Nitric oxide degradation by potato tuber mitochondria: Evidence for the involvement of external NAD(P)H dehydrogenases

The mechanisms of nitric oxide (NO) synthesis in plants have been extensively investigated. NO degradation can be just as important as its synthesis in controlling steady-state levels of NO. Here, we examined NO degradation in mitochondria isolated from potato tubers and the contribution of the respiratory chain to this process. NO degradation was faster in mitochondria energized with NAD(P)H than with succinate or malate. Oxygen consumption and the inner membrane potential were transiently inhibited by NO in NAD(P)H-energized mitochondria, in contrast to the persistent inhibition seen with succinate. NO degradation was abolished by anoxia and superoxide dismutase, which suggested that NO was consumed by its reaction with superoxide anion (O₂⁻). Antimycin-A stimulated and myxothiazol prevented NO consumption in succinate- and malate-energized mitochondria. Although favored by antimycin-A, NAD(P)H-mediated NO consumption was not abolished by myxothiazol, indicating that an additional site of O₂⁻ generation, besides complex III, stimulated NO degradation. Larger amounts of O₂⁻ were generated in NAD(P)H- compared to succinate- or malate-energized mitochondria. NAD(P)H-mediated NO degradation and O₂⁻ production were stimulated by free Ca²⁺ concentration. Together, these results indicate that Ca²⁺-dependent external NAD(P)H dehydrogenases, in addition to complex III, contribute to O₂⁻ production that favors NO degradation in potato tuber mitochondria.     

The occurrence and control of nitric oxide generation by the plant mitochondrial electron transport chain

The plant mitochondrial electron transport chain (ETC) is bifurcated such that electrons from ubiquinol are passed to oxygen via the usual cytochrome path or through alternative oxidase (AOX). We previously showed that knockdown of AOX in transgenic tobacco increased leaf concentrations of nitric oxide (NO), implying that an activity capable of generating NO had been effected. Here, we identify the potential source of this NO. Treatment of leaves with antimycin A (AA, Q_i‐site inhibitor of Complex III) increased NO amount more than treatment with myxothiazol (Myxo, Q_o‐site inhibitor) despite both being equally effective at inhibiting respiration. Comparison of nitrate‐grown wild‐type with AOX knockdown and overexpression plants showed a negative correlation between AOX amount and NO amount following AA. Further, Myxo fully negated the ability of AA to increase NO amount. With ammonium‐grown plants, neither AA nor Myxo strongly increased NO amount in any plant line. When these leaves were supplied with nitrite alongside the AA or Myxo, then the inhibitor effects across lines mirrored that of nitrate‐grown plants. Hence the ETC, likely the Q‐cycle of Complex III generates NO from nitrite, and AOX reduces this activity by acting as a non‐energy‐conserving electron sink upstream of Complex III.     

Oxidation of nitric oxide by a new heterotrophic Pseudomonas sp.

A new bacterial strain isolated from soil consumed nitric oxide (NO) under oxic conditions by oxidation to nitrate. Phenotypic and phylogenetic characterization of the new strain PS88 showed that it represents a previously unknown species of the genus Pseudomonas, closely related to Pseudomonas fluorescens and Pseudomonas putida. The heterotrophic, obligately aerobic strain PS88 was not able to denitrify or nitrify; however, strain PS88 oxidized NO to nitrate. NO was not reduced to nitrous oxide (N₂O). Nitrogen dioxide (NO₂) and nitrite (NO₂ ^–) as possible intermediates of NO oxidation to nitrate (NO₃ ^–) could not be detected. NO oxidation was inhibited under anoxic conditions and by high osmolarity, but not by nitrite. NO oxidation activity was inhibited by addition of formaldehyde, HgCl₂, and antimycin, and by autoclaving or disintegrating the cells, indicating that the process was enzyme-mediated. However, the mechanism remains unclear. A stepwise oxidation at a metalloenzyme and a radical mechanism are discussed. NO oxidation in strain PS88 seems to be a detoxification or a co-oxidation mechanism, rather than an energy-yielding process. Received: 15 November 1995 / Accepted: 24 February 1996     

