Flavohemoglobin Hmp affords inducible protection for Escherichia coli respiration, catalyzed by cytochromes bo' or bd, from nitric oxide

Respiration of Escherichia coli catalyzed either by cytochrome bo' or bd is sensitive to micromolar extracellular NO; extensive, transient inhibition of respiration increases as dissolved oxygen tension in the medium decreases. At low oxygen concentrations (25-33 microm), the duration of inhibition of respiration by 9 microm NO is increased by mutation of either oxidase. Respiration of an hmp mutant defective in flavohemoglobin (Hmp) synthesis is extremely NO-sensitive (I(50) about 0.8 microm); conversely, cells pre-grown with sodium nitroprusside or overexpressing plasmid-borne hmp(+) are insensitive to 60 microm NO and have elevated levels of immunologically detectable Hmp. Purified Hmp consumes O(2) at a rate that is instantaneously and extensively (>10-fold) stimulated by NO due to NO oxygenase activity but, in the absence of NO, Hmp does not contribute measurably to cell oxygen consumption. Cyanide binds to Hmp (K(d) 3 microm). Concentrations of KCN (100 microm) that do not significantly inhibit cell respiration markedly suppress the protection of respiration from NO afforded by Hmp and abolish NO oxygenase activity of purified Hmp. The results demonstrate the role of Hmp in protecting respiration from NO stress and are discussed in relation to the energy metabolism of E. coli in natural O(2)-depleted environments.     

Flavohemoglobin detoxifies nitric oxide in aerobic, but not anaerobic, Escherichia coli. Evidence for a novel inducible anaerobic nitric oxide-scavenging activity.

Nitric-oxide dioxygenase (NOD) and reductase (NOR) activities of flavohemoglobin (flavoHb) have been suggested as mechanisms for NO metabolism and detoxification in a variety of microbes. Mechanisms of NO detoxification were tested in Escherichia coli using flavoHb-deficient mutants and overexpressors. flavoHb showed negligible anaerobic NOR activity and afforded no protection to the NO-sensitive aconitase or the growth of anoxic E. coli, whereas the NOD activity and the protection afforded with O(2) were substantial. A NO-inducible, O(2)-sensitive, and cyanide-resistant NOR activity efficiently metabolized NO and protected anaerobic cells from NO toxicity independent of the NOR activity of flavoHb. flavoHb possesses nitrosoglutathione and nitrite reductase activities that may account for the protection it affords against these agents. NO detoxification by flavoHb occurs most effectively via O(2)-dependent NO dioxygenation.     

Anoxic function for the Escherichia coli flavohaemoglobin (Hmp): reversible binding of nitric oxide and reduction to nitrous oxide

The flavohaemoglobin Hmp of Escherichia coli is inducible by nitric oxide (NO) and provides protection both aerobically and anaerobically from inhibition of growth by NO and agents that cause nitrosative stress. Here we report rapid kinetic studies of NO binding to Fe(III) Hmp with a second order rate constant of 7.5 x 10(5) M(-1) s(-1) to generate a nitrosyl adduct that was stable anoxically but decayed in the presence of air to reform the Fe(III) protein. NO displaced CO bound to dithionite-reduced Hmp but, remarkably, CO recombined after only 2 s at room temperature indicative of NO reduction and dissociation from the haem. Addition of NO to anoxic NADH-reduced Hmp also generated a nitrosyl species which persisted while NADH was oxidised. These results are consistent with direct demonstration by membrane-inlet mass spectrometry of NO consumption and nitrous oxide production during anoxic incubation of NADH-reduced Hmp. The results demonstrate a new mechanism by which Hmp may eliminate NO under anoxic growth conditions.     

