Regulation of nitrite reductase by light and nitrate in the cotyledons of hot pepper (Capsicum annuum L.).
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Light and nitrate are the major factors regulating the nitrite reductase (NiR) amongst various environmental and metabolic cues in plants. Hot pepper was used to investigate this regulatory mechanism of the NiR gene expression and its dependency on light and nitrate. The major results from this study are: (I) the nir partial clone (581 bp) obtained from hot pepper genomic DNA by degenerative polymerase chain reaction exhibited an amino acid sequence that is highly homologous with other plants. (II) Genomic DNA blot analysis and the NiR electrophoretic assay revealed that a small multigene family encodes NiR, which exists at least in two isoforms. (III) The light-mediated increase of NiR activity is correlated with the nitrate concentration, showing saturation kinetics above 50 mM of nitrate. (IV) Exogenous nitrate was required for the appearance of nir transcripts, but not for the enzyme activity. These results suggest that the gene expression of NiR in hot pepper is determined by the presence of nitrate at the transcriptional level. Furthermore, light has a synergistic effect on the action of nitrate on NiR levels. (+info)
Mechanisms for regulating electron transfer in multi-centre redox proteins.
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Protein-mediated electron transfer is a key process in nature. Many of the proteins involved in such electron transfers are complex and contain a number of redox-active cofactors. The very complexity of these multi-centre redox proteins has made it difficult to fully understand the various electron transfer events they catalyse. This is sometimes because the electron transfer steps themselves are gated or coupled to other processes such as proton transfer. However, with the molecular structures of many of these proteins now available it is possible to probe these electron transfer reactions at the molecular level. It is becoming apparent that many of these multi-centre redox proteins have rather subtle and elegant ways for regulating electron transfer. The purpose of this article is to illustrate how nature has used different approaches to control electron transfer in a number of different systems. Illustrative examples include: thermodynamic control of electron transfer in flavocytochromes b(2) and P450 BM3; a novel control mechanism involving calmodulin-binding-dependent electron transfer in neuronal nitric oxide synthase; the probable gating of electron transfer by ATP hydrolysis in nitrogenase; conformational gating of electron transfer in cytochrome cd(1); the regulation of electron transfer by protein dynamics in the cytochrome bc(1) complex; and finally the coupling of electron transfer to proton transfer in cytochrome c oxidase. (+info)
Nitrite reductase activity is a novel function of mammalian mitochondria.
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Nitrite, which is the major stable degradation product of nitric oxide, exists in all tissues capable of nitric oxide synthesis from L-arginine. The present study provides experimental evidence that nitrite in contact with respiring mitochondria accepts reducing equivalents from the ubiquinone cycle of the respiratory chain. Univalent reduction of nitrite was totally inhibited by myxothiazol. We therefore conclude on the involvement of redox cycling that ubisemiquinone is associated with the bc1 complex. Recycling of nitric oxide degradation products via these electron carriers may become a threat to energy-linked respiration since nitric oxide in direct contact with mitochondria was shown to slow the energy-linked respiration down and to trigger a mitochondrial source for superoxide radicals. Until now, the existence of nitrite reductase activity was only demonstrated in plants and bacteria. In addition, the present observation elucidates the existence of a nitric oxide synthase-independent nitric oxide source. (+info)
Essential role of transcription factor nuclear factor-kappaB in regulation of interleukin-8 gene expression by nitrite reductase from Pseudomonas aeruginosa in respiratory epithelial cells.
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Persistent infection with Pseudomonas aeruginosa increases interleukin-8 (IL-8) levels and causes dense neutrophil infiltrations in the airways of patients with chronic airway diseases. Recently, we have reported that nitrite reductase from P. aeruginosa induces the production of IL-8 in respiratory cells, including bronchial epithelial cells. To determine the molecular mechanism(s) of nitrite reductase-induced IL-8 expression in respiratory cells, A549 epithelial cells were transfected with plasmids containing serial deletions of the 5'-flanking region of the IL-8 gene and then exposed to nitrite reductase. Nitrite reductase significantly enhanced IL-8 gene promoter-driven reporter activity. This increased IL-8 gene expression was inhibited by mutating the nuclear factor-kappaB (NF-kappaB) binding element. Nitrite reductase enhanced nuclear localization of the NF-kappaB binding complex. Furthermore, nitrite reductase induced the degradation of IkappaBalpha, the major cytoplasmic inhibitor of NF-kappaB, and the expression of IkappaBalpha mRNA. These data support the critical role of the activation of NF-kappaB in nitrite reductase-induced IL-8 gene expression in airway epithelium. (+info)
The pH-dependent changes of intramolecular electron transfer on copper-containing nitrite reductase.
