Nitric Oxide Detoxification Research Articles

Frankia alni proteome under nitrogen‐fixing and nitrogen‐replete conditions

Frankia alni induces root nodules on Alnus, in which the bacterium differentiates into nitrogen (N)‐fixing cells called vesicles. In culture, F. alni also undergoes major morphological changes as it alternates between N‐replete and N‐fixing conditions. Lack of biologically available N induces the synthesis of vesicles in which nitrogenase is protected from molecular oxygen by a thick lipid hopanoid envelope. Very little is known about the molecular basis of Frankia–host interaction as well as Frankia cell differentiation. The recent determination of the complete genome sequence of F. alni strain ACN14a has permitted us to characterize its proteome, particularly in the extracellular compartment, which could be involved in Frankia–host interaction, and in the switch from N‐replete to N‐fixing conditions. To that end, 126 bacterial proteins were analyzed by two‐dimensional protein gel electrophoresis and identified by matrix‐assisted laser desorption/ionization time of flight fingerprinting using a F. alni proteome database. Interestingly, the extracellular fraction contains some glycolytic enzymes lacking secretion signals, already reported to be extracellularly localized in some streptococci, as well as some abundant stress‐resistance proteins. As expected, several proteins involved in N assimilation and oxidative defense system were upregulated in F. alni grown under N‐fixing vs N‐replete conditions. Furthermore, two Raf kinase inhibitor protein homologs that could play a role in cellular signaling, and a hemoglobin‐like protein HbN that could be involved in detoxification of nitric oxide were also upregulated. More surprising, a succinate dehydrogenase was strongly downregulated, which could be linked to the need of pyruvate for the biosynthesis of hopanoids or to reduced oxygen diffusion in vesicles.

The role of Bradyrhizobium japonicum nitric oxide reductase in nitric oxide detoxification in soya bean root nodules

The identification of nitric oxide-bound leghaemoglobin within soya bean nodules has led to the question of how Bradyrhizobium japonicum bacteroids overcome the toxicity of this nitric oxide. It has previously been shown that one candidate for nitric oxide detoxification, the respiratory nitric oxide reductase, is expressed in soya bean nodules from plants supplied with nitrate. In this paper, the role of this enzyme in nitric oxide detoxification is assessed and discussion is provided on other possible B. japonicum nitric oxide detoxification systems.

Inhibition of Macrophage Apoptosis by Neisseria meningitidis Requires Nitric Oxide Detoxification Mechanisms

Host-driven macrophage apoptosis contributes to innate immunity during bacterial infection. Neisseria meningitidis inhibits apoptosis in a variety of cells, but its impact on macrophage apoptosis is unknown. We demonstrate that N. meningitidis prevents macrophage apoptosis via genes encoding nitric oxide detoxification and a porin, PorB.

Dissimilatory Metabolism of Nitrogen Oxides in Bacteria: Comparative Reconstruction of Transcriptional Networks

Bacterial response to nitric oxide (NO) is of major importance since NO is an obligatory intermediate of the nitrogen cycle. Transcriptional regulation of the dissimilatory nitric oxides metabolism in bacteria is diverse and involves FNR-like transcription factors HcpR, DNR, and NnrR; two-component systems NarXL and NarQP; NO-responsive activator NorR; and nitrite-sensitive repressor NsrR. Using comparative genomics approaches, we predict DNA-binding motifs for these transcriptional factors and describe corresponding regulons in available bacterial genomes. Within the FNR family of regulators, we observed a correlation of two specificity-determining amino acids and contacting bases in corresponding DNA recognition motif. Highly conserved regulon HcpR for the hybrid cluster protein and some other redox enzymes is present in diverse anaerobic bacteria, including Clostridia, Thermotogales, and delta-proteobacteria. NnrR and DNR control denitrification in alpha- and beta-proteobacteria, respectively. Sigma-54-dependent NorR regulon found in some gamma- and beta-proteobacteria contains various enzymes involved in the NO detoxification. Repressor NsrR, which was previously known to control only nitrite reductase operon in Nitrosomonas spp., appears to be the master regulator of the nitric oxides' metabolism, not only in most gamma- and beta-proteobacteria (including well-studied species such as Escherichia coli), but also in Gram-positive Bacillus and Streptomyces species. Positional analysis and comparison of regulatory regions of NO detoxification genes allows us to propose the candidate NsrR-binding motif. The most conserved member of the predicted NsrR regulon is the NO-detoxifying flavohemoglobin Hmp. In enterobacteria, the regulon also includes two nitrite-responsive loci, nipAB (hcp-hcr) and nipC (dnrN), thus confirming the identity of the effector, i.e. nitrite. The proposed NsrR regulons in Neisseria and some other species are extended to include denitrification genes. As the result, we demonstrate considerable interconnection between various nitrogen-oxides-responsive regulatory systems for the denitrification and NO detoxification genes and evolutionary plasticity of this transcriptional network.