Catalase inhibition by nitric oxide potentiates hydrogen peroxide to trigger catastrophic chromosome fragmentation in Escherichia coli

Hydrogen peroxide (H₂O₂, HP) is a universal toxin that organisms deploy to kill competing or invading cells. Bactericidal action of H₂O₂ presents several questions. First, the lethal H₂O₂ concentrations in bacterial cultures are 1000x higher than, for example, those calculated for the phagosome. Second, H₂O₂-alone kills bacteria in cultures either by mode-one, via iron-mediated chromosomal damage, or by mode-two, via unknown targets, but the killing mode in phagosomes is unclear. Third, phagosomal H₂O₂ toxicity is enhanced by production of nitric oxide (NO), but in vitro studies disagree: some show NO synergy with H₂O₂ antimicrobial action, others instead report alleviation. To investigate this “NO paradox,” we treated Escherichia coli with various concentrations of H₂O₂-alone or H₂O₂+NO, measuring survival and chromosome stability. We found that all NO concentrations make sublethal H₂O₂ treatments highly lethal, via triggering catastrophic chromosome fragmentation (mode-one killing). Yet, NO-alone is not lethal, potentiating H₂O₂ toxicity by blocking H₂O₂ scavenging in cultures. Catalases represent obvious targets of NO inhibition, and catalase-deficient mutants are indeed killed equally by H₂O₂-alone or H₂O₂+NO treatments, also showing similar levels of chromosome fragmentation. Interestingly, iron chelation blocks chromosome fragmentation in catalase-deficient mutants without blocking H₂O₂-alone lethality, indicating mode-two killing. In fact, mode-two killing of WT cells by much higher H₂O₂ concentrations is transiently alleviated by NO, reproducing the “NO paradox.” We conclude that NO potentiates H₂O₂ toxicity by promoting mode-one killing (via catastrophic chromosome fragmentation) by otherwise static low H₂O₂ concentrations, while transiently suppressing mode-two killing by immediately lethal high H₂O₂ concentrations.     

Illumination with blue light reactivates respiratory activity of mitochondria inhibited by nitric oxide, but not by glycerol trinitrate

Nitric oxide (NO) is known to inhibit mitochondrial respiration reversibly. This study aimed at clarifying whether low level illumination at specific wavelengths recovers mitochondrial respiration inhibited by NO and glycerol-trinitrate (GTN), a clinically used NO mimetic. NO fully inhibited respiration of liver mitochondria at concentrations occurring under septic shock. The respiration was completely restored by illumination at the wavelength of 430 nm while longer wavelengths were less effective. GTN inhibited mitochondrial respiration though the efficiency of GTN was lower compared to NO concentrations observed in sepsis models. However, GTN inhibition was absolutely insensitive to illumination regardless of wavelength used. Our data show that visible light of short wavelengths efficiently facilitates the recovery of mitochondria inhibited by NO-gas at the levels generated under septic conditions. The inhibition of mitochondrial respiration by GTN is not sensitive to visible light, suggesting an inhibition mechanism other that NO mediation.     

Nitric oxide formation by Escherichia coli. Dependence on nitrite reductase,the NO-sensing regulator Fnr,and flavohemoglobin Hmp

Nitric oxide (NO) is a key signaling and defense molecule in biological systems. The bactericidal effects of NO produced, for example, by macrophages are resisted by various bacterial NO-detoxifying enzymes, the best understood being the flavohemoglobins exemplified by Escherichia coli Hmp. However, many bacteria, including E. coli, are reported to produce NO by processes that are independent of denitrification in which NO is an obligatory intermediate. We demonstrate using an NO-specific electrode that E. coli cells, grown anaerobically with nitrate as terminal electron acceptor, generate significant NO on adding nitrite. The periplasmic cytochrome c nitrite reductase (Nrf) is shown, by comparing Nrf+ and Nrf- mutants, to be largely responsible for NO generation. Surprisingly, an hmp mutant did not accumulate more NO but, rather, failed to produce detectable NO. Anaerobic growth of the hmp mutant was not stimulated by nitrate, and the mutant failed to produce periplasmic cytochrome(s) c, leading to the hypothesis that accumulating NO in the absence of Hmp inactivates the global anaerobic regulator Fnr by reaction with the [4Fe-4S]2+ cluster (Cruz-Ramos, H., Crack, J., Wu, G., Hughes, M. N., Scott, C., Thomson, A. J., Green, J., and Poole, R. K. (2002) EMBO J. 21, 3235-3244). Fnr thus failed to up-regulate nitrite reductase. The model is supported by the inability of an fnr mutant to generate NO and by the restoration of NO accumulation to hmp mutants upon introducing a plasmid encoding Fnr* (D154A) known to confer activity in the presence of oxygen. A cytochrome bd-deficient mutant retained NO-generating activity. The present study reveals a critical balance between NO-generating and -detoxifying activities during anaerobic growth.     

