scholarly journals Electron transfer to nitrite reductase of Rhodobacter sphaeroides 2.4.3: examination of cytochromes c 2 and c Y

Microbiology ◽  
2006 ◽  
Vol 152 (5) ◽  
pp. 1479-1488 ◽  
Author(s):  
William P. Laratta ◽  
Michael J. Nanaszko ◽  
James P. Shapleigh
Keyword(s):  
Electron Transfer ◽  
Electron Donor ◽  
Rhodobacter Sphaeroides ◽  
Nitrite Reductase ◽  
Double Mutant ◽  
Reductase Activity ◽  
Nitrite Reduction ◽  
Nitrite Reductase Gene ◽  
Nitrite Reductase Activity

The role of cytochrome c 2, encoded by cycA, and cytochrome c Y, encoded by cycY, in electron transfer to the nitrite reductase of Rhodobacter sphaeroides 2.4.3 was investigated using both in vivo and in vitro approaches. Both cycA and cycY were isolated, sequenced and insertionally inactivated in strain 2.4.3. Deletion of either gene alone had no apparent effect on the ability of R. sphaeroides to reduce nitrite. In a cycA–cycY double mutant, nitrite reduction was largely inhibited. However, the expression of the nitrite reductase gene nirK from a heterologous promoter substantially restored nitrite reductase activity in the double mutant. Using purified protein, a turnover number of 5 s−1 was observed for the oxidation of cytochrome c 2 by nitrite reductase. In contrast, oxidation of c Y only resulted in a turnover of ∼0·1 s−1. The turnover experiments indicate that c 2 is a major electron donor to nitrite reductase but c Y is probably not. Taken together, these results suggest that there is likely an unidentified electron donor, in addition to c 2, that transfers electrons to nitrite reductase, and that the decreased nitrite reductase activity observed in the cycA–cycY double mutant probably results from a change in nirK expression.

scholarly journals Increased nitrite reductase activity of fetal versus adult ovine hemoglobin

2009 ◽  
Vol 296 (2) ◽  
pp. H237-H246 ◽  
Author(s):  
Arlin B. Blood ◽  
Mauro Tiso ◽  
Shilpa T. Verma ◽  
Jennifer Lo ◽  
Mahesh S. Joshi ◽  
...  
Keyword(s):  
Nitrite Reductase ◽  
Chronic Hypoxia ◽  
Reductase Activity ◽  
Fetal Hemoglobin ◽  
Nitrite Reduction ◽  
No Production ◽  
Low Oxygen ◽  
Nitrite Reductase Activity ◽  
Adult Hemoglobin ◽  
Severe Ischemia

Growing evidence indicates that nitrite, NO2−, serves as a circulating reservoir of nitric oxide (NO) bioactivity that is activated during physiological and pathological hypoxia. One of the intravascular mechanisms for nitrite conversion to NO is a chemical nitrite reductase activity of deoxyhemoglobin. The rate of NO production from this reaction is increased when hemoglobin is in the R conformation. Because the mammalian fetus exists in a low-oxygen environment compared with the adult and is exposed to episodes of severe ischemia during the normal birthing process, and because fetal hemoglobin assumes the R conformation more readily than adult hemoglobin, we hypothesized that nitrite reduction to NO may be enhanced in the fetal circulation. We found that the reaction was faster for fetal than maternal hemoglobin or blood and that the reactions were fastest at 50–80% oxygen saturation, consistent with an R-state catalysis that is predominant for fetal hemoglobin. Nitrite concentrations were similar in blood taken from chronically instrumented normoxic ewes and their fetuses but were elevated in response to chronic hypoxia. The findings suggest an augmented nitrite reductase activity of fetal hemoglobin and that the production of nitrite may participate in the regulation of vascular NO homeostasis in the fetus.

scholarly journals The functional nitrite reductase activity of the heme-globins

Blood ◽  
2008 ◽  
Vol 112 (7) ◽  
pp. 2636-2647 ◽  
Author(s):  
Mark T. Gladwin ◽  
Daniel B. Kim-Shapiro
Keyword(s):  
Small Molecules ◽  
Nitrite Reductase ◽  
Reductase Activity ◽  
Vital Role ◽  
Nitrite Reduction ◽  
Cellular Respiration ◽  
Oxygen Binding ◽  
Nitrite Reductase Activity ◽  
Hypoxic Vasodilation ◽  
Anaerobic Conversion