Novel nirK Cluster Genes in Nitrosomonas europaea Are Required for NirK-Dependent Tolerance to Nitrite

Nitrite reductase (NirK) of Nitrosomonas europaea confers tolerance to nitrite (NO2-). The nirK gene is clustered with three genes of unknown physiological function: ncgABC. At present, this organization is unique to nitrifying bacteria. Here we report that the ncgABC gene products facilitate NirK-dependent NO2- tolerance by reversing the negative physiological effect that is associated with the activity of NirK in their absence. We hypothesize that the ncg gene products are involved in the detoxification of nitric oxide that is produced by NirK.

Transcriptional regulation of the flavohemoglobin gene for aerobic nitric oxide detoxification by the second nitric oxide-responsive regulator of Pseudomonas aeruginosa.

The regulatory gene for a sigma54-dependent-type transcriptional regulator, fhpR, is located upstream of the fhp gene for flavohemoglobin in Pseudomonas aeruginosa. Transcription of fhp was induced by nitrate, nitrite, nitric oxide (NO), and NO-generating reagents. Analysis of the fhp promoter activity in mutant strains deficient in the denitrification enzymes indicated that the promoter was regulated by NO or related reactive nitrogen species. The NO-responsive regulation was operative in a mutant strain deficient in DNR (dissimilatory nitrate respiration regulator), which is the NO-responsive regulator required for expression of the denitrification genes. A binding motif for sigma54 was found in the promoter region of fhp, but an FNR (fumarate nitrate reductase regulator) box was not. The fhp promoter was inactive in the fhpR or rpoN mutant strain, suggesting that the NO-sensing regulation of the fhp promoter was mediated by FhpR. The DNR-dependent denitrification promoters (nirS, norC, and nosR) were active in the fhpR or rpoN mutants. These results indicated that P. aeruginosa has at least two independent NO-responsive regulatory systems. The fhp or fhpR mutant strains showed sensitivity to NO-generating reagents under aerobic conditions but not under anaerobic conditions. These mutants also showed significantly low aerobic NO consumption activity, indicating that the physiological role of flavohemoglobin in P. aeruginosa is detoxification of NO under aerobic conditions.

Transcriptional Responses of Escherichia coli to S-Nitrosoglutathione under Defined Chemostat Conditions Reveal Major Changes in Methionine Biosynthesis

Nitric oxide and nitrosating agents exert powerful antimicrobial effects and are central to host defense and signal transduction. Nitric oxide and S-nitrosothiols can be metabolized by bacteria, but only a few enzymes have been shown to be important in responses to such stresses. Glycerol-limited chemostat cultures in defined medium of Escherichia coli MG1655 were used to provide bacteria in defined physiological states before applying nitrosative stress by addition of S-nitrosoglutathione (GSNO). Exposure to 200 microm GSNO for 5 min was sufficient to elicit an adaptive response as judged by the development of NO-insensitive respiration. Transcriptome profiling experiments were used to investigate the transcriptional basis of the observed adaptation to the presence of GSNO. In aerobic cultures, only 17 genes were significantly up-regulated, including genes known to be involved in NO tolerance, particularly hmp (encoding the NO-consuming flavohemoglobin Hmp) and norV (encoding flavorubredoxin). Significantly, none of the up-regulated genes were members of the Fur regulon. Six genes involved in methionine biosynthesis or regulation were significantly up-regulated; metN, metI, and metR were shown to be important for GSNO tolerance, because mutants in these genes exhibited GSNO growth sensitivity. Furthermore, exogenous methionine abrogated the toxicity of GSNO supporting the hypothesis that GSNO nitrosates homocysteine, thereby withdrawing this intermediate from the methionine biosynthetic pathway. Anaerobically, 10 genes showed significant up-regulation, of which norV, hcp, metR, and metB were also up-regulated aerobically. The data presented here reveal new genes important for nitrosative stress tolerance and demonstrate that methionine biosynthesis is a casualty of nitrosative stress.