Mutagenicity of nitric oxide and its inhibition by antioxidants

Nitric oxide (NO) is produced both by macrophages in vivo as a physiological response to infection and by a variety of cell types as an intercellular messenger. In addition, NO and nitrogen dioxide (NO₂) are significant components of many combustion processes. The ubiquitous exposure of humans to nitrogen oxides (NO_x), both endogenously and exogenously, may play a significant role in the carcinogenic process due to nitrosation of amines by NO_x. We report here that exposure to low concentrations of NO, alone or in combination with NO₂, results in significantly enhanced mutation in Salmonella typhimurium TA1535 using a modified Ames Salmonella reversion assay. The observed mutagenicity requires that the bacteria be actively dividing at the time of exposure to NO or NO₂, suggesting that the nitrogen oxides, or their reaction products, function as direct-acting mutagens and that the induced lesion is easily repairable by non-dividing cells. Exposure to NO resulted in a time- and dose-dependent increase in the number of revertants approximately proportional to the square of the NO concentration from 0 to 20 ppm. NO was a more effective mutagen relative to NO₂, however, the observed requirement for 0₂ suggests limited oxidation of NO (presumably to NO₂) is necessary. Numerous lipid- and aqueous-phase inhibitors of nitrosation, as well as a number of other general antioxidants and free-radical trapping agents, were examined for their effectiveness in blocking the mutagenic effects of NO. The mutagenic activity of NO was most effectively inhibited by β-carotene and tocopherols. BHT, dimethyl sulfoxide and mannitol also blocked the mutagenic effects of NO_x but appeared less effective than β-carotene or vitamin E, while ascorbate was ineffective as an inhibitor of mutation resulting from NO exposure.     

Evidence for mutagenesis by nitric oxide during nitrate metabolism in Escherichia coli

In Escherichia coli, nitrosative mutagenesis may occur during nitrate or nitrite respiration. The endogenous nitrosating agent N2O3 (dinitrogen trioxide, nitrous anhydride) may be formed either by the condensation of nitrous acid or by the autooxidation of nitric oxide, both of which are metabolic by-products. The purpose of this study was to determine which of these two agents is more responsible for endogenous nitrosative mutagenesis. An nfi (endonuclease V) mutant was grown anaerobically with nitrate or nitrite, conditions under which it has a high frequency of A:T-to-G:C transition mutations because of a defect in the repair of hypoxanthine (nitrosatively deaminated adenine) in DNA. These mutations could be greatly reduced by two means: (i) introduction of an nirB mutation, which affects the inducible cytoplasmic nitrite reductase, the major source of nitric oxide during nitrate or nitrite metabolism, or (ii) flushing the anaerobic culture with argon (which should purge it of nitric oxide) before it was exposed to air. The results suggest that nitrosative mutagenesis occurs during a shift from nitrate/nitrite-dependent respiration under hypoxic conditions to aerobic respiration, when accumulated nitric oxide reacts with oxygen to form endogenous nitrosating agents such as N2O3. In contrast, mutagenesis of nongrowing cells by nitrous acid was unaffected by an nirB mutation, suggesting that this mutagenesis is mediated by N2O3 that is formed directly by the condensation of nitrous acid.     

