scholarly journals The nitric oxide reductase of Paracoccus denitrificans

1990 ◽  
Vol 269 (2) ◽  
pp. 423-429 ◽  
Author(s):  
G J Carr ◽  
S J Ferguson
Keyword(s):  
Nitric Oxide ◽  
Specific Activity ◽  
Paracoccus Denitrificans ◽  
Reductase Activity ◽  
Cytochrome Bc1 Complex ◽  
Horse Heart Cytochrome ◽  
Dodecyl Maltoside ◽  
Solubilized Enzyme ◽  
Horse Heart Cytochrome C ◽  
Good Retention

The nitric oxide (NO) reductase activity of the cytoplasmic membrane of Paracoccus denitrificans can be solubilized in dodecyl maltoside with good retention of activity. The solubilized enzyme lacks NADH-dependent activity, but can be assayed with isoascorbate plus 2,3,5,6-tetramethylphenylene-1,4-diamine as electron donor and with horse heart cytochrome c as mediator. Reduction of NO was measured with an amperomeric electrode. The solubilized enzyme could be separated from other electron-transport components, including the cytochrome bc1 complex and nitrite reductase, by several steps of chromatography. The purified enzyme had a specific activity of 11 mumols.min-1.mg of protein-1 and the Km(NO) was estimated as less than 10 microM. The enzyme formed N2O from NO with the expected stoichiometry. These observations support the view that NO reductase is a discrete enzyme that participates in the denitrification process. The enzyme contained both b- and c-type haems. The former was associated with a polypeptide of apparent molecular mass 37 kDa and the latter with a polypeptide of 18 kDa. Polypeptides of 29 and 45 kDa were also identified in the purified protein which showed variable behaviour on electrophoresis in polyacrylamide gels.

scholarly journals Characterization of the second prosthetic group of the flavoenzyme NADH–acceptor reductase (component C) of the methane mono-oxygenase from Methylococcus capsulatus (Bath)

1979 ◽  
Vol 177 (3) ◽  
pp. 903-908 ◽  
Author(s):  
J Colby ◽  
H Dalton
Keyword(s):  
Reductase Activity ◽  
Methylococcus Capsulatus ◽  
Prosthetic Group ◽  
Horse Heart Cytochrome ◽  
Oxygenase Activity ◽  
Component C ◽  
Hexamethyl Phosphoramide ◽  
Horse Heart Cytochrome C ◽  
Acid Labile

1. A new two-step purification is described that routinely yields 100mg quantities of component C for biochemical studies. 2. Chemical analyses show component C purified by this procedure to contain 2 g-atoms of iron, 2 mol of acid-labile sulphide (S) and 1 mol of FAD per mol of protein. 3. The Fe-S core of component C was extruded by treating the protein with p-methoxybenzenethiol in hexamethyl phosphoramide/50mM-Tris/HCl buffer, pH 8.5 (4:1, v/v), under anaerobic conditions. The spectral properties of the extruded core suggest that component C contains 1 mol of [2Fe-2S(S-Cys)4] centre per mol of protein. 4. E.p.r. spectroscopy confirms the presence of a Fe-S centre in component C. 5. Component C catalyses the reduction by NADH of ferricyanide, 2,6-dichlorophenol-indophenol or horse heart cytochrome c, with specific activities of 50–230 units/mg of protein. 6. The optimum pH for the NADH-acceptor reductase activity is 8.5–9.0, and the apparent Km values for NADH and NADPH are 0.05mM and 15.5mM respectively. 7. Unlike methane mono-oxygenase activity, NADH-acceptor reductase activity of component C is not inhibited by 8-hydroxyquinoline or by acetylene.

scholarly journals Identification and characterization of a calmodulin-dependent nitric oxide synthase from GH3 pituitary cells

1992 ◽  
Vol 285 (1) ◽  
pp. 201-206 ◽  
Author(s):  
D J Wolff ◽  
G A Datto
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Cytochrome C ◽  
Specific Activity ◽  
Reductase Activity ◽  
Deae Cellulose ◽  
Bovine Brain ◽  
Pituitary Cells ◽  
Cytochrome C Reductase ◽  
Oxide Synthase

