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Query: EC:1.7.1.4 (nitrite reductase)
 1,847 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The Neurospora crassa assimilatory NADPH-nitrite reductase (NAD(P)H: nitrite oxidoreductase, EC 1.6.6.4), which catalyzes the NADPH-dependent formation of ammonia from nitrite, has been purified to homogeneity as judged by polyacrylamide gel electrophoresis. The specific activity of the purified enzyme is 26.9 mumol nitrite reduced/min per mg protein, which corresponds to a turnover number of 7800 min(-1). The enzyme also has associated NADH-nitrite reductase, NADPH-hydroxylamine reductase and NADH-hydroxylamine reductase activities. The stoichiometry of 3 mol NADPH oxidized per mol nitrite reduced and ammonia formed has been confirmed. The visible absorption spectrum of the nitrite reductase reveals maxima at 280,390 (Soret) and 580 (alpha) nm. The latter bands are indicative of the occurrence of siroheme as a prosthetic group. The A280nm/A390nm ratio of 7.0 and the Soret/alpha ratio of 3.8 are compatible with values reported for other purified siroheme-containing enzymes. These results are discussed in terms of the comparative biochemistry of various enzymes involved in nitrite, hydroxylamine and sulfite metabolism in Neurospora crassa and other organisms.
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PMID:Preparation and some properties of homogeneous Neurospora crassa assimilatory NADPH-nitrite reductase. 15 Aug 63

Cytochrome c552, which has been implicated as an electron carrier for nitrite reduction by Escherichia coli, has been separated from NADH-nitrite oxidoreductase activity. The cytochrome is therefore not required for the reduction of nitrite by NADH in vitro. Nevertheless, some mutants which were selected by their inability to use nitrite as a nitrogen source during anaerobic growth synthesize neither NADH-nitrite oxidoreductase nor cytochrome c552. The defects in these mutants are due to mutations in a single gene, nirA, which is located at about minute 29 on the recalibrated linkage map. Experiments with an F' plasmid which carries a nirA+ allele established that nirA+ is dominant to the defective allele. Other mutants, defective in nitrate reductase activity because of mutations in the chlA or chlB genes, synthesized nitrite reductase and cytochrome c552 in the absence of nitrate or nitrite. A mutant with a defective fnr gene was also NirA- and, conversely, nirA mutants were Fnr-. In a series of transduction experiments, attempts to separate the nirA and fnr defects were unsuccessful. Furthermore, no complementation was observed when an F' plasmid carrying a defective nirA allele was transferred into the fnr strain. It is concluded that the fnr gene described by Lambden & Guest (1976) is identical to the nirA gene and that its product affects the synthesis or assembly of a variety of anaerobic redox enzymes which include nitrite reductase, cytochrome c552, nitrate reductase, fumarate reductase and formate hydrogenlyase.
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PMID:The chromosomal location and pleiotropic effects of mutations of the nirA+ gene of Escherichia coli K12: the essential role of nirA+ in nitrite reduction and in other anaerobic redox reactions. 20 51

The reaction of bovine heart ferrocytochrome c with nitrite was studied under various conditions. The reaction product was ferricytochrome c at around pH 5, whereas at around pH 3 it was Compound I, characterized by twin peaks at 529 and 563 nm of equal intensity. However, ferrocytochrome c decreased obeying first-order kinetics over the pH range examined, irrespective of the presence or absence of molecular oxygen. The apparent first-order rate constant was proportional to the square of the nitrite concentration at pH 4.4 and it increased as the pH was lowered. At pH 3 the reaction was so rapid that it had to be followed by stopped-flow and rapid-scanning techniques. The apparent rate constant at this pH was found to increase linearly with the nitrite concentration. Based on these results the active species of nitrite was concluded to be dinitrogen trioxide at pH 4.4 and nitrosonium ion, no+, at pH 3. Compound II was formed by reaction of ferrocytochrome c and NO gas at acidic and alkaline pH values. The absorption peaks were at 533 and 563 nm at pH 3, and at 538 and 567 nm at pH 12.9. This compound was also formed by reducing Compound I with reductants. Compound I prepared from ferricytochrome c and NO was stable below pH 6. However, appreciable absorption peaks for ferrocytochrome c appeared between pH 8 and 10, because Compound I was dissociated into ferrocytochrome c and NO+, and because ferrocytochrome c thus formed reacted with NO very slowly in this pH region. Saccharomyces ferricytochrome c under NO gas behaved differently from mammalian cytochrome, indicating the significance of the nature of the heme environment in determing the reactivity. Only at extreme pH values was Compound II formed exclusively and persisted. A model system for dissimilatory nitrite reductase was constructed by using bovine heart cytochrome c, nitrite and NADH plus PMS at pH 3.3, and a scheme involving cyclic turnover of ferrocytochrome c, Compound I and Compound II is presented, with kinetic parameters.
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PMID:Reaction of cytochrome c with nitrite and nitric oxide. A model of dissimilatory nitrite reductase. 21 67