The impact of organic matter on nitric oxide formation by Nitrosomonas europaea

Chemolithoautotrophically growing cells of Nitrosomonas europaea quantitatively oxidized ammonia to nitrite under aerobic conditions with no loss of inorganic nitrogen. Significant inorganic nitrogen losses occurred when cells were growing mixotrophically with ammonium, pyruvate, yeast extract and peptone. Under oxygen limitation the nitrogen losses were even higher. In the absence of oxygen pyruvate was metabolized slowly while nitrite was consumed concomitantly. Nitrogen losses were due to the production of nitric oxide and nitrous oxide. In mixed cultures of Nitrosomonas and Nitrobacter, strong inhibition of nitrite oxidation was reproducibly measured. NO and ammonium were not inhibitory to Nitrobacter. First evidence is given that hydroxylamine, the intermediate of the Nitrosomonas monooxygenase-reaction, is formed. 0.2 to 1.7 M NH₂OH were produced by mixotrophically growing cells of Nitrosomonas and Nitrosovibrio. Hydroxylamine was both a selective inhibitory agent to Nitrobacter cells and a strong reductant which reduced nitrite to NO and N₂O. It is discussed whether chemodenitrification or denitrification is the most abundant process for NO and N₂O production of Nitrosomonas.     

Nitric oxide gas stimulates germination of dormant Arabidopsis seeds: use of a flow-through apparatus for delivery of nitric oxide

Nitric oxide (NO) is a gaseous free radical that reacts with O₂ in air and aqueous solution. NO donors have been widely used to circumvent the difficulties inherent in working with a reactive gas, but NO donors do not deliver NO at a constant rate for prolonged periods of time. Furthermore, some of the most commonly used NO donors produce additional, bioactive decomposition products. We designed and built an apparatus that allowed for the precise mixing of gaseous NO with air and the delivery of gas through sample vials at fixed rates. This experimental setup has the added advantage that continuous flow of gas over the sample reduces the buildup of volatile breakdown products. To show that this experimental setup was suitable for studies on the dormancy and germination of Arabidopsis thaliana seeds, we introduced vapors from water or sodium nitroprusside (SNP) into the gas stream. Seeds remained dormant when treated with water vapor, but gases generated by SNP increased germination to 90%. When pure NO was mixed with air and passed over dormant seeds, ∼ ∼30% of the seeds germinated. Because nitrite accumulates in aqueous solutions exposed to NO gas, we measured the accumulation of nitrite under our experimental conditions and found that it did not exceed 100 µM. Nitrite or nitrate at concentrations of up to 500 µM did not increase germination of C24 ecotype Arabidopsis seeds to more than 10%. These data support the hypothesis that NO participates in the loss of Arabidopsis seed dormancy, and they show that for some dormant seeds, exposure to exogenous NO is sufficient to trigger germination.     

Nitric oxide degradation by potato tuber mitochondria: evidence for the involvement of external NAD(P)H dehydrogenases

The mechanisms of nitric oxide (NO) synthesis in plants have been extensively investigated. NO degradation can be just as important as its synthesis in controlling steady-state levels of NO. Here, we examined NO degradation in mitochondria isolated from potato tubers and the contribution of the respiratory chain to this process. NO degradation was faster in mitochondria energized with NAD(P)H than with succinate or malate. Oxygen consumption and the inner membrane potential were transiently inhibited by NO in NAD(P)H-energized mitochondria, in contrast to the persistent inhibition seen with succinate. NO degradation was abolished by anoxia and superoxide dismutase, which suggested that NO was consumed by its reaction with superoxide anion (O2(-)). Antimycin-A stimulated and myxothiazol prevented NO consumption in succinate- and malate-energized mitochondria. Although favored by antimycin-A, NAD(P)H-mediated NO consumption was not abolished by myxothiazol, indicating that an additional site of O2(-) generation, besides complex III, stimulated NO degradation. Larger amounts of O2(-) were generated in NAD(P)H- compared to succinate- or malate-energized mitochondria. NAD(P)H-mediated NO degradation and O2(-) production were stimulated by free Ca2+ concentration. Together, these results indicate that Ca2+-dependent external NAD(P)H dehydrogenases, in addition to complex III, contribute to O2(-) production that favors NO degradation in potato tuber mitochondria.     

