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Query: EC:1.7.1.1 (nitrate reductase)
 3,728 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The -proteobacterium Wolinella succinogenes grows anaerobically by respiratory nitrite ammonification but not by denitrification. Nevertheless, it is capable of N(2)O reduction to N(2). Recently, the genome sequence of W. succinogenes revealed a nos gene cluster with intriguing features encoding a new type of N(2)O reductase. The predicted enzyme is similar to other N(2)O reductases exhibiting conservation of all residues ligating the two multinuclear copper centers but carries an unprecedented C-terminal monoheme cytochrome c domain. Notably, the N(2)O reductase pre-protein is synthesized with a Sec-dependent signal peptide, rather than the usually observed twin-arginine signal sequence, implying that the copper and heme cofactors are both incorporated in the periplasm. The nos gene cluster further consists of four adjacent open reading frames which are predicted to encode two monoheme c-type cytochromes as well as homologs of NapG and NapH. The latter proteins are thought to function in quinol oxidation coupled to cytochrome c reduction in electron transport to periplasmic nitrate reductase. While the accessory genes nosD, -F, -Y and -L are present in W. succinogenes, homologs of nosR and nosX are absent from the genome. We hypothesize that the nos gene cluster of W. succinogenes encodes a complete electron transport chain catalyzing N(2)O reduction by menaquinol, a pathway which might also be relevant to other bacteria.
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PMID:The unprecedented nos gene cluster of Wolinella succinogenes encodes a novel respiratory electron transfer pathway to cytochrome c nitrous oxide reductase. 1522

Rate-limiting processes of catalysis by eukaryotic molybdenum-containing nitrate reductase (NaR, EC 1.7.1.1-3) were investigated using two viscosogens (glycerol and sucrose) and observing their impact on NAD(P)H:NaR activity of corn leaf NaR and recombinant Arabidopsis and yeast NaR. Holo-NaR has two "hinge" sequences between stably folded regions housing its internal electron carriers: 1) Hinge 1 between the molybdenum-containing nitrate reducing module and cytochrome b domain containing heme and 2) Hinge 2 between cytochrome b and cytochrome b reductase (CbR) module containing FAD. Solution viscosity negatively impacted the activity of these holo-NaR forms, which suggests that the rate-limiting events in catalysis were likely to involve large conformational changes that restrict or "gate" internal electron-proton transfers (IET). Little effect of viscosity was observed on recombinant CbR module and methyl viologen nitrate reduction by holo-NaR, suggesting that these activities involved no large conformational changes. To determine whether Hinge 2 is involved in gating the first step in IET, the effects of viscosogen on cytochrome c and ferricyanide reductase activities of holo-NaR and ferricyanide reductase activity of the recombinant molybdenum reductase module (CbR, Hinge 2, and cytochrome b) were analyzed. Solution viscosity negatively impacted these partial activities, as if Hinge 2 were involved in gating IET in both enzyme forms. We concluded that both Hinges 1 and 2 appear to be involved in gating IET steps by restricting the movement of the cytochrome b domain relative to the larger nitrate-reducing and electron-donating modules of NaR.
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PMID:Viscosity effects on eukaryotic nitrate reductase activity. 1589 95

Vibrio cholerae lives in different habitats, varying from aquatic ecosystems to the human intestinal tract. The organism has acquired a set of electron transport pathways for aerobic and anaerobic respiration that enable adaptation to the various environmental conditions. We have inactivated the V. cholerae ccmE gene, which is required for cytochrome c biogenesis. The resulting strain is deficient of all c-type cytochromes and allows us to characterize the physiological role of these proteins. Under aerobic conditions in rich medium, V. cholerae produces at least six c-type cytochromes, none of which is required for growth. Wild-type V. cholerae produces active fumarate reductase, trimethylamine N-oxide reductase, cbb3 oxidase, and nitrate reductase, of which only the fumarate reductase does not require maturation of c-type cytochromes. The reduction of nitrate in the medium resulted in the accumulation of nitrite, which is toxic for the cells. This suggests that V. cholerae is able to scavenge nitrate from the environment only in the presence of other nitrite-reducing organisms. The phenotypes of cytochrome c-deficient V. cholerae were used in a transposon mutagenesis screening to search for additional genes required for cytochrome c maturation. Over 55,000 mutants were analyzed for nitrate reductase and cbb3 oxidase activity. No transposon insertions other than those within the ccm genes for cytochrome c maturation and the dsbD gene, which encodes a disulphide bond reductase, were found. In addition, the role of a novel CcdA-like protein in cbb3 oxidase assembly is discussed.
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PMID:Cytochrome c maturation and the physiological role of c-type cytochromes in Vibrio cholerae. 1610 41