Dissimilatory Metabolism of Nitrogen Oxides in Bacteria: Comparative Reconstruction of Transcriptional Networks

Bacterial response to nitric oxide (NO) is of major importance since NO is an obligatory intermediate of the nitrogen cycle. Transcriptional regulation of the dissimilatory nitric oxides metabolism in bacteria is diverse and involves FNR-like transcription factors HcpR, DNR and NnrR, two-component systems NarXL and NarQP, NO-responsive activator NorR, and nitrite sensitive repressor NsrR. Using comparative genomics approaches we predict DNA-binding signals for these transcriptional factors and describe corresponding regulons in available bacterial genomes. Within the FNR family of regulators, we observed a correlation of two specificity-determining amino acids and contacting bases in corresponding DNA signal. Highly conserved regulon HcpR for the hybrid cluster protein and some other redox enzymes is present in diverse anaerobic bacteria including Clostridia, Thermotogales and delta-proteobacteria. NnrR and DNR control denitrification in alpha- and beta-proteobacteria, respectively. Sigma-54-dependent NorR regulon found in some gamma- and beta-proteobacteria contains various enzymes involved in the NO detoxification. Repressor NsrR, which was previously known to control only nitrite reductase operon in Nitrosomonas spp., appears to be the master regulator of the nitric oxides metabolism not only in most gamma- and beta-proteobacteria (including well-studied species like Escherichia coli), but also in Gram-positive Bacillus and Streptomyces species. Positional analysis and comparison of regulatory regions of NO detoxification genes allows us to propose the candidate NsrR-binding signal. The most conserved member of the predicted NsrR regulon is the NO-detoxifying flavohemoglobin Hmp. In enterobacteria, the regulon includes also two nitrite-responsive loci, nipAB (hcp-hcr) and nipC (dnrN), thus confirming the identity of the effector, i.e., nitrite. The proposed NsrR regulons in Neisseria and some other species are extended to include denitrification genes. As the result, we demonstrate considerable interconnection between various nitrogen-oxides-responsive regulatory systems for the denitrification and NO detoxification genes and evolutionary plasticity of this transcriptional network.

New Genes Implicated in the Protection of Anaerobically Grown Escherichia coli against Nitric Oxide

Nitric oxide produced by activated macrophages plays a key role as one of the immune system's weapons against pathogens. Because the lifetime of nitric oxide is short in aerobic conditions, whereas in anaerobic conditions the cytotoxic effects of nitric oxide are greatly increased as in the infection/inflammation processes, it is important to establish which systems are able to detoxify nitric oxide under anaerobic conditions. In the present work a new set of Escherichia coli K-12 genes conferring anaerobic resistance to nitric oxide is presented, namely the gene product of YtfE and a potential transcriptional regulator of the helix-turn-helix LysR-type (YidZ). The crucial role of flavohemoglobin for anaerobic nitric oxide protection is also demonstrated. Furthermore, nitric oxide is shown to cause a significant alteration of the global E. coli gene transcription profile that includes the increase of the transcript level of genes encoding for detoxification enzymes, iron-sulfur cluster assembly systems, DNA-repairing enzymes, and stress response regulators.

Nitric oxide detoxification – a new era for bacterial globins in biotechnology?