NADH-dependent metabolism of nitric oxide in alfalfa root cultures expressing barley hemoglobin

Transgenic alfalfa (Medicago sativa L.) root cultures expressing sense and antisense barley (Hordeum vulgare L.) hemoglobin were examined for their ability to metabolize NO. Extracts from lines overexpressing hemoglobin had approximately twice the NO conversion rate of either control or antisense lines under normoxic conditions. Only the control line showed a significant increase in the rate of NO degradation when placed under anaerobic conditions. The decline in NO was dependent on the presence of reduced pyridine nucleotide, with the NADH-dependent rate being about 2.5 times faster than the NADPH-dependent rate. Most of the activity was found in the cytosolic fraction of the extracts, while only small amounts were found in the cell wall, mitochondria, and 105,000-g membrane fraction. The NADH-dependent NO conversion exhibited a broad pH optimum in the range 7–8 and a strong affinity to NADH and NADPH (K _m 3 M for both). It was sensitive to diphenylene iodonium, an inhibitor of flavoproteins. The activity was strongly reduced by applying antibodies raised against recombinant barley hemoglobin. Extracts of Escherichia coli overexpressing barley hemoglobin showed a 4-fold higher rate of NO metabolism as compared to non-transformed cells. The NADH/NAD and NADPH/NADP ratios were higher in lines underexpressing hemoglobin, indicating that the presence of hemoglobin has an effect on these ratios. They were increased under hypoxia and antimycin A treatment. Alfalfa root extracts exhibited methemoglobin reductase activity, using either cytochrome c or recombinant barley hemoglobin as substrates. There was a correspondence between NO degradation and nitrate formation. The activity was eluted from a Superose 12 column as a single peak with molecular weight of 35±4 kDa, which corresponds to the size of the hemoglobin dimer. The results are consistent with an NO dioxygenase-like activity, with hemoglobin acting in concert with a flavoprotein, to metabolize NO to nitrate utilizing NADH as the electron donor.Abbreviation Hb Hemoglobin     

NAD(P)H- and superoxide-dependent nitric oxide degradation by rat liver mitochondria

Mitochondria consume nitric oxide (NO) mainly through reaction with superoxide anion (). Here, we analyzed the sources for NO degradation by isolated rat liver mitochondria. Electron leakage from complex III and reverse electron transport to complex I accounted for -dependent NO degradation by mitochondria in the presence of a protonmotive force. Mitochondria incubated with NAD(P)H also presented intense generation and NO degradation rates that were insensitive to respiratory inhibitors and abolished after proteinase treatment. These results suggest that an outer membrane-localized NAD(P)H oxidase activity, in addition to the electron leakage from the respiratory chain, promotes -dependent NO degradation in rat liver mitochondria.     

Inhibition of macrophage nitric oxide production by arachidonate-cascade inhibitors

We examined whether inhibitors of the arachidonic acid cascade inhibited nitric oxide (NO) production, as measured by nitrite concentration, either in macrophages or by their cytosolic fractions. Nitrite production by peritoneal macrophages from mice receiving OK-432 treatment was significantly inhibited by phospholipase A₂ inhibitors [dexamethasone and 4-bromophenacyl bromide (4-BPB)], lipoxygenase inhibitors [nordihydroguaiaretic acid (NDGA) and ketoconazole] and a glutathioneS-transferase (leukotrienes LTA₄-LTC₄) inhibitor (ethacrynic acid). However, caffeic acid and esculetin, inhibitors of 5- and 12-lipoxygenase respectively, were not inhibitory. On the other hand, indomethacin, a cyclooxygenase inhibitor, slightly inhibited whereas another inhibitor, ibuprofen, did not. Inhibition of the nitrite production by dexamethasone, 4-BPB, NDGA and ethacrynic acid was also demonstrated when the macrophages were restimulated ex vivo with OK-432 or with lipopolysaccharide. The inhibitory activity of dexamethasone, NDGA and ethacrynic acid was significantly reduced by ex vivo restimulation with OK-432, whereas that of 4-BPB was hardly affected. Furthermore, the inhibitory activity of dexamethasone, NDGA and ethacrynic acid was much higher when the macrophages were continuously exposed to the agents than when they were pulsed. Meanwhile, inhibition by 4-BPB was almost the same with either treatment. In addition, the inhibitory activity of these agents was not blocked withl-arginine, a substrate of NO synthases, or with arachidonate metabolites (LTB₄, LTC₄ and LTE₄). Ethacrynic acid and 4-BPB, but not dexamethasone and NDGA, also inhibited nitrite production by the cytosolic fractions from OK-432-restimulated peritoneal macrophages, and the inhibitory activity of 4-BPB was superior to that of ethacrynic acid. These agents, however, did not inhibit nitrite production from sodium nitroprusside, a spontaneous NO-releasing compound. These results indicate that dexamethasone, 4-BPB, NDGA and ethacrynic acid inhibited the production of NO by macrophages through at least two different mechanisms: one was inhibited by dexamethasone, NDGA and ethacrynic acid and the other by 4-BPB. Furthermore, 4-BPB and ethacrynic acid directly inhibited the activity of the NO synthase in macrophages, suggesting that the agents work by binding to the active site(s) of the enzyme.     