AbstractHemoglobin and myoglobin are among the most extensively studied proteins, and nitrite is one of the most studied small molecules. Recently, multiple physiologic studies have surprisingly revealed that nitrite represents a biologic reservoir of NO that can regulate hypoxic vasodilation, cellular respiration, and signaling. These studies suggest a vital role for deoxyhemoglobin- and deoxymyoglobin-dependent nitrite reduction. Biophysical and chemical analysis of the nitrite-deoxyhemoglobin reaction has revealed unexpected chemistries between nitrite and deoxyhemoglobin that may contribute to and facilitate hypoxic NO generation and signaling. The first is that hemoglobin is an allosterically regulated nitrite reductase, such that oxygen binding increases the rate of nitrite conversion to NO, a process termed R-state catalysis. The second chemical property is oxidative denitrosylation, a process by which the NO formed in the deoxyhemoglobin-nitrite reaction that binds to other deoxyhemes can be released due to heme oxidation, releasing free NO. Third, the reaction undergoes a nitrite reductase/anhydrase redox cycle that catalyzes the anaerobic conversion of 2 molecules of nitrite into dinitrogen trioxide (N2O3), an uncharged molecule that may be exported from the erythrocyte. We will review these reactions in the biologic framework of hypoxic signaling in blood and the heart.

scholarly journals Nitrite reductase activity of hemoglobin as a systemic nitric oxide generator mechanism to detoxify plasma hemoglobin produced during hemolysis

2008 ◽  
Vol 295 (2) ◽  
pp. H743-H754 ◽  
Author(s):  
Peter C. Minneci ◽  
Katherine J. Deans ◽  
Sruti Shiva ◽  
Huang Zhi ◽  
Steven M. Banks ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Nitrite Reductase ◽  
Reductase Activity ◽  
Vascular Complications ◽  
Blood Substitutes ◽  
Intravascular Hemolysis ◽  
No Donor ◽  
Nitrite Reductase Activity

Hemoglobin (Hb) potently inactivates the nitric oxide (NO) radical via a dioxygenation reaction forming nitrate (NO3−). This inactivation produces endothelial dysfunction during hemolytic conditions and may contribute to the vascular complications of Hb-based blood substitutes. Hb also functions as a nitrite (NO2−) reductase, converting nitrite into NO as it deoxygenates. We hypothesized that during intravascular hemolysis, nitrite infusions would limit the vasoconstrictive properties of plasma Hb. In a canine model of low- and high-intensity hypotonic intravascular hemolysis, we characterized hemodynamic responses to nitrite infusions. Hemolysis increased systemic and pulmonary arterial pressures and systemic vascular resistance. Hemolysis also inhibited NO-dependent pulmonary and systemic vasodilation by the NO donor sodium nitroprusside. Compared with nitroprusside, nitrite demonstrated unique effects by not only inhibiting hemolysis-associated vasoconstriction but also by potentiating vasodilation at plasma Hb concentrations of <25 μM. We also observed an interaction between plasma Hb levels and nitrite to augment nitroprusside-induced vasodilation of the pulmonary and systemic circulation. This nitrite reductase activity of Hb in vivo was recapitulated in vitro using a mitochondrial NO sensor system. Nitrite infusions may promote NO generation from Hb while maintaining oxygen delivery; this effect could be harnessed to treat hemolytic conditions and to detoxify Hb-based blood substitutes.

scholarly journals Identification, Functional Studies, and Genomic Comparisons of New Members of the NnrR Regulon in Rhodobacter sphaeroides

2009 ◽  
Vol 192 (4) ◽  
pp. 903-911 ◽  
Author(s):  
Angela Hartsock ◽  
James P. Shapleigh
Keyword(s):  
Nitric Oxide ◽  
Rhodobacter Sphaeroides ◽  
Nitrite Reductase ◽  
Reductase Activity ◽  
Nitrite Reduction ◽  
No Production ◽  
Nitric Oxide Reductase ◽  
New Members ◽  
Functional Studies ◽  
The Status

