in the yeast Hansenula polymorpha.

The role of nitrate reductase (NR) in the regulation of the nitrate assimilation pathway was evaluated in the yeast Hansenula polymorpha. Posttranscriptional regulation of NR in response to reduced nitrogen sources and the effect of a heterologous NR on the transcriptional regulation of nitrate-assimilatory gene expression was examined. The strain bearing YNR1 (nitrate reductase gene) under the control of the methanol-induced MOX (methanol oxidase) promoter showed that NR is active in the presence of reduced nitrogen sources. In cells incubated with glutamine plus nitrate, rapamycin abolished nitrogen catabolite repression, NR activity being very similar to that in cells induced by nitrate alone. This reveals the involvement of the Tor-signalling pathway in the transcriptional regulation of H. polymorpha nitrate assimilation genes. To assess the role of NR in nitrate-assimilatory gene expression, different strains lacking YNR1, or both YNR1 and YNT1 (high-affinity nitrate transporter) genes, or expressing the tobacco NR under the YNR1 promoter, were used. Tobacco NR abolished the constitutive nitrate-assimilatory gene induction shown by an NR gene disruptant strain. Moreover, in strains lacking the high-affinity nitrate transporter and NR this deregulation disappeared. These facts discard the role of NR protein in the transcriptional induction of the nitrate-assimilatory genes and point out the involvement of the high-affinity nitrate transporter as a part of the nitrate-signalling pathway.

Antifungal Agents↗
PubMed1989

Induction and synthesis of nitrate reductase in Chlorella vulgaris.

Assimilatory nitrate reductase is an inducible, eukaryotic enzyme that responds to a variety of environmental cues. When higher plants and green algae are grown with ammonia as a nitrogen source, low levels of nitrate reductase activity are present. Transfer to nitrate-containing medium is accompanied by substantial increase of nitrate reductase activity. Here it is shown immunologically that, in the green algae Chlorella vulgaris, nitrate reductase protein is over-produced as activity appears during induction. Immunoreactive protein is also found in cells grown on ammonia. Low levels of translatable mRNA for nitrate reductase are present in ammonia-grown cells. These data suggest that: (i) nitrate reductase appearance is controlled primarily on a transcriptional level, but that transcription is not completely halted under repressing conditions; (ii) there is an overproduction of nitrate reductase protein early during the induction period as previously suggested; and (iii) nascent protein, from in vitro translation, is of approximately the same molecular size as the nitrate reductase subunit and therefore little posttranslational modification is necessary to generate the functional enzyme. Insertion of cofactors and assembly are probably the only post-translational events.

Chlorella↗
PubMed1979

Partial purification and some properties of the Staphylococcus aureus cytoplasmic nitrate reductase.

The cytoplasmic nitrate reductase in heme mutant H-14 of Staphylococcus aureus was partially purified by steps which included ammonium sulfate fractionation and chromatography on Bio-Gel A 1.5m and ion-exchange columns. The active fractions from the ion-exchange columns showed two forms of the enzyme upon electrophoresis in nondenaturing gels of polyacrylamide; these corresponded to proteins of R(f) 0.16 and 0.28. Each form contained a predominant polypeptide of molecular weight 140,000, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The R(f) 0.16 form contained another major polypeptide of molecular weight 57,000, but the R(f) 0.28 form contained several other polypeptides. The sedimentation properties of the enzyme were examined after partial purification on Bio-Gel A 1.5m. In sucrose gradients containing Triton X-100 the enzyme sedimented as a homogeneous peak with an estimated molecular weight of 225,000; without detergent a heterogeneous profile was observed of molecular weight greater than 250,000. Treatment of the enzyme with trypsin increased the specific activity, and the enzyme sedimented as a homogeneous peak in sucrose gradients without Triton X-100, with an estimated molecular weight of 202,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that trypsin treatment converted the polypeptide of molecular weight 140,000 to a polypeptide of molecular weight 112,000. We conclude that the cytoplasmic nitrate reductase of S. aureus has a large subunit of molecular weight 140,000, which can be modified by trypsin to a polypeptide of molecular weight 112,000 without loss of catalytic activity.