THE ASSIMILATORY NITRATE REDUCTASE (NADH: nitrate oxidoreductase, E.C. 1.6.6.2.) from the marine diatom Thalassiosira pseudonana, Hasle and Heimdal, has been purified 200-fold and characterized. The regulation of nitrate reductase in response to various conditions of nitrogen nutrition has been investigated.Nitrate reductase activity is repressed by the presence of ammonium in vivo, and its synthesis is derepressed when ammonium is absent. The derepression process is sensitive to cycloheximide and apparently requires protein synthesis. Repression of enzyme activity by ammonium is neither inhibited nor delayed by the presence of cycloheximide. In vitro, ammonium does not inhibit enzyme activity.NADH is the physiological electron donor for the enzyme in a flavin-dependent reaction. Spectral studies have indicated the presence of a b-type cytochrome associated with the enzyme. It is possible to observe enzymatic oxidation-reduction reactions which represent partial functions of the over-all electron transport capacity of this enzyme. Nitrate reductase will accept electrons from artificial electron donors such as reduced methyl viologen in a flavin-independent reaction. Further, dithionitereduced flavin adenine dinucleotide can donate electrons to the enzyme to reduce nitrate to nitrite. Finally, the nitrate reductase will exhibit a diaphorase activity and reduce the artificial electron acceptor mammalian cytochrome c in flavin-adeninedinucleotide-dependent reaction.Inhibition studies with potassium cyanide, sodium azide, and o-phenanthroline have yielded indirect evidence for metal component (s) of the enzyme.The inhibition of the NADH-requiring enzyme activities by p-hydroxymercuribenzoate has shown that an essential sulfhydryl group is involved in the initial portion of the electron transport. Heat treatment exerts an effect similar to the p-hydroxymercuribenzoate inhibition; namely, the NADH-requiring activities are rapidly inactivated, whereas the terminal nitrate-reducing activities are relatively stable to heat.The T. pseudonana nitrate reductase molecule has the hydrodynamic properties of an ellipsoid with a frictional coefficient of 1.69 and a molecular weight of 330,000.
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PMID:Purification and Characterization of the Nitrate Reductase from the Diatom Thalassiosira pseudonana. 1665 41

The 4S cytochrome c (Cyt c) reductase activity of several plant species was markedly stimulated by cyanide and ferrocyanide but those of the 8S nitrate reductase component and other particulate components of the maize (Zea mays L.) scutellum by comparison, were increased only slightly. The effect of cyanide and ferrocyanide was not due to elimination of cytochrome oxidase interference but resulted from the stimulation of NADH-dependent reduction of Cyt c. A 4S Cyt c reductase component which could be isolated by ammonium sulfate fractionation and diethyl-aminoethyl-cellulose chromatography was found to be stimulated markedly by cyanide and ferrocyanide. The remaining 4S Cyt c reductase, which was insensitive to cyanide and ferrocyanide, was also fractionated with ammonium sulfate into two components. One of these, like the 8S Cyt c reductase, was sensitive to a protease from the maize roots which is relatively specific for nitrate reductase. This 4S Cyt c reductase species could be a subunit of nitrate reductase.
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PMID:Nitrate Reductase and Soluble Cytochrome c Reductase(s) in Higher Plants. 1666 Mar 77

