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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)

Assimilatory nitrite reductase was purified 1,700-fold with a yield of 22% from spinach leaves with a procedure involving ammonium sulfate fractionation, DEAE-cellulose and DEAE-Sephadex chromatography, gel filtration and ferredoxin-Sepharose affinity chromatography. The purified enzyme was apparently homogeneous as shown by disc and SDS-gel electrophoresis with a specific activity (mumol NO2-reduced/min/mg protein) of 140.
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PMID:Purification to homogeneity of spinach nitrite reductase by ferredoxin-sepharose affinity chromatography. 91 14

The nucleotide sequence encoding the structural gene (651 bp) and flanking regions for the assimilatory-type sulfite reductase from the sulfate-reducing bacterium Desulfovibrio vulgaris (Hildenborough) was determined after cloning a 1.4 kb HindIII/SalI genomic fragment possessing the gene into Bluescript pBS(+)KS. The primary structure of the protein was deduced, and the molecular mass of the apoprotein was estimated as 24 kDa. The amino acid sequence of the polypeptide shows some similarities at putative [Fe4S4] cluster binding sites in comparison with the heme protein subunit of the larger Escherichia coli and Salmonella typhimurium sulfite reductases and spinach nitrite reductase. This is the first reported sequence of a member of a new class of low molecular weight assimilatory sulfite-reducing enzymes recently identified in a number of anaerobic bacteria [Moura, I., Lina, A. R., Moura, J. J. G., Xavier, A. V., Fauque, G., Peck, H. D., & Le Gall, J. (1986) Biochem. Biophys. Res. Commun. 141, 1032-1041].
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PMID:Primary structure of the assimilatory-type sulfite reductase from Desulfovibrio vulgaris (Hildenborough): cloning and nucleotide sequence of the reductase gene. 191 81

Dissimilatory nitrite reductase was isolated from anaerobically nitrate-grown Vibrio fischeri cells and purified to electrophoretic homogeneity. The enzyme catalyzes the six-electron reduction of nitrite to ammonia. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, under either nonreducing or reducing conditions, the purified nitrite reductase migrated as a single protein band of Mr 57,000. Gel filtration chromatography revealed a native molecular weight of 58,000, indicating the enzyme as isolated to be present in the monomeric form. Purified nitrite reductase exhibited typical c-type cytochrome absorption spectra with the reduced alpha-band at 552.5 nm. Heme content analysis using the purified preparation indicated the enzyme to contain 5.5 heme c groups per molecule. Iron analysis showed the presence of 5.62 g iron atoms per mole of enzyme and no nonheme irons were detected. These results clearly indicate that, similar to the dissimilatory nitrite reductases from Desulfovibrio desulfuricans, Wolinella succinogenes, and Escherichia coli, the V. fischeri nitrite reductase is a hexaheme c-type cytochrome. Amino acid composition of V. fischeri also revealed close similarities to those of the other three hexaheme nitrite reductases previously studied. Based on this information, it is concluded that the four ammonia-forming, dissimilatory nitrite reductases isolated to date represent a homologous group of proteins with the distinct property of being hexaheme c-type cytochromes.
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PMID:Purification of Vibrio fischeri nitrite reductase and its characterization as a hexaheme c-type cytochrome. 283 68

Anaerobic cytochrome c552 was purified to electrophoretic homogeneity by ion-exchange chromatography and gel filtration from a mutant of Escherichia coli K 12 that synthesizes an increased amount of this pigment. Several molecular and enzymatic properties of the cytochrome were investigated. Its relative molecular mass was determined to be 69 000 by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. It was found to be an acidic protein that existed in the monomeric form in the native state. From its heme and iron contents, it was concluded to be a hexaheme protein containing six moles of heme c/mole protein. The amino-acid composition and other properties of the purified cytochrome c552 indicated its similarity to Desulfovibrio desulfuricans hexaheme cytochrome. The cytochrome c552 showed nitrite and hydroxylamine reductase activities with benzyl viologen as an artificial electron donor. It catalyzed the reduction of nitrite to ammonia in a six-electron transfer. FMN and FAD also served as electron donors for the nitrite reduction. The apparent Michaelis constants for nitrite and hydroxylamine were 110 microM and 18 mM, respectively. The nitrite reductase activity of the cytochrome c552 was inhibited effectively by cupric ion and cyanide.
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PMID:Purification of a hexaheme cytochrome c552 from Escherichia coli K 12 and its properties as a nitrite reductase. 300 98