For more than a decade, the expression of Vitreoscilla hemoglobin (VHb) has been used to improve the growth and/or productivity of various organisms that are important for the production of valuable metabolites and recombinant proteins by biotechnological processes. Extensive experimental data have shown that VHb enhances the energy status of the cell under oxygen-limited conditions, presumably by improving the supply of intracellular oxygen. Recently, bacterial globin proteins have gained more attention in research because of their ability to detoxify nitric oxide (NO) in vivo. These new results have increased our knowledge, encouraging us to reconsider the role of VHb in vivo. The expression of heterologous globins might improve cellular protection against nitrosative stress under oxygen-limited conditions.

Arabidopsis nonsymbiotic hemoglobin AHb1 modulates nitric oxide bioactivity.

Nitric oxide (NO) is a widespread signaling molecule, and numerous targets of its action exist in plants. Whereas the activity of NO in erythrocytes, microorganisms, and invertebrates has been shown to be regulated by several hemoglobins, the function of plant hemoglobins in NO detoxification has not yet been elucidated. Here, we show that Arabidopsis thaliana nonsymbiotic hemoglobin AHb1 scavenges NO through production of S-nitrosohemoglobin and reduces NO emission under hypoxic stress, indicating its role in NO detoxification. However, AHb1 does not affect NO-mediated hypersensitive cell death in response to avirulent Pseudomonas syringae, suggesting that it is not involved in the removal of NO bursts originated from acute responses when NO mediates crucial defense signaling functions.

Inducible defense mechanism against nitric oxide in Candida albicans.

The yeast Candida albicans is an opportunistic pathogen that threatens patients with compromised immune systems. Immune cell defenses against C. albicans are complex but typically involve the production of reactive oxygen species and nitrogen radicals such as nitric oxide (NO) that damage the yeast or inhibit its growth. Whether Candida defends itself against NO and the molecules responsible for this defense have yet to be determined. The defense against NO in various bacteria and the yeast Saccharomyces cerevisiae involves an NO-scavenging flavohemoglobin. The C. albicans genome contains three genes encoding flavohemoglobin-related proteins, CaYHB1, CaYHB4, and CaYHB5. To assess their roles in NO metabolism, we constructed strains lacking each of these genes and demonstrated that just one, CaYHB1, is responsible for NO consumption and detoxification. In C. albicans, NO metabolic activity and CaYHB1 mRNA levels are rapidly induced by NO and NO-generating agents. Loss of CaYHB1 increases the sensitivity of C. albicans to NO-mediated growth inhibition. In mice, infections with Candida strains lacking CaYHB1 still resulted in lethality, but virulence was decreased compared to that in wild-type strains. Thus, C. albicans possesses a rapid, specific, and highly inducible NO defense mechanism involving one of three putative flavohemoglobin genes.

Redox-dependent Open and Closed Forms of the Active Site of the Bacterial Respiratory Nitric-oxide Reductase Revealed by Cyanide Binding Studies

The bacterial respiratory nitric-oxide reductase (NOR) catalyzes the respiratory detoxification of nitric oxide in bacteria and Archaea. It is a member of the well known super-family of heme-copper oxidases but has a [heme Fe-non-heme Fe] active site rather than the [heme Fe-Cu(B)] active site normally associated with oxygen reduction. Paracoccus denitrificans NOR is spectrally characterized by a ligand-to-metal charge transfer absorption band at 595 nm, which arises from the high spin ferric heme iron of a micro-oxo-bridged [heme Fe(III)-O-Fe(III)] active site. On reduction of the nonheme iron, the micro-oxo bridge is broken, and the ferric heme iron is hydroxylated or hydrated, depending on the pH. At present, the catalytic cycle of NOR is a matter of much debate, and it is not known to which redox state(s) of the enzyme nitric oxide can bind. This study has used cyanide to probe the nature of the active site in a number of different redox states. Our observations suggest that the micro-oxo-bridged [heme Fe(III)-O-Fe(III)] active site represents a closed or resting state of NOR that can be opened by reduction of the non-heme iron.