Centrifugation, Density Gradient↗
PubMed1981

Molybdenum insertion in vitro in demolybdo nitrate reductase of Chlorella vulgaris.

Demolybdo nitrate reductase (also called cyt c reductase) of Chlorella vulgaris has been converted to active nitrate reductase by insertion of Mo from Na2MoO4 in vitro. A procedure is described which consistently gives about 0.3 unit of nitrate reductase from about 6 units of cyt c reductase, a yield of 30% of the maximum expected, if we calculate on a basis of a ratio of 6 to 1 for the cyt c reductase/nitrate reductase of purified normal enzyme. The demolybdoenzyme is incubated for 30 s at 31 degrees C with molybdate and reduced glutathione (GSH) at pH 4.8, and the pH is then raised to 7, and the incubation continued for 20 min. At the acid pH, there must be a partial denaturation or unfolding which permits Mo insertion, with a refolding to active enzyme at the higher pH. The GSH is not essential for activation, but in its absence the yield of active enzyme was about 50% lower. Experiments with labeled GSH showed that no GSH was incorporated into the protein during the activation procedure. Although the enzyme activity measurements suggested that only 30% of the enzyme was activated, measurements with 99Mo showed that there was one Mo incorporated per subunit weight of 90,000. The Km for nitrate of the activated nitrate reductase was identical with the Km for nitrate of the normal enzyme. On gradient centrifugation, activated nitrate reductase, cyt c reductase, and normal nitrate reductase all behaved identically.

Chlorella↗
PubMed1977

Reactions of the Neurospora crassa nitrate reductase with NAD(P) analogs.

The assimilatory NADPH-nitrate reductase (NADPH:nitrate oxidoreductase, EC 1.6.6.3) from Neurospora crassa is competitively inhibited by 3-aminopyridine adenine dinucleotide (AAD) and 3-aminopyridine adenine dinucleotide phosphate (AADP) which are structural analogs of NAD and NADP, respectively. The amino group of the pyridine ring of AAD(P) can react with nitrous acid to yield the diazonium derivative which may covalently bind at the NAD(P) site. As a result of covalent attachment, diazotized AAD(P) causes time-dependent irreversible inactivation of nitrate reductase. However, only the NADPH-dependent activities of the nitrate reductase, i.e. the overall NADPH-nitrate reductase and the NADPH-cytochrome c reductase activities, are inactivated. The reduced methyl viologen- and reduced FAD-nitrate reductase activities which do not utilize NADPH are not inhibited. This inactivation by diazotized AADP is prevented by 1 mM NADP. The inclusion of 1 muM FAD can also prevent inactivation, but the FAD effect differs from the NADP protection in that even after removal of the exogenous FAD by extensive dialysis or Sephadex G-25 filtration chromatography, the enzyme is still protected against inactivation. The FAD-generated protected form of nitrate reductase could again be inactivated if the enzyme was treated with NADPH, dialyzed to remove the NADPH, and then exposed to diazotized AADP. When NADP was substituted for NADPH in this experiment, the enzyme remained in the FAD-protected state. Difference spectra of the inactivated nitrate reductase demonstrated the presence of bound AADP, and titration of the sulfhydryl groups of the inactivated enzyme revealed that a loss of accessible sulfhydryls had occurred. The hypothesis generated by these experiments is that diazotized AADP binds at the NADPH site on nitrate reductase and reacts with a functional sulfhydryl at the site. FAD protects the enzyme against inactivation by modifying the sulfhydryl. Since NADPH reverses this protection, it appears the modifications occurring are oxidation-reduction reactions. On the basis of these results, the physiological electron flow in the nitrate reductase is postulated to be from NADPH via sulfhydryls to FAD and then the remainder of the electron carriers as follows: NADPH leads to -SH leads to FAD leads to cytochrome b-557 leads to Mo leads to NO-3.