Homogeneous squash cotyledon reduced nicotinamide-adenine dinucleotide (NADH):nitrate reductase (NR) was isolated using blue-Sepharose and polyacrylamide gel electrophoresis. Gel slices containing NR were pulverized and injected into a previously unimmunized rabbit. This process was repeated weekly and antiserum to NR was obtained after four weeks. Analysis of the antiserum by Ouchterlony double diffusion using a blue-Sepharose preparation of NR resulted in a single precipitin band while immunoelectrophoresis revealed two minor contaminants. The antiserum was found to inhibit the NR reaction and the partial reactions to different degrees. When the NADH:NR and the reduced methyl viologen:NR activities were inhibited 90% by specifically diluted antiserum, the reduction of cytochrome c was inhibited 50%, and the reduction of ferricyanide was inhibited only 30%. Antiserum was also used to compare the cross reactivities of NR from squash cotyledons, spinach, corn, and soybean leaves, Chlorella vulgaris, and Neurospora crassa. These tests revealed a high degree of similarity between NADH:NR from the squash and spinach, while NADH:NR from corn and soybean and the NAD(P)H:NR from soybean were less closely related to the squash NADH:NR. The green algal (C. vulgaris) NADH:NR and the fungal (N. crassa) NADPH:NR were very low in cross reactivity and are apparently quite different from squash NADH:NR in antigenicity. Antiserum to N. crassa NADPH:NR failed to give a positive Ouchterlony result with higher plant or C. vulgaris NADH:NR, but this antiserum did inhibit the activity of squash NR. Thus, it can be concluded from these immunological comparisons that all seven forms of assimilatory NR studied here have antigenic determinants in common and are probably derived from a common ancestor. Although these assimilatory NR have similar catalytic characteristics, they appear to have diverged to a great degree in their structural features.
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PMID:Immunological approach to structural comparisons of assimilatory nitrate reductases. 1666 83

Sixty-five Nicotiana plumbaginifolia mutants affected in the nitrate reductase structural gene (nia mutants) have been analyzed and classified. The properties evaluated were: (a) enzyme-linked immunosorbent assay (two-site ELISA) using a monoclonal antibody as coating reagent and (b) presence of partial catalytic activities, namely nitrate reduction with artificial electron donors (reduced methyl viologen, reduced flavin mononucleotide, or reduced bromphenol blue), and cytochrome c (Cyt c) reduction with NADH. Four classes have been defined: 40 mutants fall within class 1 which includes all mutants that have no protein detectable in ELISA and no partial activities; mutants of classes 2 and 3 exhibit an ELISA-detectable nitrate reductase protein and lack either Cyt c reductase activity (class 2: fourteen mutants) or the terminal nitrate reductase activities (class 3: eight mutants) of the enzyme. Three mutants (class 4) are negative in the ELISA test, lack Cyt c reductase activity, and lack or have a very low level of reduced methyl viologen or reduced flavin mononucleotide-nitrate reductase activities; however, they retain the reduced bromphenol blue nitrate reductase activity. Variations in the degrees of terminal nitrate reductase activities among the mutants indicated that the flavin mononucleotide and methyl viologen-dependent activities were linked while the bromphenol blue-dependent activity was independent of the other two. The putative positions of the lesions in the mutant proteins and the nature of structural domains of nitrate reductase involved in each partial activity are discussed.
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PMID:Biochemical and Immunological Characterization of Nitrate Reductase Deficient nia Mutants of Nicotiana plumbaginifolia. 1666 31