A nitric oxide synthase activity stimulated more than 30-fold by the concurrent presence of Ca2+ and calmodulin (CaM), and inhibited by trifluoperazine (50 microM), has been identified in extracts of GH3 pituitary cells. The CaM-dependent nitric oxide synthase of the crude extract was stimulated more than 9-fold by (6R)-5,6,7,8-tetrahydro-L-biopterin with half-maximal stimulation occurring at a concentration of 300 nM. Fractionation of the extract on DEAE-cellulose enhanced nitric oxide synthase specific activity up to 300-fold and provided a preparation which on Western blot analysis possessed a 152 kDa protein which cross-reacted with antibodies to homogeneous bovine brain nitric oxide synthase. The DEAE-cellulose-purified enzyme exhibited apparent Km values of 4.3 microM, 0.4 microM, 0.3 microM and 4 nM for L-arginine, NADPH, Ca2+ and CaM respectively. The CaM-dependent nitric oxide synthase of GH3 extract bound to 2′,5′-ADP-agarose and was eluted by NADPH with a 500-fold increased specific activity. Citrulline formation by the ADP-agarose-purified enzyme was inhibited by NG-nitro-L-arginine, NG-methyl-L-arginine and Nitro Blue Tetrazolium with apparent Ki values of 0.2, 1.8 and 7 microM respectively. The ADP-agarose-purified enzyme displayed cytochrome c reductase activity which was stimulated more than 18-fold by the concurrent presence of Ca2+ and CaM and inhibited by trifluoperazine. NG-Nitro-L-arginine and NG-methyl-L-arginine did not inhibit the cytochrome c reductase activity.

Cardiolipin modulates allosterically the nitrite reductase activity of horse heart cytochrome c

2014 ◽  
Vol 19 (7) ◽  
pp. 1195-1201 ◽  
Author(s):  
Paolo Ascenzi ◽  
Maria Marino ◽  
Fabio Polticelli ◽  
Roberto Santucci ◽  
Massimo Coletta
Keyword(s):  
Cytochrome C ◽  
Nitrite Reductase ◽  
Reductase Activity ◽  
Horse Heart Cytochrome ◽  
Nitrite Reductase Activity ◽  
Horse Heart Cytochrome C

scholarly journals Regulation and Symbiotic Role of nirK and norC Expression in Rhizobium etli

2011 ◽  
Vol 24 (2) ◽  
pp. 233-245 ◽  
Author(s):  
Nicolás Gómez-Hernández ◽  
Alma Reyes-González ◽  
Cristina Sánchez ◽  
Yolanda Mora ◽  
María J. Delgado ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Nitrate Reductase ◽  
Specific Activity ◽  
Reductase Activity ◽  
Functional Characterization ◽  
Regulatory Region ◽  
Rhizobium Etli ◽  
Bean Plants ◽  
Genes Encoding ◽  
Significant Expression

Rhizobium etli CFN42 is unable to use nitrate for respiration and lacks nitrate reductase activity as well as the nap or nar genes encoding respiratory nitrate reductase. However, genes encoding proteins closely related to denitrification enzymes, the norCBQD gene cluster and a novel nirKnirVnnrRnnrU operon are located on pCFN42f. In this study, we carried out a genetic and functional characterization of the reductases encoded by the R. etli nirK and norCB genes. By gene fusion expression analysis in free-living conditions, we determined that R. etli regulates its response to nitric oxide through NnrR via the microaerobic expression mediated by FixKf. Interestingly, expression of the norC and nirK genes displays a different level of dependence for NnrR. A null mutation in nnrR causes a drastic drop in the expression of norC, while nirK still exhibits significant expression. A thorough analysis of the nirK regulatory region revealed that this gene is under both positive and negative regulation. Functional analysis carried out in this work demonstrated that reduction of nitrite and nitric oxide in R. etli requires the reductase activities encoded by the norCBQD and nirK genes. Levels of nitrosylleghemoglobin complexes in bean plants exposed to nitrate are increased in a norC mutant but decreased in a nirK mutant. The nitrate-induced decline in nitrogenase-specific activity observed in both the wild type and the norC mutant was not detected in the nirK mutant. This data indicate that bacterial nitrite reductase is an important contributor to the formation of NO in bean nodules in response to nitrate.

Nitric oxide binding to ferrous native horse heart cytochrome c and to its carboxymethylated derivative: A spectroscopic and thermodynamic Study

1994 ◽  
Vol 53 (4) ◽  
pp. 273-280 ◽  
Author(s):  
Paolo Ascenzi ◽  
Massimo Coletta ◽  
Roberto Santucci ◽  
Francesca Polizio ◽  
Alessandro Desideri
Keyword(s):  
Nitric Oxide ◽  
Cytochrome C ◽  
Thermodynamic Study ◽  
Horse Heart Cytochrome ◽  
Horse Heart Cytochrome C

scholarly journals Heterologous NNR-Mediated Nitric Oxide Signaling inEscherichia coli

2000 ◽  
Vol 182 (22) ◽  
pp. 6434-6439 ◽  
Author(s):  
Matthew I. Hutchings ◽  
Neil Shearer ◽  
Sarah Wastell ◽  
Rob J. M. van Spanning ◽  
Stephen Spiro
Keyword(s):  
Nitric Oxide ◽  
Paracoccus Denitrificans ◽  
Culture Media ◽  
Reductase Activity ◽  
Site Directed Mutagenesis ◽  
Anaerobic Growth ◽  
Reporter System ◽  
Nitric Oxide Signaling ◽  
Cell Extracts ◽  
Nitrate And Nitrite