The role of nitrite in nitric oxide homeostasis: A comparative perspective

Nitrite is endogenously produced as an oxidative metabolite of nitric oxide, but it also functions as a NO donor that can be activated by a number of cellular proteins under hypoxic conditions. This article discusses the physiological role of nitrite and nitrite-derived NO in blood flow regulation and cytoprotection from a comparative viewpoint, with focus on mammals and fish. Constitutive nitric oxide synthase activity results in similar plasma nitrite levels in mammals and fish, but nitrite can also be taken up across the gills in freshwater fish, which has implications for nitrite/NO levels and nitrite utilization in hypoxia. The nitrite reductase activity of deoxyhemoglobin is a major mechanism of NO generation from nitrite and may be involved in hypoxic vasodilation. Nitrite is readily transported across the erythrocyte membrane, and the transport is enhanced at low O₂ saturation in some species. Also, nitrite preferentially reacts with deoxyhemoglobin rather than oxyhemoglobin at intermediate O₂ saturations. The hemoglobin nitrite reductase activity depends on heme O₂ affinity and redox potential and shows species differences within mammals and fish. The NO forming capacity is elevated in hypoxia-tolerant species. Nitrite-induced vasodilation is well documented, and many studies support a role of erythrocyte/hemoglobin-derived NO. Vasodilation can, however, also originate from nitrite reduction within the vessel wall, and at present there is no consensus regarding the relative importance of competing mechanisms. Nitrite reduction to NO provides cytoprotection in tissues during ischemia-reperfusion events by inhibiting mitochondrial respiration and limiting reactive oxygen species. It is argued that the study of hypoxia-tolerant lower vertebrates and diving mammals may help evaluate mechanisms and a full understanding of the physiological role of nitrite.     

Production of nitric oxide in Nitrosomonas europaea by reduction of nitrite

Nitrosomonas europaea and Nitrosovibrio sp. produced NO and N₂O during nitrification of ammonium. Less then 15% of the produced NO was due to chemical decomposition of nitrite. Production of NO and especially of N₂O increased when the bacteria were incubated under anaerobic conditions at decreasing flow rates of air, or at increasing cell densities. Low concentrations of chlorite (10 M) inhibited the production of NO and N₂, but not of nitrite indicating that NO and N₂O were not produced during the oxidative conversion of ammonium to nitrite. NO and N₂O were produced during reduction of nitrite with hydrazine as electron donor in almost stoichiometric quantities indicating that reduction of nitrite was the main source of NO and N₂O.     