A cDNA clone was isolated from a maize (Zea mays L. cv W64AxW183E) scutellum lambdagt11 library using maize leaf NADH:nitrate reductase Zmnr1 cDNA clone as a hybridization probe; it was designated Zmnr1S. Zmnr1S was shown to be an NADH:nitrate reductase clone by nucleotide sequencing and comparison of its deduced amino acid sequence to Zmnr1. Zmnr1S, which is 1.8 kilobases in length and contains the code for both the cytochrome b and flavin adenine dinucleotide domains of nitrate reductase, was cloned into the EcoRI site of the Escherichia coli expression vector pET5b and expressed. The cell lysate contained NADH:cytochrome c reductase activity, which is a characteristic partial activity of NADH:nitrate reductase dependent on the cytochrome b and flavin adenine dinucleotide domains. Recombinant cytochrome c reductase was purified by immunoaffinity chromatography on monoclonal antibody Zm2(69) Sepharose. The purified cytochrome c reductase, which had a major size of 43 kilodaltons, was inhibited by polyclonal antibodies for maize leaf NADH:nitrate reductase and bound these antibodies when blotted to nitrocellulose. Ultraviolet and visible spectra of oxidized and NADH-reduced recombinant cytochrome c reductase were nearly identical with those of maize leaf NADH:nitrate reductase. These two enzyme forms also had very similar kinetic properties with respect to NADH-dependent cytochrome c and ferricyanide reduction.
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PMID:Expression in Escherichia coli of Cytochrome c Reductase Activity from a Maize NADH:Nitrate Reductase Complementary DNA. 1666 41

Bradyrhizobium japonicum cytochrome c(550), encoded by cycA, has been previously suggested to play a role in denitrification, the respiratory reduction of nitrate to dinitrogen. However, the exact role of this cytochrome in the denitrification process is unknown. This study shows that cytochrome c(550) is involved in electron transfer to the copper-containing nitrite reductase of B. japonicum, as revealed by the inability of a cycA mutant strain to consume nitrite and, consequently, to grow under denitrifying conditions with nitrite as the electron acceptor. Mutation of cycA had no apparent effect on methylviologen-dependent nitrite reductase activity. However, succinate-dependent nitrite reduction was largely inhibited, suggesting that c(550) is the in vivo electron donor to copper-containing nitrite reductase. In addition, this study demonstrates that a cytochrome c(550) mutation has a negative effect on expression of the periplasmic nitrate reductase. This phenotype can be rescued by extending the growth period of the cells. A model is proposed whereby a mutation in cycA reduces expression of the cbb(3)-type oxidase, affecting oxygen consumption rate by the cells and consequently preventing maximal expression of the periplasmic nitrate reductase during the first days of the growth period.
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PMID:Role of Bradyrhizobium japonicum cytochrome c550 in nitrite and nitrate respiration. 1817 91

Nitrate respiration catalysed by the epsilon-proteobacterium Wolinella succinogenes relies on the NapAGHBFLD system that comprises periplasmic nitrate reductase (NapA) and various other Nap proteins required for electron transport from menaquinol to NapA or maturation of Nap components. The W. succinogenes Nap system is unusual as electron transfer to NapA was shown previously to depend on both subunits of the predicted menaquinol dehydrogenase complex NapGH but did not require a cytochrome c of the NapC/NrfH family. Nonetheless, minor residual growth by nitrate respiration was observed in napG and napH gene inactivation mutants. Here, the question is addressed whether alternative membrane-bound menaquinol dehydrogenases, like NrfH and NosGH, involved in nitrite or N2O reduction systems, are able to functionally replace NapGH. The phenotypes of various gene deletion mutants as well as strains expressing chimeric nap/nos operons demonstrate that NosH is able to donate electrons to the respiratory chain of nitrate respiration at a physiologically relevant rate, whereas NrfH and NosG are not. The iron-sulphur protein NapG was shown to form a complex with NapH in the membrane but was detected in the periplasmic cell fraction in the absence of NapH. Likewise, NosH is able to bind NapG. Each of the eight poly-cysteine motifs present in either NapG or NapH was shown to be essential for nitrate respiration. The NapG homologue NosG could not substitute for NapG, even after adjusting the cysteine spacing to that of NapG, implying that NapG and NosG are specific adapter proteins that channel electrons into either the Nap or Nos system. The current model on the structure and function of the NapGH menaquinol dehydrogenase complex is presented and the composition of the electron transport chains that deliver electrons to periplasmic reductases for either nitrate, nitrite or N2O is discussed.
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PMID:Characterization of the NapGH quinol dehydrogenase complex involved in Wolinella succinogenes nitrate respiration. 1863 Dec 38


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