ABSTRACT The transcription factor NNR from Paracoccus denitrificans was expressed in a strain of Escherichia coli carrying a plasmid-borne fusion of the melRpromoter to lacZ, with a consensus FNR-binding site 41.5 bp upstream of the transcription start site. This promoter was activated by NNR under anaerobic growth conditions in media containing nitrate, nitrite, or the NO+ donor sodium nitroprusside. Activation by nitrate was abolished by a mutation in the molybdenum cofactor biosynthesis pathway, indicating a requirement for nitrate reductase activity. Activation by nitrate was modulated by the inclusion of reduced hemoglobin in culture media, because of the ability of hemoglobin to sequester nitric oxide and nitrite. The ability of nitrate and nitrite to activate NNR is likely due to the formation of NO (or related species) during nitrate and nitrite respiration. Amino acids potentially involved in NNR activity were replaced by site-directed mutagenesis, and the activities of NNR derivatives were tested in the E. coli reporter system. Substitutions at Cys-103 and Tyr-35 significantly reduced NNR activity but did not abolish the response to reactive nitrogen species. Substitutions at Phe-82 and Tyr-93 severely impaired NNR activity, but the altered proteins retained the ability to repress an FNR-repressible promoter, so these mutations have a “positive control” phenotype. It is suggested that Phe-82 and Tyr-93 identify an activating region of NNR that is involved in an interaction with RNA polymerase. Replacement of Ser-96 with alanine abolished NNR activity, and the protein was undetectable in cell extracts. In contrast, NNR in which Ser-96 was replaced with threonine retained full activity.

scholarly journals Nitric and nitrous oxide reductases are active under aerobic conditions in cells of Thiosphaera pantotropha

1991 ◽  
Vol 273 (2) ◽  
pp. 423-427 ◽  
Author(s):  
L C Bell ◽  
S J Ferguson
Keyword(s):  
Nitrous Oxide ◽  
Reductase Activity ◽  
Nitrous Oxide Reductase ◽  
Nitric Oxide Reductase ◽  
Mutual Inhibition ◽  
Horse Heart Cytochrome ◽  
Aerobic Conditions ◽  
Thiosphaera Pantotropha ◽  
Horse Heart Cytochrome C ◽  
In Cells

Use of Clark-type electrodes has shown that, in cells of Thiosphaera pantotropha, the nitrous oxide reductase is active in the presence of O2, and that the two gases involved (N2O, O2) are reduced simultaneously, but with mutual inhibition. Reduction of nitrate, or nitrite, to N2O under aerobic conditions involves NO as an intermediate, as judged by trapping experiments with the ferric form of extracellular horse heart cytochrome c and the demonstration that the cells possess a nitric oxide reductase activity. The overall conversion of nitrate to N2, the process of denitrification, under aerobic conditions, is thus not prevented by reaction of NO with O2 and depends upon a nitrous oxide reductase system which differs from that in other organisms by being neither directly inhibited nor inactivated by O2.

scholarly journals Oscillations of nitric oxide concentration in the perturbed denitrification pathway of Paracoccus denitrificans

1992 ◽  
Vol 286 (1) ◽  
pp. 111-116 ◽  
Author(s):  
I Kučera
Keyword(s):  
Nitric Oxide ◽  
Paracoccus Denitrificans ◽  
Reductase Activity ◽  
Membrane Vesicles ◽  
Inhibitory Effect ◽  
Nitric Oxide Reductase ◽  
Sustained Oscillations ◽  
Carbonyl Cyanide ◽  
Oxide Concentration ◽  
Nitric Oxide Concentration

The metabolism of nitric oxide in Paracoccus denitrificans has been studied using a Clark-type electrode. The uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) and the SH reagent N-ethylmaleimide, both of which released nitric oxide from cells respiring nitrite, were found to be efficient inhibitors of nitric oxide reductase activity. Control experiments with another uncoupler, pentachlorophenol, showed that the inhibitory effect of CCCP was not the result of a decrease in membrane potential. The denitrification pathway in cells with partly inhibited nitric oxide reductase, or in a reconstituted system containing purified nitric reductase and membrane vesicles, exhibited marked sustained oscillations of nitric oxide concentration. The occurrence of the oscillations was strictly dependent on the initial concentration of nitrite. The observed oscillatory kinetics is considered to reflect two regulatory signals destabilizing the denitrification pathway, namely the inhibition of nitric oxide reductase by nitric oxide and/or by nitrite.

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