A theoretical study of myoglobin working as a nitric oxide scavenger

The mechanism for the reaction between nitric oxide (NO) and O₂ bound to the heme iron of myoglobin (Mb), including the following isomerization to nitrate, has been investigated using hybrid density functional theory (B3LYP). Myoglobin working as a NO scavenger could be of importance, since NO reversibly inhibits the terminal enzyme in the respiration chain, cytochrome c oxidase. The concentration of NO in the cell will thus affect the respiration and thereby the synthesis of ATP. The calculations show that the reaction between NO and the heme-bound O₂ gives a peroxynitrite intermediate whose O–O bond undergoes a homolytic cleavage, forming a NO₂ radical and myoglobin in the oxo-ferryl state. The NO₂ radical then recombines with the oxo-ferryl, forming heme-bound nitrate. Nine different models have been used in the present study to examine the effect on the reaction both by the presence and the protonation state of the distal His64, and by the surroundings of the proximal His93. The barriers going from the oxy-Mb and nitric oxide reactant to the peroxynitrite intermediate and further to the oxo-ferryl and NO₂ radical are around 10 and 7 kcal/mol, respectively. Forming the product, nitrate bound to the heme iron has a barrier of less than ~7 kcal/mol. The overall reaction going from a free nitric oxide and oxy-Mb to the heme bound nitrate is exergonic by more than 30 kcal/mol.     

Function of nitric oxide and superoxide anion in the adventitious root development and antioxidant defence in <Emphasis Type="Italic">Panax ginseng</Emphasis>

The involvement of NO in O₂ ^·− generation, rootlet development and antioxidant defence were investigated in the adventitious root cultures of mountain ginseng. Treatments of NO producers (SNP, sodium nitroprusside; SNAP, S-nitroso-N-acetylpenicillamine; and sodium nitrite with ascorbic acid), and NO scavenger (PTIO, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl3-oxide) revealed that NO is involved in the induction of new rootlets. Severe decline in number of new rootlets compared to the control under PTIO treatment indicates that NO acts downstream of auxin action in the process. NO producers (SNP, SNAP and sodium nitrite with ascorbic acid) activated NADPH oxidase activity, resulting in greater O₂ ^·− generation and higher number of new rootlets in the adventitious root explants. Moreover, treatment of diphenyliodonium chloride, a NADPH oxidase inhibitor, individually or along with SNP, inhibited root growth, NADPH oxidase activity and O₂ ^·− anion generation. NO supply also enhanced the activities of antioxidant enzymes that are likely to be responsible for reducing H₂O₂ levels and lipid peroxidation as well as modulation of ascorbate and non-protein thiol concentrations in the adventitious roots. Our results suggest that NO-induced generation of O₂ ^·− by activating NADPH oxidase activity is related to adventitious root formation in mountain ginseng.     

Acetylcholine inhibits nitric oxide (NO) synthesis in the gastropod nervous system

Acetylcholine (ACh) is one of the main signals regulating nitric oxide synthase (NOS) expression and nitric oxide (NO) biosynthesis in mammals. However, few comparative studies have been performed on the role of ACh on NOS activity in non-mammalian animals. We have therefore studied the cholinergic control of NOS in the snail Helix pomatia and compared the effects of ACh on NO synthesis in the enteric nervous system of the snail and rat. Analyses by the NADPH-diaphorase reaction, immunocytochemistry, purification with ion-exchange chromatography, Western-blot, and quantitative polymerase chain reaction have revealed the expression of neuronal NOS in the rat intestine and of a 60-kDa subunit of NOS in the enteric nerve plexus of H. pomatia. In H. pomatia, quantification of the NO-derived nitrite ions has established that NO formation is confined to the NOS-containing midintestine. Nitrite production can be elevated by L-arginine but inhibited by N_ω-nitro-L-arginine. In rats, ACh moderately elevates nitrite production, whereas ACh, the nicotinic receptor agonists (nicotine, acetyl thiocholine iodide, metacholine) and the cholinesterase inhibitor eserine reduce enteric nitrite formation in snails. The nicotinic receptor antagonist tubocurarine also provokes nitrite liberation, whereas the muscarinic receptor agonists or antagonists have no significant effect in snails. In the presence of EDTA or tetrodotoxin, ACh fails to inhibit nitrite production. In pharmacological studies, we have found that ACh contracts the midintestinal muscles and, in snails, simultaneously reduces the antagonistic muscle relaxant effect of L-arginine. Our experiments provide the first evidence for an inhibitory regulation of neuronal NO synthesis by ACh in an invertebrate species. This article is dedicated to Dr. Gábor Hollósi on the 50th anniversary of his graduation and being a teacher at the University of Debrecen.     