Staining of sodium dodecyl sulfate or lithium dodecyl sulfate gels with 3,3',5,5'-tetramethylbenzidine (TMBZ)/H2O2 after electrophoresis has frequently been used as a specific method of detecting heme proteins. That TMBZ is an electron donor for O2 reduction by the nonheme-soluble cytochrome oxidase/nitrite reductase from Nitrosomonas europaea is now shown; this protein is detected by the TMBZ/H2O2 method. A method for the determination of TMBZ oxidase activity is given; hence, the detection of artifactual staining due to proteins of this type is possible.
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PMID:3,3',5,5'-tetramethylbenzidine/H2O2 staining is not specific for heme proteins separated by gel electrophoresis. 620 49

Neurospora crassa nitrite reductase (Mr = 290,000) catalyzes the NAD(P)H-dependent 6-electron reduction of nitrite to ammonia via flavin and siroheme prosthetic groups. Homogeneous N. crassa nitrite reductase has been prepared employing conventional purification methods followed by affinity chromatography on blue dextran-Sepharose 4B. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of homogeneous nitrite reductase reveals a single subunit band of Mr = 140,000. Isoelectric focusing of dissociated enzyme followed by sodium dodecyl sulfate-gel electrophoresis in the second dimension yields a single subunit spot with an isoelectric point at pH 6.8-6.9. Two-dimensional thin layer chromatography of acid-hydrolyzed nitrite reductase treated with 5-dimethylaminoaphthalene-1-sulfonyl chloride yields a single reactive NH2-terminal corresponding to glycine. An investigation of the prosthetic groups of nitrite reductase reveals little or no flavin associated with the purified protein, although exogenously added FAD is required for activity in vitro. An iron content of 9-10 Fe eq/mol suggests the presence of nonheme iron in addition to the siroheme moieties. Amino acid analysis yields 43 cysteinyl residues and sulfhydryl reagents react with 50 thiol eq/mol of nitrite reductase. The non-cysteinyl sulfur content, determined as 8.1 acid-labile sulfide eq/mol, is presumably associated with nonheme iron to form iron-sulfur centers. We conclude that N. crassa nitrite reductase is a homodimer of large molecular weight subunits housing an electron transfer complex of FAD, iron-sulfur centers, and siroheme to mediate the reduced pyridine nucleotide-dependent reduction of nitrite to ammonia.
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PMID:Neurospora crassa NAD(P)H-nitrite reductase. Studies on its composition and structure. 645 37

A dissimilatory nitrite reductase from the facultatively phototrophic bacterium, Rhodopseudomonas palustris strain 1a1 was studied. A basic level of the enzyme (10-50 mU/mg protein) was measured in dark, aerated and anaerobic, photosynthetic cultures. A marked derepression of enzyme synthesis occurred under conditions of oxygen limitation (200-300 mU/mg protein). The addition of nitrite (or nitrate) to the culture medium had only a slight effect on the maximal nitrite reductase titer of cells. The enzyme was purified from photosynthetically grown cells by precipitation with ammonium sulfate, gel filtration through Sepharose 6B and repeated chromatography on DE 52-cellulose. As estimated by gel filtration, the nitrite reductase had a molecular weight of about 120 000 +/- 12 000 and yielded only one band (mol. wt. of about 68 000 +/- 7000) in SDS-gel electrophoresis. The isoelectric point of the enzyme was at pH 5.1. Nitric oxide (NO) was identified as the reaction product of nitrite reduction. The enzyme also exhibited cytochrome c-oxidase activity and was active with chemically reduced viologen dyes, FMN and cytochrome c as electron donors. Highly purified nitrite reductase preparations contained 10 mol% of a c-type cytochrome. Trace metal analyses indicated the presence of Cu in the enzyme. Consistent with the detection of Cu was the finding that the Cu-chelator, diethyldithiocarbamate, strongly inhibited the nitrite reductase.
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PMID:Purification and characterization of a dissimilatory nitrite reductase from the phototrophic bacterium Rhodopseudomonas palustris. 667 Mar 57