Flavin-Adenine Dinucleotide↗
PubMed2000

Recombinant expression of molybdenum reductase fragments of plant nitrate reductase at high levels in Pichia pastoris.

Mo reductase (MoR; formerly cytochrome c reductase) fragments of NADH:NO(3) reductase (NR; EC1.6.6.1) were cytosolically expressed in Pichia pastoris, a methylotrophic yeast, using spinach (Spinacia oleracea) and corn (Zea maize) cDNAs. In fermenter cultures, spinach MoR was expressed at 420 mg L(-1), corn MoR at 32 mg L(-1), and corn MoR plus with putative NR interface domain N terminus (MoR+) at 17 mg L(-1). Constitutively expressed MoR+ was structurally stable while it was degraded when expressed by methanol induction, which suggests methanol growth produces more proteinase. Methanol-induced expression yielded more target protein. All three MoR were purified to homogeneity and their polypeptides were approximately 41 (MoR) and approximately 66 (MoR+) kD. MoR was monomeric and MoR+ dimeric, confirming the predicted role for dimer interface domain of NR. MoR+, although differing in quaternary structure from MoR, has similar kinetic properties for ferricyanide and cytochrome c reductase activities and visible spectra, which were like NR. Redox potentials of MoR and MoR+ were similar for flavin, whereas MoR+ had a more negative potential for heme-iron. Reaction schemes for MoR catalyzed reactions were proposed based on fast-reaction rapid-scan stopped-flow kinetic analysis of MoR. P. pastoris is an excellent system for producing the large amounts of NR fragments needed for detailed biochemical studies.

Aldehyde Oxidoreductases↗
PubMed2002

The molybdate-responsive Escherichia coli ModE transcriptional regulator coordinates periplasmic nitrate reductase (napFDAGHBC) operon expression with nitrate and molybdate availability.

Expression of the Escherichia coli napFDAGHBC operon (also known as aeg46.5), which encodes the periplasmic molybdoenzyme for nitrate reduction, is increased in response to anaerobiosis and further stimulated by the addition of nitrate or to a lesser extent by nitrite to the cell culture medium. These changes are mediated by the transcription factors Fnr and NarP, respectively. Utilizing a napF-lacZ operon fusion, we demonstrate that napF gene expression is impaired in strain defective for the molybdate-responsive ModE transcription factor. This control abrogates nitrate- or nitrite-dependent induction during anaerobiosis. Gel shift and DNase I footprinting analyses establish that ModE binds to the napF promoter with an apparent K(d) of about 35 nM at a position centered at -133.5 relative to the start of napF transcription. Although the ModE binding site sequence is similar to other E. coli ModE binding sites, the location is atypical, because it is not centered near the start of transcription. Introduction of point mutations in the ModE recognition site severely reduced or abolished ModE binding in vitro and conferred a modE phenotype (i.e., loss of molybdate-responsive gene expression) in vivo. In contrast, deletion of the upstream ModE region site rendered napF expression independent of modE. These findings indicate the involvement of an additional transcription factor to help coordinate nitrate- and molybdate-dependent napF expression by the Fnr, NarP, NarL, and ModE proteins. The upstream ModE regulatory site functions to override nitrate control of napF gene expression when the essential enzyme component, molybdate, is limiting in the cell environment.

Bacterial Proteins↗
PubMed1997

Constitutive non-inducible expression of the Arabidopsis thaliana Nia 2 gene in two nitrate reductase mutants of Nicotiana plumbaginifolia.