Isolation of flavohemoglobin from the actinomycete Streptomyces antibioticus grown without external nitric oxide stress.

A flavocytochrome protein was isolated from the actinomycete Streptomyces antibioticus. The purified protein contained protoheme and FAD, and its M(r) was estimated to be 52000. The absorption spectra in its resting oxidized, dithionite-reduced, carbon monoxide-bound, and oxygenated (O(2)-bound) forms were characteristic of those of flavohemoglobin (Fhb). The N-terminal amino acid sequence showed high identities to those of other Fhb's. Furthermore, the actinomycete flavocytochrome scavenged nitric oxide in the presence of NADH. These results demonstrated that the flavocytochrome is the first Fhb purified from actinomycetes. The actinomycete Fhb was produced in S. antibioticus cells in large amounts without any external nitric oxide (NO) stress, which is indicative of a physiological function of Fhb other than detoxification of NO.

Flavohemoglobin Hmp, but Not Its Individual Domains, Confers Protection from Respiratory Inhibition by Nitric Oxide in Escherichia coli

Escherichia coli possesses a two-domain flavohemoglobin, Hmp, implicated in nitric oxide (NO) detoxification. To determine the contribution of each domain of Hmp toward NO detoxification, we genetically engineered the Hmp protein and separately expressed the heme (HD) and the flavin (FD) domains in a defined hmp mutant. Expression of each domain was confirmed by Western blot analysis. CO-difference spectra showed that the HD of Hmp can bind CO, but the CO adduct showed a slightly blue-shifted peak. Overexpression of the HD resulted in an improvement of growth to a similar extent to that observed with the Vitreoscilla hemeonly globin Vgb, whereas the FD alone did not improve growth. Viability of the hmp mutant in the presence of lethal concentrations of sodium nitroprusside was increased (to 30% survival after 2 h in 5 mM sodium nitroprusside) by overexpressing Vgb or the HD. However, maximal protection was provided only by holo-Hmp (75% survival under the same conditions). Cellular respiration of the hmp mutant was instantaneously inhibited in the presence of 13.5 microM NO but remained insensitive to NO inhibition when these cells overexpressed Hmp. When HD or FD was expressed separately, no significant protection was observed. By contrast, overexpression of Vgb provided partial protection from NO respiratory inhibition. Our results suggest that, despite the homology between the HD from Hmp and Vgb (45% identity), their roles seem to be quite distinct.

Neuroglobin, a novel member of the globin family, is expressed in focal regions of the brain.

Hemoproteins are widely distributed among unicellular eukaryotes, plants, and animals. In addition to myoglobin and hemoglobin, a third hemoprotein, neuroglobin, has recently been isolated from vertebrate brain. Although the functional role of this novel member of the globin family remains unclear, neuroglobin contains a heme-binding domain and may participate in diverse processes such as oxygen transport, oxygen storage, nitric oxide detoxification, or modulation of terminal oxidase activity. In this study we utilized in situ hybridization (ISH) and RT-PCR analyses to examine the expression of neuroglobin in the normoxic and hypoxic murine brain. In the normoxic adult mouse, neuroglobin expression was observed in focal regions of the brain, including the lateral tegmental nuclei, the preoptic nucleus, amygdala, locus coeruleus, and nucleus of the solitary tract. Using ISH and RT-PCR techniques, no significant changes in neuroglobin expression in the adult murine brain was observed in response to chronic 10% oxygen. These results support the hypothesis that neuroglobin is a hemoprotein that is expressed in the brain and may have diverse functional roles.

The NorR protein of Escherichia coli activates expression of the flavorubredoxin gene norV in response to reactive nitrogen species.

The Escherichia coli norVW genes encode a flavorubredoxin and NADH:(flavo)rubredoxin reductase, respectively, which are involved in nitric oxide detoxification under anaerobic growth conditions. Here it is shown that the norVW genes also have a role in protection against reactive nitrogen intermediates generated from nitroprusside. Transcription from the norV promoter is activated by the presence of nitroprusside in the growth medium; activation requires the product of a divergently transcribed regulatory gene, norR.