NADH-nitrite oxidoreductase (EC 1.6.4) was purified to better than 95% homogeneity from batch cultures of Escherichia coli strain OR75Ch15, which is partially constitutive for nitrite reductase synthesis. Yields of purified enzyme were low, mainly because of a large loss of activity during chromatography on DEAE-cellulose. The quantitative separation of cytochrome c-552 from nitrite reductase activity resulted in an increase in the specific activity of the enzyme: this cytochrome is not therefore an integral part of nitrite reductase. The subunit molecular weights of nitrite reductase and of a haemoprotein contaminant, as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, were 88000 and 80000 respectively. The sedimentation coefficient was calculated to be in the range 8.5-9.5S, consistent with a mol.wt. of 190000. It is suggested therefore that the native enzyme is a dimer with two identical or similar-sized subunits. Purest samples contained 0.4 mol of flavin/mol of enzyme, but no detectable haem. Catalytic activity was totally inhibited by 20 micron-p-chloromercuribenzoate and 1 mM-cyanide, slightly inhibited by 1 micron-sulphite and 10mM-arsenite, but insensitive to 1 mM-2,2'-bipyridine, 4mM-1,10-phenanthroline and 10mM-NaN3. Three molecules of NADH were oxidized for each NO2-ion reduced: the product of the reaction is therefore assumed to be NH4+. The specific activity of hydroxylamine reductase increased at each step in the purification of nitrite reductase, and the elution profiles for these two activities during chromatography on DEAE-Sephadex were coincident. It is likely that a single enzyme is responsible for both activities.
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PMID:Purification and properties of nitrite reductase from Escherichia coli K12. 21 42

Five mutants of Escherichia coli impaired on nitrite reduction were studied. All have lost NADH-nitrite reductase activity but have retained the capacity to synthesize all or part of their cytochrome c552. Three genes, nir C, nir D, and nir E were mapped at 26, 72.5 and 49.5 min, respectively. Another gene, nir F was tentatively localized around 52 min.
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PMID:Nitrite reduction in Escherichia coli: genetic analysis of nir mutants. 36 84

Five open reading frames designated nirB, nirD, nirE, nirC and cysG have been identified from the DNA sequence of the Escherichia coli nir operon. Complementation experiments established that the NirB, NirD and CysG polypeptides are essential and sufficient for NADH-dependent nitrite reductase activity (EC 1.6.6.4). A series of plasmids has been constructed in which each of the open reading frames has been fused in-phase with the beta-galactosidase gene, lacZ. Rates of beta-galactosidase synthesis during growth in different media revealed that nirB, -D, -E and -C are transcribed from the FNR-dependent promoter, p-nirB, located just upstream of the nirB gene: expression is co-ordinately repressed by oxygen and induced during anaerobic growth. Although the nirB, -D and -C open reading frames are translated into protein, no translation of nirE mRNA was detected. The cysG gene product is expressed from both p-nirB and a second, FNR-independent promoter, p-cysG, located within the nirC gene. No NADH-dependent nitrite reductase activity was detected in extracts from bacteria lacking either NirB or NirD, but a mixture of the two was as active as an extract from wild-type bacteria. Reconstitution of enzyme activity in vitro required stoichiometric quantities of NirB and NirD and was rapid and independent of the temperature during mixing. NirD remained associated with NirB during the initial stages of purification of the active enzyme, suggesting that NirD is a second structural subunit of the enzyme.
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PMID:Transcriptional control, translation and function of the products of the five open reading frames of the Escherichia coli nir operon. 143 59

