Myoglobin Is a Nitrite Reductase That Generates NO and Regulates Mitochondrial Respiration.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1561-1561
Author(s):  
Sruti Shiva ◽  
Zhi Huang ◽  
Peter H. MacArthur ◽  
Lorna A. Ringwood ◽  
Mark T. Gladwin
Keyword(s):  
Rate Constant ◽  
Nitrite Reductase ◽  
Mitochondrial Respiration ◽  
Reductase Activity ◽  
Oxidase Inhibitor ◽  
Nitrite Reduction ◽  
Rat Liver Mitochondria ◽  
Xanthine Oxidase Inhibitor ◽  
Maximum Reaction Rate ◽  
Bimolecular Rate Constant

Abstract Previous studies have demonstrated that nitrite is an endocrine store of NO that may play an important role in hypoxic vasodilation. Nitrite can be converted to NO enzymatically by deoxyhemoglobin, in a reaction that is under allosteric control with a maximum reaction rate near the hemoglobin P50. In this study, we characterize the nitrite reductase activity of deoxymyoglobin, which reduces nitrite approximately 50-times faster than deoxyhemoglobin due to its lower redox potential. Spectrophotometric measurements show the deoxymyoglobin-nitrite reaction to be second order and linearly dependent on deoxymyoglobin, nitrite and proton concentration with a bimolecular rate constant of 11.7 M−1s−1 at pH 7.4 at 37 degrees Celsius. Using this bimolecular rate constant, we calculate that at physiological concentrations of nitrite (20uM) and myoglobin (25uM), NO will be generated at a rate of 2.85 nM/sec. Since the IC50 of inhibition of mitochondrial respiration is approximately 100nM at physiological oxygen tensions (5–10 μM), we hypothesized that the myoglobin-dependent reduction of nitrite could regulate mitochondrial respiration. Indeed, the addition of deoxymyoglobin in conjunction with nitrite to isolated rat liver mitochondria resulted in the inhibition of respiration, while myoglobin or nitrite alone had no effect. The addition of nitrite to rat heart homogenate, which contains both myoglobin and mitochondria, resulted in a measurable production of NO (with 1mM nitrite addition) and the inhibition of mitochondrial respiration (with 25μM nitrite addition) that was not significantly changed by the addition of the xanthine oxidase inhibitor allopurinol. These data corroborate previous studies demonstrating that NO generated from nitrite reduction can escape heme autocapture to mediate biological responses and confirm that the regulation of mitochondrial respiration by the nitrite-myoglobin reaction is relevant even in a physiological milieu. These data have implications for the modulation of hypoxic respiration, nitrite-dependent hypoxic signaling in tissue, and the regulation of oxygen gradients in the microcirculation.

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 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.

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.

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.

Beneficial Effects of Xanthine Oxidase Inhibitor, Topiloxostat, on Experimental Diabetic Neuropathy in Mice

Diabetes ◽  
2018 ◽  
Vol 67 (Supplement 1) ◽  
pp. 549-P
Author(s):  
HIROKI MIZUKAMI ◽  
REMINA KOYAMA ◽  
KAZUHISA TAKAHASHI ◽  
SHO OSONOI ◽  
SAORI OGASAWARA ◽  
...  
Keyword(s):  
Diabetic Neuropathy ◽  
Xanthine Oxidase ◽  
Oxidase Inhibitor ◽  
Xanthine Oxidase Inhibitor ◽  
Beneficial Effects ◽  
Experimental Diabetic Neuropathy

Simultaneous Monitoring of Febuxostat and Uric Acid in Human Serum Samples Using the Direct Square-Wave Voltammetric Method

2019 ◽  
Vol 15 (6) ◽  
pp. 678-684
Author(s):  
Biljana Nigović ◽  
Jakov Vlak
Keyword(s):  
Uric Acid ◽  
Human Serum ◽  
Oxidase Inhibitor ◽  
Serum Samples ◽  
Boron Doped Diamond ◽  
Fast Analysis ◽  
Xanthine Oxidase Inhibitor ◽  
Voltammetric Method ◽  
Square Wave ◽  
Human Serum Samples

Background: High uric acid serum level, hyperuricemia, is now associated with many diseases such as gout, chronic kidney disease, hypertension, coronary artery disease and diabetes. Febuxostat is a novel selective xanthine oxidase inhibitor approved for the treatment of hyperuricemia. Objective: The aim of this study was to develop a first analytical method for the simultaneous determination of febuxostat and uric acid. Methods: An unmodified boron-doped diamond electrode provided concurrent quantitation of drug at low levels and uric acid, which has clinical significance in the diagnosis and therapy of hyperuricemia, at relatively high concentrations. The direct square-wave voltammetric method was applied to the analysis of both analytes in human serum samples. Results: Under the optimized conditions, the linear response of peak current on febuxostat concentration was achieved in the range from 7.5 × 10-7 to 3 × 10-5 M, while uric acid showed two linear ranges of 5 × 10-6 - 5 × 10-5 M and 5 × 10-5 - 2 × 10-4 M. The method was successfully utilised for quantification of both analytes in human serum samples. Good recoveries were obtained without interference from common inorganic cations and anions as well as glucose, dopamine, ascorbic and folic acids at concentrations expected in physiological conditions. Conclusion: The great benefits of developed method are fast analysis (only 7.5 s for run), low cost and simplicity of performance.

Allopurinol and Loss of Consciousness in a 78-old Year Man Suffering from Gout

2020 ◽  
Vol 20 (2) ◽  
pp. 253-256 ◽  
Author(s):  
Mahnaz Arian ◽  
Mina AkbariRad ◽  
Ahmad Bagheri Moghaddam ◽  
Abdollah Firoozi ◽  
Mohammad Jami
Keyword(s):  
Uric Acid ◽  
Xanthine Oxidase ◽  
Kidney Stones ◽  
Oxidase Inhibitor ◽  
Loss Of Consciousness ◽  
Drug Induced ◽  
Xanthine Oxidase Inhibitor ◽  
Case Presentation ◽  
Maculopapular Rashes ◽  
Reduced State

: Allopurinol is an FDA -Approved xanthine oxidase inhibitor, which is effective in the treatment of gout, hyperuricemia and uremic kidney stones in patients with an increased level of uric acid excretion. Xanthine oxidase acts by converting hypoxanthine and xanthine into uric acid, and therefore its inhibition results in decreased production of uric acid. The most common side effects of this medication are as follows: maculopapular rashes, hives, itching, headache, dizziness, abnormal hair loss, fever and hypersensitivity reaction. Case Presentation: This report represents a case of drug-induced meningitis of a senile man who ended up in the ICU due to the remarkably reduced state of consciousness.

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