Resveratrol inhibition of inducible nitric oxide synthase in skeletal muscle involves AMPK but not SIRT1

The plant-derived polyphenol resveratrol (RSV) modulates life span and metabolism, and it is thought that these effects are largely mediated by activating the deacetylase enzyme SIRT1. However, RSV also activates the cell energy sensor AMP-activated protein kinase (AMPK). We have previously reported that AMPK activators inhibit inducible nitric oxide synthase (iNOS), a key proinflammatory mediator of insulin resistance in endotoxemia and obesity. The aim of this study was to evaluate whether RSV inhibits iNOS induction in insulin target tissues and to determine the role of SIRT1 and AMPK activation in this effect. We found that RSV (40 mg/kg ip) treatment decreased iNOS induction and NO production in skeletal muscle and white adipose tissue, but not in liver, of endotoxin (LPS)-challenged mice. This effect of the polyphenol was recapitulated in vitro, where RSV (10-80 μM) robustly inhibited iNOS protein induction and NO production in cytokine/LPS-treated L6 myocytes and 3T3-L1 adipocytes. However, no effect of RSV was observed on iNOS induction in FAO hepatocytes. Further studies using inhibitors of SIRT1 revealed that the deacetylase enzyme is not involved in RSV action on iNOS. In marked contrast, RSV activates AMPK in L6 myocytes, and blunting its activation using Compound C or RNA interference partly blocked the inhibitory effect of RSV on NO production. These results show that RSV specifically inhibits iNOS induction in muscle through a mechanism involving AMPK but not SIRT1 activation. This anti-inflammatory action of RSV likely contributes to the therapeutic effect of this plant polyphenol.     

Mutagenicity of nitric oxide and its inhibition by antioxidants.

Nitric oxide (NO) is produced both by macrophages in vivo as a physiological response to infection and by a variety of cell types as an intercellular messenger. In addition, NO and nitrogen dioxide (NO2) are significant components of many combustion processes. The ubiquitous exposure of humans to nitrogen oxides (NOx), both endogenously and exogenously, may play a significant role in the carcinogenic process due to nitrosation of amines by NOx. We report here that exposure to low concentrations of NO, alone or in combination with NO2, results in significantly enhanced mutation in Salmonella typhimurium TA1535 using a modified Ames Salmonella reversion assay. The observed mutagenicity requires that the bacteria be actively dividing at the time of exposure to NO or NO2, suggesting that the nitrogen oxides, or their reaction products, function as direct-acting mutagens and that the induced lesion is easily repairable by non-dividing cells. Exposure to NO resulted in a time- and dose-dependent increase in the number of revertants approximately proportional to the square of the NO concentration from 0 to 20 ppm. NO was a more effective mutagen relative to NO2, however, the observed requirement for O2 suggests limited oxidation of NO (presumably to NO2) is necessary. Numerous lipid- and aqueous-phase inhibitors of nitrosation, as well as a number of other general antioxidants and free-radical trapping agents, were examined for their effectiveness in blocking the mutagenic effects of NO. The mutagenic activity of NO was most effectively inhibited by beta-carotene and tocopherols. BHT, dimethyl sulfoxide and mannitol also blocked the mutagenic effects of NOx but appeared less effective than beta-carotene or vitamin E, while ascorbate was ineffective as an inhibitor of mutation resulting from NO exposure.     

Nitrosative stress in Escherichia coli: reduction of nitric oxide

The ability of enteric bacteria to protect themselves against reactive nitrogen species generated by their own metabolism, or as part of the innate immune response, is critical to their survival. One important defence mechanism is their ability to reduce NO (nitric oxide) to harmless products. The highest rates of NO reduction by Escherichia coli K-12 were detected after anaerobic growth in the presence of nitrate. Four proteins have been implicated as catalysts of NO reduction: the cytoplasmic sirohaem-containing nitrite reductase, NirB; the periplasmic cytochrome c nitrite reductase, NrfA; the flavorubredoxin NorV and its associated oxidoreductase, NorW; and the flavohaemoglobin, Hmp. Single mutants defective in any one of these proteins and even the mutant defective in all four proteins reduced NO at the same rate as the parent. Clearly, therefore, there are mechanisms of NO reduction by enteric bacteria that remain to be characterized. Far from being minor pathways, the currently unknown pathways are adequate to sustain almost optimal rates of NO reduction, and hence potentially provide significant protection against nitrosative stress.     