We have isolated a haploid cell line of N. plumbaginifolia, hNP 588, that is constitutive and not inducible for nitrate reductase. Nitrate reductase mutants were isolated from hNP 588 protoplasts upon UV irradiation. Two of these nitrate reductase-deficient cell lines, nia 3 and nia 25, neither of which contained any detectable nitrate reductase activity, were selected for complementation studies. A cloned Arabidopsis thaliana nitrate reductase gene Nia 2 was introduced into each of the two mutants resulting in 56 independent kanamycin-resistant cell lines. Thirty of the 56 kanamycin-resistant cell lines were able to grow on nitrate as the sole nitrogen source. Eight of these were further analyzed for nitrate reductase enzyme activity and nitrate reductase mRNA production. All eight lines had detectable nitrate reductase activity ranging from 7% to 150% of wild-type hNP 588 callus. The enzyme activity levels were not influenced by the nitrogen source in the medium. The eight lines examined expressed a constitutive, non-inducible 3.2 kb mRNA species that was not present in untransformed controls.

Arabidopsis↗
PubMed2001

Maximal expression of membrane-bound nitrate reductase in Paracoccus is induced by nitrate via a third FNR-like regulator named NarR.

Respiratory reduction of nitrate to nitrite is the first key step in the denitrification process that leads to nitrate loss from soils. In Paracoccus pantotrophus, the enzyme system that catalyzes this reaction is encoded by the narKGHJI gene cluster. Expression of this cluster is maximal under anaerobic conditions in the presence of nitrate. Upstream from narK is narR, a gene encoding a member of the FNR family of transcriptional activators. narR is transcribed divergently from the other nar genes. Mutational analysis reveals that NarR is required for maximal expression of the membrane-bound nitrate reductase genes and narK but has no other regulatory function related to denitrification. NarR is shown to require nitrate and/or nitrite is order to activate gene expression. The N-terminal region of the protein lacks the cysteine residues that are required for formation of an oxygen-sensitive iron-sulfur cluster in some other members of the FNR family. Also, NarR lacks a crucial residue involved in interactions of this family of regulators with the sigma(70) subunit of RNA polymerase, indicating that a different mechanism is used to promote transcription. narR is also found in Paracoccus denitrificans, indicating that this species contains at least three FNR homologues.

Amino Acid Sequence↗
PubMed1969

The occurrence of nitrate reductase in apple leaves.

Nitrate reductase utilizing NADH or reduced flavin mononucleotide (FMNH(2)) as electron donor was extracted from the leaves, stems and petioles, and roots of apple seedlings. Successful extraction was made possible by the use of insoluble polyvinylpyrrolidone (Polyclar AT) which forms insoluble complexes with polyphenols and tannins. The level of nitrate reductase per gram fresh weight was highest in the leaf tissue although the nitrate content of the roots was much higher than that of the leaves. Nitrite reductase activity was detected only in leaf extracts and was 4 times higher than nitrate reductase activity. Nitrate was found in all parts of young apple trees and trace amounts were also detected in mature leaves from mature trees. Nitrate reductase was induced in young leaves of apple seedlings and in mature leaves from 3 fruit-bearing varieties. An inhibitor of polyphenoloxidase, 2-mercaptobenzothiazole was used in both the inducing medium and the extracting medium in concentrations from 10(-3) to 10(-5)m with no effect upon nitrate reductase activity.

Journal Article↗
PubMed1986

Purification and properties of nitrate reductase from Mitsuokella multiacidus.