ABSTRACT Analysis of the Rhodobacter sphaeroides 2.4.3 genome revealed four previously unidentified sequences similar to the binding site of the transcriptional regulator NnrR. Expression studies demonstrated that three of these sequences are within the promoters of genes, designated paz, norEF, and cdgA, in the NnrR regulon, while the status of the fourth sequence, within the tat operon promoter, remains uncertain. nnrV, under control of a previously identified NnrR site, was also identified. paz encodes a pseudoazurin that is a donor of electrons to nitrite reductase. paz inactivation did not decrease nitrite reductase activity, but loss of pseudoazurin and cytochrome c2 together reduced nitrite reduction. Inactivation of norEF reduced nitrite and nitric oxide reductase activity and increased the sensitivity to nitrite in a taxis assay. This suggests that loss of norEF increases NO production as a result of decreased nitric oxide reductase activity. 2.4.3 is the only strain of R. sphaeroides with norEF, even though all four of the strains whose genomes have been sequenced have the norCBQD operon and nnrR. norEF was shown to provide resistance to nitrite when it was mobilized into R. sphaeroides strain 2.4.1 containing nirK. Inactivation of the other identified genes did not reveal any detectable denitrification-related phenotype. The distribution of members of the NnrR regulon in R. sphaeroides revealed patterns of coselection of structural genes with the ancillary genes identified here. The strong coselection of these genes indicates their functional importance under real-world conditions, even though inactivation of the majority of them does not impact denitrification under laboratory conditions.

Separation of soluble denitrifying enzymes and cytochromes from Pseudomonas perfectomarinus

1973 ◽  
Vol 19 (7) ◽  
pp. 861-872 ◽  
Author(s):  
C. D. Cox Jr. ◽  
W. J. Payne
Keyword(s):  
Nitric Oxide ◽  
Electron Donor ◽  
Nitrite Reductase ◽  
Reductase Activity ◽  
Electron Flow ◽  
Deae Cellulose ◽  
The Other ◽  
Nitric Oxide Reductase ◽  
Effective Electron ◽  
Nitrite Reductase Activity

Nitrite and nitric oxide reductases were found soluble in extracts of Pseudomonas perfectomarinus cultured anaerobically at the expense of nitrate and ruptured with the French pressure cell. Malic enzyme, transhydrogenase, and flavin reductase that provided electron flow for these reductases were soluble as well. Nitrous oxide reductase remained particle-bound. Exogenous NADH was a poor electron donor for crude extracts, but a combination of malate, NADP, and NAD served well in the reduction of nitrite and nitric oxide. Nitrite reductase activity lost on dialysis of crude extract was restored by addition of this combination. Addition of free flavins was required for reduction of nitrite and nitric oxide. A nitrite reductase complex was separated from the nitric oxide reductase by gel filtration and DEAE-cellulose chromatography. NADH was an effective electron donor for this system with flavins provided as well. A c-type cytochrome with a split-α peak (perhaps associated with a d type) and two additional c-type cytochromes were separated from the nitrite reductase fraction. One of the latter (RI) emerged oxidized, the other (RII) reduced. Only nitric oxide oxidized RII. When these cytochromes were added to reaction mixtures containing nitrite reductase, activity was increased most by the split-α fraction. After reduction with dithionite, the absorption spectrum of the split-α cytochrome was returned to the oxidized spectrum by addition of nitrite but not the other oxides. A significant amount of a c-type cytochrome remained bound to the nitric oxide reductase fraction. A combination of malic acid, NAD, and NADP was more effective than NADH as electron donor for this system with free flavins provided as well. Addition of RI increased the rate of nitric oxide reduction by this fraction.

scholarly journals Involvement of the PrrB/PrrA Two-Component System in Nitrite Respiration in Rhodobacter sphaeroides 2.4.3: Evidence for Transcriptional Regulation

2002 ◽  
Vol 184 (13) ◽  
pp. 3521-3529 ◽  
Author(s):  
William P. Laratta ◽  
Peter S. Choi ◽  
Ivan E. Tosques ◽  
James P. Shapleigh
Keyword(s):  
Rhodobacter Sphaeroides ◽  
Nitrite Reductase ◽  
Response Regulator ◽  
Reductase Activity ◽  
Wild Type ◽  
Gene Encoding ◽  
Wild Type Phenotype ◽  
Two Component ◽  
Nitrite Reductase Activity ◽  
Nitrite Respiration