Role of nitric oxide in the SOS response in Escherichia coli

Having one electron with unpaired spin, nitric oxide (NO) shows high reactivity and activates or inhibits free radical chain reactions. NO toxic and genotoxic effects appear to be the result of intracellular formation of peroxinitrite that can induce some cellular damages, including DNA strand breaks, DNA base oxidation, destruction of the key enzymes, etc. Taking into account the character of DNA damages being formed under NO activity, we proposed a formation of the SOS signal and induction the SOS DNA repair response in E. coli cells treated with NO physiological donors--DNIC and GSNO. The ability of NO donor compounds to induce the SOS DNA response in E. coli PQ37 with sfiA::lacZ operon fusion is reported here at the first time. So, the SOS DNA repair response induction is one of the function of nitric oxide.     

Exogenous Hsc70, but not thermal preconditioning, confers protection to motoneurons subjected to oxidative stress

Proper sensing of stress and the initiation of the stress response are critical to maintaining cell viability in response to noxious stimuli. Induction of the stress response prior to the exposure of a lethal stress (preconditioning) can be protective. Heat shock proteins (Hsps), the main products of the stress response, are considered to be responsible for this protective effect. Most cells readily initiate a stress response, but some neuronal phenotypes, including motoneurons (MNs), have a diminished capacity to do so. We have found that, given a proper stimulus, MNs can execute a heat stress response; but, it does not protect them from death caused by hydrogen peroxide (H(2)O(2)) induced oxidative stress, despite inhibiting H(2)O(2)-induced caspase activation. Conversely, we demonstrate that incubation with the heat shock cognate 70 (Hsc70) protein prior to oxidative insult can protect MNs from oxidative stress. This survival promoting effect may be mediated through the substrate binding domain (SBD) of Hsc70. Our data suggest that stress preconditioning may not be beneficial to MNs, but that pharmacological interventions and alternative means of acquiring components of the stress response are an effective means of ameliorating lethal stress in MNs and may be potentially useful therapeutically in preventing pathological MN loss.     

Kinetics of electron transfer from NADH to the Escherichia coli nitric oxide reductase flavorubredoxin

Escherichia coli flavorubredoxin (FlRd) belongs to the family of flavodiiron proteins (FDPs), microbial enzymes that are expressed to scavenge nitric oxide (NO) under anaerobic conditions. To degrade NO, FlRd has to be reduced by NADH via the FAD-binding protein flavorubredoxin reductase, thus the kinetics of electron transfer along this pathway was investigated by stopped-flow absorption spectroscopy. We found that NADH, but not NADPH, quickly reduces the FlRd-reductase (k = 5.5 +/- 2.2 x 10(6) M(-1).s(-1) at 5 degrees C), with a limiting rate of 255 +/- 17 s(-1). The reductase in turn quickly reduces the rubredoxin (Rd) center of FlRd, as assessed at 5 degrees C working with the native FlRd enzyme (k = 2.4 +/- 0.1 x 10(6) m(-1).s(-1)) and with its isolated Rd-domain (k approximately 1 x 10(7) M(-1).s(-1)); in both cases the reaction was found to be dependent on pH and ionic strength. In FlRd the fast reduction of the Rd center occurs synchronously with the formation of flavin mononucleotide semiquinone. Our data provide evidence that (a) FlRd-reductase rapidly shuttles electrons between NADH and FlRd, a prerequisite for NO reduction in this detoxification pathway, and (b) the electron accepting site in FlRd, the Rd center, is in very fast redox equilibrium with the flavin mononucleotide.