Nitrate reductase of Mitsuokella multiacidus (formerly Bacteroides multiacidus) was solublized from the membrane fraction with 1% sodium deoxycholate and purified 40-fold by immunoaffinity chromatography on the antibody-Affi-Gel 10 column. The preparation showed a major band (86% of total protein) with enzyme activity and a minor band on polyacrylamide gel after disc electrophoresis in the presence of 0.1% Triton X-100. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave a major band, the relative mobility of which corresponded to a molecular weight of 160,000, and two minor bands. The molecular weight of the enzyme was determined to be 160,000 by gel filtration on Bio-Gel A-1.5 m in the presence of 0.1% deoxycholate. Molybdenum cofactor was detected in the enzyme by fluorescence spectroscopy and by complementation of nitrate reductase from the nit-1 mutant of Neurospora crassa. The M. multiacidus enzyme catalyzed reduction of nitrate, chlorate, and bromate using methyl viologen as an electron donor. The maximal activity was found at pH 6.2-7.5 for nitrate reduction. Either methyl or benzyl viologen served well as the electron donor, but FAD, FMN, and horse heart cytochrome c were not effective. Ferredoxin from Clostridium pasteurianum supplied electron to the nitrate reductase. The purified enzyme had Km values of 0.13 mM, 0.12 mM, and 0.22 mM for nitrate, methyl viologen, and ferredoxin, respectively. The enzyme activity was inhibited by cyanide (85% at 1 mM), azide (88% at 0.1 mM), and thiocyanate (75% at 10 mM).

Bacteroides↗
PubMed1992

Studies of the uptake of nitrate in barley : v. Estimation of root cytoplasmic nitrate concentration using nitrate reductase activity-implications for nitrate influx.

The cytoplasmic NO(3) (-) concentration ([NO(3) (-)](c)) was estimated for roots of barley (Hordeum vulgare L. cv Klondike) using a technique based on measurement of in vivo nitrate reductase activity. At zero external NO(3) (-) concentration ([NO(3) (-)](o)), [NO(3) (-)](c) was estimated to be 0.66 mm for plants previously grown in 100 mum NO(3) (-). It increased linearly with [NO(3) (-)](o) between 2 and 20 mm, up to 3.9 mm at 20 mm [NO(3) (-)](o). The values obtained are much lower than previous estimates from compartmental analysis of barley roots. These observations support the suggestion (MY Siddiqi, ADM Glass, TJ Ruth [1991] J Exp Bot 42: 1455-1463) that the nitrate reductase-based technique and compartmental analysis determine [NO(3) (-)](c) for two separate pools; an active, nitrate reductase-containing pool (possibly located in the epidermal cells) and a larger, slowly metabolized storage pool (possibly in the cortical cells), respectively. Given the values obtained for [NO(3) (-)](c) and cell membrane potentials of -200 to -300 mV (ADM Glass, JE Schaff, LV Kochian [1992] Plant Physiol 99: 456-463), it is very unlikely that passive influx of NO(3) (-) is possible via the high-concentration, low-affinity transport system for NO(3) (-). This conclusion is consistent with the suggestion by Glass et al. that this system is thermodynamically active and capable of transporting NO(3) (-) against its electrochemical potential gradient.

Journal Article↗
PubMed2005

Structural basis of eukaryotic nitrate reduction: crystal structures of the nitrate reductase active site.

Nitrate assimilation in autotrophs provides most of the reduced nitrogen on earth. In eukaryotes, reduction of nitrate to nitrite is catalyzed by the molybdenum-containing NAD(P)H:nitrate reductase (NR; EC 1.7.1.1-3). In addition to the molybdenum center, NR contains iron-heme and flavin adenine dinucleotide as redox cofactors involved in an internal electron transport chain from NAD(P)H to nitrate. Recombinant, catalytically active Pichia angusta nitrate-reducing, molybdenum-containing fragment (NR-Mo) was expressed in P. pastoris and purified. Crystal structures for NR-Mo were determined at 1.7 and 2.6 angstroms. These structures revealed a unique slot for binding nitrate in the active site and identified key Arg and Trp residues potentially involved in nitrate binding. Dimeric NR-Mo is similar in overall structure to sulfite oxidases, with significant differences in the active site. Sulfate bound in the active site caused conformational changes, as compared with the unbound enzyme. Four ordered water molecules located in close proximity to Mo define a nitrate binding site, a penta-coordinated reaction intermediate, and product release. Because yeast NAD(P)H:NR is representative of the family of eukaryotic NR, we propose a general mechanism for nitrate reduction catalysis.

Animals↗
PubMed1978

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, FADH2-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 b557 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 b557-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.

Copper↗