An open reading frame from Rhizobium leguminosarum bv. viciae strain VF39, previously identified and found to be similar to Escherichia coli fnr and Rhizobium meliloti fixK (orf240, thereafter called fnrN), was further analysed. Analysis of the expression of an fnrN-lacZ transcriptional fusion revealed that fnrN is preferentially expressed under oxygen limitation. Using R. meliloti fixN-lacZ fusions it was shown that the fnrN gene product only mediates transcriptional activation under microaerobiosis, indicating that the FnrN protein responds, directly or indirectly, to oxygen. Plasmids which expressed fnrN under the control of an E. coli promoter were able to complement an E. coli fnr mutant with respect to anaerobic growth on nitrate but not fumarate, and to promote anaerobic but not aerobic activation of the Fnr-dependent E. coli genes narGHJI, nirB and fdnGHI coding for nitrate reductase, NADH-dependent nitrite reductase and formate dehydrogenase-N, respectively. Fumarate and DMSO reductase activities were not induced by FnrN. The E. coli fnr gene substituted for fnrN in oxygen-regulated transcription of nirB- and fixN-lacZ fusions in R. leguminosarum. The results indicate that Fnr and FnrN are functionally very similar and share a common mode of oxygen-dependent transcriptional activation. From hybridization studies, it appeared that fnrN-like genes are present in a number of different R. leguminosarum strains.
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PMID:The Rhizobium leguminosarum FnrN protein is functionally similar to Escherichia coli Fnr and promotes heterologous oxygen-dependent activation of transcription. 148 91

Nitrosation activity was measured in Escherichia coli isolates and a range of nitrite reductase (nir) mutants. Activity was only detected in intact cells and could be inhibited by a number of treatments such as sonication and osmotic shock. Aerobically-grown cells had highest nitrosation activity compared to oxygen-limited ones. Inclusion of nitrite in growth media induced high activities of nitrite reductase and for some isolates, nitrosation. Analysis of nir mutants identified two which were unable to nitrosate. This result suggested that NADH-dependent nitrite reductase was implicated either directly or indirectly in nitrosation.
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PMID:Nitrosating activity in Escherichia coli. 151 11

A chromosomal locus of Salmonella typhimurium which complements S. typhimurium asr (anaerobic sulfite reduction) mutants and confers on Escherichia coli the ability to produce hydrogen sulfide from sulfite was recently cloned (C. J. Huang and E. L. Barrett, J. Bacteriol. 172:4100-4102, 1990). The DNA sequence and the transcription start site have been determined. Analysis of the sequence and gene products revealed a functional operon containing three genes which have been designated asrA, asrB, and asrC, encoding peptides of 40, 31, and 37 kDa, respectively. The predicted amino acid sequences of both asrA and asrC contained arrangements of cysteines characteristic of [4Fe-4S] ferredoxins. The sequence of asrB contained a typical nucleotide-binding region. The sequence of asrC contained, in addition to the ferredoxinlike cysteine clusters, two other cysteine clusters closely resembling the proposed siroheme-binding site in biosynthetic sulfite reductase. Expression of lacZ fused to the asr promoter was repressed by oxygen and induced by sulfite. Analysis of promoter deletions revealed a region specific for sulfite regulation and a second region required for anaerobic expression. Computer-assisted DNA sequence analysis revealed a site just upstream of the first open reading frame which had significant homology to the FNR protein-binding site of E. coli NADH-linked nitrite reductase. However, asr expression by the fusion plasmid was not affected by site-specific mutations within the apparent FNR-binding site.
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PMID:Sequence analysis and expression of the Salmonella typhimurium asr operon encoding production of hydrogen sulfide from sulfite. 170 86

From conditions for production in Fusarium oxysporum of the unique nitrate/nitrite-inducible cytochrome P-450, tentatively called P-450dNIR, it was expected that the fungus is capable of metabolizing nitrate dissimilatively. Here we report that F. oxysporum exhibits a distinct denitrifying ability which results in the anaerobic evolution of nitrous oxide (N2O) from nitrate or nitrite. Comparison of the cell growth during denitrification indicated that the dissimilatory reduction of nitrate to nitrite is an energetically favorable process in F. oxysporum; however, further reduction of nitrite to N2O might be energy-exhausting and may function as a detoxification mechanism. A potent nitrite reductase activity to form N2O could be reconstituted by combination of the cell-free extract prepared from the denitrifying cells and an NADH-phenadinemethosulfate-dependent reducing system. The activity was strongly inhibited by carbon monoxide, cyanide, oxygen (O2), and the antibody against P-450dNIR. The results, along with those concerning inducing conditions of P-450dNIR, were highly indicative that the cytochrome is involved in the denitrifying nitrite reduction. This work has thus presented not only the first demonstration that a eukaryote exhibits a marked denitrifying ability, but also the first instance of a cytochrome P-450 that is involved in a reducing reaction with a distinct physiological significance against a hydrophilic, inorganic substrate.
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PMID:Denitrification by the fungus Fusarium oxysporum and involvement of cytochrome P-450 in the respiratory nitrite reduction. 204 Jun 19


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