Dormancy of<Emphasis Type="Italic"> Arabidopsis</Emphasis> seeds and barley grains can be broken by nitric oxide

Seeds of Arabidopsis thaliana (L.) Heynh. and grains of barley (Hordeum vulgare L.) were used to characterize the affects of nitric oxide (NO) on seed dormancy. Seeds of the C24 and Col-1 ecotypes of Arabidopsis are almost completely dormant when freshly harvested, but dormancy was broken by stratification for 3 days at 4°C or by imbibition of seeds with the NO donor sodium nitroprusside (SNP). This effect of SNP on dormancy of Arabidopsis seeds was concentration dependent. SNP concentrations as low as 25 M reduced dormancy and stimulated germination, but SNP at 250 M or more impaired seedling development, including root growth, and inhibited germination. Dormancy was also reduced when Arabidopsis seeds were exposed to gasses that are generated by solutions of SNP. Nitrate and nitrite, two other oxides of nitrogen, reduced the dormancy of Arabidopsis seeds, but much higher concentrations of these were required compared to SNP. Furthermore, the kinetics of germination were slower for seeds imbibed with either nitrate or nitrite than for seeds imbibed with SNP. Although seeds imbibed with SNP had reduced dormancy, seeds imbibed with SNP and abscisic acid (ABA) remained strongly dormant. This may indicate that the effects of ABA action on germination are downstream of NO action. The NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3 oxide (cPTIO) strengthened dormancy of unstratified and briefly stratified Arabidopsis seeds. Dormancy of three cultivars of barley was also reduced by SNP. Furthermore, dormancy in barley grain was strengthened by imbibition of grain with cPTIO. The data presented here support the conclusion that NO is a potent dormancy breaking agent for seeds and grains. Experiments with the NO scavenger suggest that NO is an endogenous regulator of seed dormancy.Abbreviations ABA Abscisic acid - cPTIO 2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3 oxide - GA Gibberellin - SNP Sodium nitroprusside - NO_x Gaseous oxides of nitrogen     

The arachidonic acid effect on platelet nitric oxide level

Arachidonic acid can act as a second messenger regulating many cellular processes among which is nitric oxide (NO) formation. The aim of the present study was to investigate the molecular mechanisms involved in the arachidonic acid effect on platelet NO level. Thus NO, cGMP and superoxide anion level, the phosphorylation status of nitric oxide synthase, the protein kinase C (PKC), and NADPH oxidase activation were measured. Arachidonic acid dose-dependently reduced NO and cGMP level. The thromboxane A₂ mimetic U46619 behaved in a similar way. The arachidonic acid or U46619 effect on NO concentration was abolished by the inhibitor of the thromboxane A₂ receptor SQ29548 and partially reversed by the PKC inhibitor GF109203X or by the phospholipase C pathway inhibitor U73122. Moreover, it was shown that arachidonic acid activated PKC and decreased nitric oxide synthase (eNOS) activities. The phosphorylation of the inhibiting eNOSthr495 residue mediated by PKC was increased by arachidonic acid, while no changes at the activating ser1177 residue were shown. Finally, arachidonic acid induced NADPH oxidase activation and superoxide anion formation. These effects were greatly reduced by GF109203X, U73122, and apocynin. Likely arachidonic acid reducing NO bioavailability through all these mechanisms could potentiate its platelet aggregating power.     

The alternative oxidase (AOX) gene in Vibrio fischeri is controlled by NsrR and upregulated in response to nitric oxide

Alternative oxidase (AOX) is a respiratory oxidase found in certain eukaryotes and bacteria; however, its role in bacterial physiology is unclear. Exploiting the genetic tractability of the bacterium Vibrio fischeri, we explore the regulation of aox expression and AOX function. Using quantitative PCR and reporter assays, we demonstrate that aox expression is induced in the presence of nitric oxide (NO), and that the NO‐responsive regulatory protein NsrR mediates the response. We have identified key amino acid residues important for NsrR function and experimentally confirmed a bioinformatically predicted NsrR binding site upstream of aox. Microrespirometry demonstrated that oxygen consumption by V. fischeri CydAB quinol oxidase is inhibited by NO treatment, whereas oxygen consumption by AOX is less sensitive to NO. NADH oxidation assays using inverted membrane vesicles confirmed that NO directly inhibits CydAB, and that AOX is resistant to NO inhibition. These results indicate a role for V. fischeri AOX in aerobic respiration during NO stress.     