ABSTRACT Rhodobacter sphaeroides strain 2.4.3 is capable of diverse metabolic lifestyles, including denitrification. The regulation of many Rhodobacter genes involved in redox processes is controlled, in part, by the PrrBA two-component sensor-regulator system, where PrrB serves as the sensor kinase and PrrA is the response regulator. Four strains of 2.4.3 carrying mutations within the prrB gene were isolated in a screen for mutants unable to grow anaerobically on medium containing nitrite. Studies revealed that the expression of nirK, the structural gene encoding nitrite reductase, in these strains was significantly decreased compared to its expression in 2.4.3. Disruption of prrA also eliminated the ability to grow both photosynthetically and anaerobically in the dark on nitrite-amended medium. Complementation with prrA restored the wild-type phenotype. The PrrA strain exhibited a severe decrease in both nitrite reductase activity and expression of a nirK-lacZ fusion. Nitrite reductase activity in the PrrA strain could be restored to wild-type levels by using nirK expressed from a heterologous promoter, suggesting that the loss of nitrite reductase activity in the PrrA and PrrB mutants was not due to problems with enzyme assembly or the supply of reductant. Inactivation of prrA had no effect on the expression of the gene encoding NnrR, a transcriptional activator required for the expression of nirK. Inactivation of ccoN, part of the cbb 3-type cytochrome oxidase shown to regulate the kinase activity of PrrB, also caused a significant decrease in both nirK expression and Nir activity. This was unexpected, since PrrA-P accumulates in the ccoN strain. Together, these results demonstrate that PrrBA plays an essential role in the regulation of nirK.

scholarly journals Identification of the sources of nitrous oxide produced by oxidative and reductive processes in Nitrosomonas europaea

1972 ◽  
Vol 126 (5) ◽  
pp. 1181-1191 ◽  
Author(s):  
G. A. F. Ritchie ◽  
D. J. D. Nicholas
Keyword(s):  
Nitrite Reductase ◽  
Ammonia Oxidation ◽  
Reductase Activity ◽  
Close Association ◽  
Nitrosomonas Europaea ◽  
Nitrite Reduction ◽  
Anaerobic Conditions ◽  
Cell Extracts ◽  
Artificial Electron Acceptor ◽  
Nitrite Reductase Activity

1. Cells of Nitrosomonas europaea produced N2O during the oxidation of ammonia and hydroxylamine. 2. The end-product of ammonia oxidation, nitrite, was the predominant source of N2O in cells. 3. Cells also produced N2O, but not N2 gas, by the reduction of nitrite under anaerobic conditions. 4. Hydroxylamine was oxidized by cell-free extracts to yield nitrite and N2O aerobically, but to yield N2O and NO anaerobically. 5. Cell extracts reduced nitrite both aerobically and anaerobically to NO and N2O with hydroxylamine as an electron donor. 6. The relative amounts of NO and N2O produced during hydroxylamine oxidation and/or nitrite reduction are dependent on the type of artificial electron acceptor utilized. 7. Partially purified hydroxylamine oxidase retained nitrite reductase activity but cytochrome oxidase was absent. 8. There is a close association of hydroxylamine oxidase and nitrite reductase activities in purified preparations.

Characteristics of nitrite reductase activity in Lactobacillus lactis TS4

1985 ◽  
Vol 31 (6) ◽  
pp. 558-562 ◽  
Author(s):  
Karen L. Dodds ◽  
David L. Collins-Thompson
Keyword(s):  
Nitrite Reductase ◽  
Nadh Oxidase ◽  
Specific Activity ◽  
Reductase Activity ◽  
Cell Extract ◽  
Nitrite Reduction ◽  
Ph Optimum ◽  
Cell Extracts ◽  
Nitrite Reductase Activity ◽  
Lactobacillus Lactis

Nitrite reductase activity in Lactobacillus lactis TS4 was induced by the presence of nitrite and was active under anaerobic conditions. An electron donor was required. Glucose was the most efficient donor in whole cells, while NADH was the most efficient in cell extracts. The optimum nitrite concentration for reduction was 2.0 mM, with higher levels sharply inhibiting activity. The pH optimum for nitrite reduction by resting cell suspensions was 7.2, and the temperature optimum was 30 °C. High levels of NADH oxidase activity in cell extracts interfered with nitrite reductase activity. Fractionation of the cell extract by ultracentrifugation and ammonium sulphate precipitation decreased the specific activity of NADH oxidase by 40 and 41%, respectively. Nitrite reductase activity was detected in the supernatant fluid after centrifugation of cell extract at 226 000 × g for 1 h.

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