By use of a membrane fraction prepared from Desulfovibrio gigas grown in a lactate-sulfate medium, synthesis of ATP was demonstrated to be coupled to the oxidation of molecular hydrogen and reduction of either nitrite or hydroxylamine. This phosphorylation was uncoupled from electron transport by pentachlorophenol, methyl viologen, and gramicidin, but not by oligomycin. The extrusion of protons from the cells was shown to be coupled to the hydrogen-nitrite respiratory system, and, assuming the localization of nitrite reductase on the outer side of the plasma membrane, H+/2e- values of 2.0 +/- 0.3 were obtained. Energy coupling observed with this system appears to be due to electron transfer-coupled proton translocation rather than vectorial electron transfer associated with hydrogen oxidation.
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PMID:Energy coupling to nitrite respiration in the sulfate-reducing bacterium Desulfovibrio gigas. 682 77

Various dehydrogenases, reductases, and electron transfer proteins involved in respiratory sulfate reduction by Desulfovibrio gigas have been localized with respect to the periplasmic space, membrane, and cytoplasm. This species was grown on a lactate-sulfate medium, and the distribution of enzyme activities and concentrations of electron transfer components were determined in intact cells, cell fractions prepared with a French press, and lysozyme spheroplasts. A significant fraction of formate dehydrogenase was demonstrated to be localized in the periplasmic space in addition to hydrogenase and some c-type cytochrome. Cytochrome b, menaquinone, fumarate reductase, and nitrite reductase were largely localized on the cytoplasmic membrane. Fumarate reductase was situated on the inner aspect on the membrane, and the nitrite reductase appeared to be transmembraneous. Adenylylsulfate reductase, bisulfite reductase (desulfoviridin), pyruvate dehydrogenase, and succinate dehydrogenase activities were localized in the cytoplasm. Significant amounts of hydrogenase and c-type cytochromes were also detected in the cytoplasm. Growth of D. gigas on a formate-sulfate medium containing acetate resulted in a 10-fold increase in membrane-bound formate dehydrogenase and a doubling of c-type cytochromes. Growth on fumarate with formate resulted in an additional increase in b-type cytochrome compared with lactate-sulfate-grown cells.
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PMID:Localization of dehydrogenases, reductases, and electron transfer components in the sulfate-reducing bacterium Desulfovibrio gigas. 724 92

Desulfovibrio desulfuricans (ATCC 27774), a strictly anaerobic sulfate-reducing bacteria, is able to perform anaerobic nitrate respiration in which nitrate is first reduced to nitrite by the action of nitrate reductase, and nitrite reductase then catalyzes the six-electron reduction of nitrite to ammonia. The nitrite reductase was found to be a membrane-bound enzyme and has been purified to electrophoretic homogeneity. The purified enzyme has a minimal Mr = 66,000 as judged by sodium dodecyl sulfate gel electrophoresis and contains 6 c-type heme groups/molecule. Pure nitrite reductase exhibits a typical c-type cytochrome absorption spectrum with reduced alpha-band at 552.5 nm. NADH and NADPH do not function as direct electron donors for the nitrite reductase. Desulfovibrio vulgaris hydrogenase, however, is able to transfer electrons from H2 to the nitrite reductase using FAD as the electron transfer mediator. The dithionite-reduced nitrite reductase was demonstrated to be auto-oxidizable even in the presence of potassium cyanide. On addition of nitrite, the dithionite-reduced enzyme is re-oxidized immediately. Hydroxylamine, however, can only partially re-oxidize the reduced enzyme. Ascorbate reduces the enzyme to a limited extent and the partially reduced enzyme is neither auto-oxidizable nor re-oxidizable by nitrite or hydroxylamine. Purified nitrite reductase has a pH optimum in the range of 8.0-9.5 and optimal activity at 57 degrees C. Purified nitrite reductase also has hydroxylamine reductase activity, and the Km for nitrite was determined to be 1.14 mM and that for hydroxylamine is 113.5 mM. The difference in Km values seems to exclude the possibility of hydroxylamine being a free intermediate in the reduction of nitrite.
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PMID:The isolation of a hexaheme cytochrome from Desulfovibrio desulfuricans and its identification as a new type of nitrite reductase. 730 57


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