Inhibition of nitrogenase by nitrite and nitric oxide in Rhodopseudomonas sphaeroides f. sp. denitrificans

In cells of Rhodopseudomonas sphaeroides f. sp. denitrificans nitrite and nitric oxide, the products of denitrification, inhibit activity of nitrogenase enzyme.Ferredoxin-linked CO₂ fixation, with H₂ as a reductant, was also inhibited by nitrite and NO in denitrifying cells.EPR spectroscopy of cell preparations treated with NO showed that it reacts with non-haem iron-sulphur proteins to form iron-nitrosyl complexes. Nitrite also reacts with these iron-sulphur proteins, but the formation of ironnitrosyl complexes was dependent on the presence of dithionite. Since nitrite is reduced to NO by dithionite it is likely that nitrogenase and CO₂ fixation reactions are inhibited not only by nitrite itself, but also by nitric oxide.Abbreviation DPPH 1,1-diphenyl-2-picrylhydrazyl     

Nitrite exerts potent negative inotropy in the isolated heart via eNOS-independent nitric oxide generation and cGMP-PKG pathway activation

The ubiquitous anion nitrite (NO₂⁻) has recently emerged as an endocrine storage form of nitric oxide (NO) and a signalling molecule that mediates a number of biological responses. Although the role of NO in regulating cardiac function has been investigated in depth, the physiological signalling effects of nitrite on cardiac function have only recently been explored. We now show that remarkably low concentrations of nitrite (1 nM) significantly modulate cardiac contractility in isolated and perfused Langendorff rat heart. In particular, nitrite exhibits potent negative inotropic and lusitropic activities as evidenced by a decrease in left ventricular pressure and relaxation, respectively. Furthermore, we demonstrate that the nitrite-dependent effects are mediated by NO formation but independent of NO synthase (NOS) activity. Specifically, nitrite infusion in the Langendorff system produces NO and cGMP/PKG-dependent negative inotropism, as evidenced by the formation of cellular iron-nitrosyl complexes and inhibition of biological effect by NO scavengers and by PKG inhibitors. These data are consistent with the hypothesis that nitrite represents an eNOS-independent source of NO in the heart which modulates cardiac contractility through the NO-cGMP/PKG pathway. The observed high potency of nitrite supports a physiological function of nitrite as a source of cardiomyocyte NO and a fundamental signalling molecule in the heart.     

Reduction of nitric oxide in bacterial nitric oxide reductase—a theoretical model study

The mechanism of the nitric oxide reduction in a bacterial nitric oxide reductase (NOR) has been investigated in two model systems of the heme-b₃-Fe_B active site using density functional theory (B3LYP). A model with an octahedral coordination of the non-heme Fe_B consisting of three histidines, one glutamate and one water molecule gave an energetically feasible reaction mechanism. A tetrahedral coordination of the non-heme iron, corresponding to the one of Cu_B in cytochrome oxidase, gave several very high barriers which makes this type of coordination unlikely. The first nitric oxide coordinates to heme b₃ and is partly reduced to a more nitroxyl anion character, which activates it toward an attack from the second NO. The product in this reaction step is a hyponitrite dianion coordinating in between the two irons. Cleaving an NO bond in this intermediate forms an Fe_B (IV)O and nitrous oxide, and this is the rate determining step in the reaction mechanism. In the model with an octahedral coordination of Fe_B the intrinsic barrier of this step is 16.3 kcal/mol, which is in good agreement with the experimental value of 15.9 kcal/mol. However, the total barrier is 21.3 kcal/mol, mainly due to the endergonic reduction of heme b₃ taken from experimental reduction potentials. After nitrous oxide has left the active site the ferrylic Fe_B will form a μ-oxo bridge to heme b₃ in a reaction step exergonic by 45.3 kcal/mol. The formation of a quite stable μ-oxo bridge between heme b₃ and Fe_B is in agreement with this intermediate being the experimentally observed resting state in oxidized NOR. The formation of a ferrylic non-heme Fe_B in the proposed reaction mechanism could be one reason for having an iron as the non-heme metal ion in NOR instead of a Cu as in cytochrome oxidase.