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Electron transfer over 12.6 A from the type 1 copper (T1Cu) to the type 2 copper (T2Cu) was investigated in the copper-containing nitrite reductases from two denitrifying bacteria (Alcaligenes xylosoxidans GIFU 1051 and Achromobacter cycloclastes IAN 1013), following pulse radiolytical reduction of T1Cu. In the presence of nitrite, the rate constant for the intramolecular electron transfer of the enzyme from A. xylosoxidans decreased 1/2 fold to 9 x 10(2) s-1 (20 degrees C, pH 7.0) as compared to that for the same process in the absence of nitrite. However, the rate constant increased with decreasing pH to become the same (2 x 10(3) s-1) as that in the absence of nitrite at pH 6.0. A similar result was obtained for the enzyme from A. cycloclastes. The pH profiles of the two enzymes in the presence of nitrite are almost the same as that of the enzyme activity of nitrite reduction. This suggests that the intramolecular electron transfer process is closely linked to the following process of catalytic reduction of nitrite. The difference in redox potential (DeltaE) of T2Cu minus T1Cu was calculated from equilibrium data for the electron transfer. The pH-dependence of DeltaE was in accord with the equation: DeltaE = DeltaE(0)+0.058 log (Kr[H+]+[H+]2)/(K(0)+[H+]), where K(r) and K(0) are the proton dissociation constants for the oxidized and reduced states of T2Cu, respectively. These results raise the possibility that amino acid residues linked by the redox of T2Cu play important roles in the enzyme reaction, being located near T2Cu. (+info)
Chloramphenicol inhibition of denitrifying enzyme activity in two agricultural soils.
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Chloramphenicol, at concentrations greater than 0.1 g/liter (0.3 mM), inhibited the denitrifying enzyme activity (DEA) of slurries of humisol and sandy loam soils by disrupting the activity of existing nitrate reductase enzymes. When the concentration of chloramphenicol was increased from 0.1 to 2.0 g/liter (6.0 mM), the rate of nitrite production from nitrate decreased by 25 to 46%. The rate of NO production from nitrate decreased by 20 to 39%, and the rate of N(2)O production from nitrate, in the presence of acetylene (DEA), decreased by 21 to 61%. The predicted values of DEA at 0 g of chloramphenicol/liter computed from linear regressions of DEA versus chloramphenicol concentration were 18 to 43% lower than DEA measurements made in the absence of chloramphenicol and within a few per cent of DEA rates measured in the presence of 0.1 g of chloramphenicol/liter. We conclude that DEA assays should be carried out with a single (0.1-g/liter) chloramphenicol concentration. Chloramphenicol at concentrations greater than 0.1 g/liter inhibits the activity of existing denitrifying enzymes and should not be used in DEA assays. (+info)
Identification of nitrite-oxidizing bacteria with monoclonal antibodies recognizing the nitrite oxidoreductase.
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Immunoblot analyses performed with three monoclonal antibodies (MAbs) that recognized the nitrite oxidoreductase (NOR) of the genus Nitrobacter were used for taxonomic investigations of nitrite oxidizers. We found that these MAbs were able to detect the nitrite-oxidizing systems (NOS) of the genera Nitrospira, Nitrococcus, and Nitrospina. The MAb designated Hyb 153-2, which recognized the alpha subunit of the NOR (alpha-NOR), was specific for species belonging to the genus Nitrobacter. In contrast, Hyb 153-3, which recognized the beta-NOR, reacted with nitrite oxidizers of the four genera. Hyb 153-1, which also recognized the beta-NOR, bound to members of the genera Nitrobacter and Nitrococcus. The molecular masses of the beta-NOR of the genus Nitrobacter and the beta subunit of the NOS (beta-NOS) of the genus Nitrococcus were identical (65 kDa). In contrast, the molecular masses of the beta-NOS of the genera Nitrospina and Nitrospira were different (48 and 46 kDa). When the genus-specific reactions of the MAbs were correlated with 16S rRNA sequences, they reflected the phylogenetic relationships among the nitrite oxidizers. The specific reactions of the MAbs allowed us to classify novel isolates and nitrite oxidizers in enrichment cultures at the genus level. In ecological studies the immunoblot analyses demonstrated that Nitrobacter or Nitrospira cells could be enriched from activated sludge by using various substrate concentrations. Fluorescence in situ hybridization and electron microscopic analyses confirmed these results. Permeated cells of pure cultures of members of the four genera were suitable for immunofluorescence labeling; these cells exhibited fluorescence signals that were consistent with the location of the NOS. (+info)
Differential regulation of the high affinity nitrite transport systems III and IV in Chlamydomonas reinhardtii.
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Two high affinity nitrite transporters have been identified in Chlamydomonas reinhardtii. They have been named system III and system IV and shown to be differentially regulated by nitrogen and carbon supply. System III was induced under high CO(2) and required a micromolar nitrate signal for optimal expression, was inhibited by ammonium, and was not affected by either chloride or the chloride channel inhibitor 5-nitro-2-(3-phenylpropylamino)benzoic acid. System IV was induced optimally under limiting CO(2) and did not require nitrate signal, was inhibited by chloride and 5-nitro-2-(3-phenylpropylamino)benzoic acid, but was not affected by ammonium. Two transcripts that shared the expression pattern of systems III and IV activities were detected with an Nrt2;3 gene probe. In addition, a mutant defective in both the activity of system III and the expression of Nrt2;3 gene has been isolated. Genetic crosses and in vivo complementation studies indicate that this mutant is defective in a locus that is closely linked to the regulatory gene Nit2. (+info)