Respiratory Detoxification of Nitric Oxide by the Cytochromec Nitrite Reductase of Escherichia coli

Nitric oxide is a key element in host defense against invasive pathogens. The periplasmic cytochrome c nitrite reductase (NrfA) of Escherichia coli catalyzes the respiratory reduction of nitrite, but in vitro studies have shown that it can also reduce nitric oxide. The physiological significance of the latter reaction in vivo has never been assessed. In this study the reduction of nitric oxide by Escherichia coli was measured in strains active or deficient in periplasmic nitrite reduction. Nrf(+) cells, harvested from cultures grown anaerobically, possessed a nitric-oxide reductase activity with physiological electron donation of 60 nmol min(-1) x mg dry wt(-1), and an in vivo turnover number of NrfA of 390 NO* s(-1) was calculated. Nitric-oxide reductase activity could not be detected in Nrf(-) strains. Comparison of the anaerobic growth of Nrf(+) and Nrf(-) strains revealed a higher sensitivity to nitric oxide in the NrfA(-) strains. A higher sensitivity to the nitrosating agent S-nitroso-N-acetyl penicillamine (SNAP) was also observed in agar plate disk-diffusion assays. Oxygen respiration by E. coli was also more sensitive to nitric oxide in the Nrf(-) strains compared with the Nrf(+) parent strain. The results demonstrate that active periplasmic cytochrome c nitrite reductase can confer the capacity for nitric oxide reduction and detoxification on E. coli. Genomic analysis of many pathogenic enteric bacteria reveals the presence of nrf genes. The present study raises the possibility that this reflects an important role for the cytochrome c nitrite reductase in nitric oxide management in oxygen-limited environments.

Flavorubredoxin, an Inducible Catalyst for Nitric Oxide Reduction and Detoxification in Escherichia coli

Nitric oxide (NO) is a poison, and organisms employ diverse systems to protect against its harmful effects. In Escherichia coli, ygaA encodes a transcription regulator (b2709) controlling anaerobic NO reduction and detoxification. Adjacent to ygaA and oppositely transcribed are ygaK (encoding a flavorubredoxin (flavoRb) (b2710) with a NO-binding non-heme diiron center) and ygbD (encoding a NADH:(flavo)Rb oxidoreductase (b2711)), which function in NO reduction and detoxification. Mutation of either ygaA or ygaK eliminated inducible anaerobic NO metabolism, whereas ygbD disruption partly impaired the activity. NO-sensitive [4Fe-4S] (de)hydratases, including the Krebs cycle aconitase and the Entner-Doudoroff pathway 6-phosphogluconate dehydratase, were more susceptible to inactivation in ygaK or ygaA mutants than in the parental strain, and these metabolic poisonings were associated with conditional growth inhibitions. flavoRb (NO reductase) and flavohemoglobin (NO dioxygenase) maximally metabolized and detoxified NO in anaerobic and aerobic E. coli, respectively, whereas both enzymes scavenged NO under microaerobic conditions. We suggest designation of the ygaA-ygaK-ygbD gene cluster as the norRVW modulon for NO reduction and detoxification.

Human glutathione transferase P1-1 and nitric oxide carriers; a new role for an old enzyme.

S-Nitrosoglutathione and the dinitrosyl-diglutathionyl iron complex are involved in the storage and transport of NO in biological systems. Their interactions with the human glutathione transferase P1-1 may reveal an additional physiological role for this enzyme. In the absence of GSH, S-nitrosoglutathione causes rapid and stable S-nitrosylation of both the Cys(47) and Cys(101) residues. Ion spray ionization-mass spectrometry ruled out the possibility of S-glutathionylation and confirms the occurrence of a poly-S-nitrosylation in GST P1-1. S-Nitrosylation of Cys(47) lowers the affinity 10-fold for GSH, but this negative effect is minimized by a half-site reactivity mechanism that protects one Cys(47)/dimer from nitrosylation. Thus, glutathione transferase P1-1, retaining most of its original activity, may act as a NO carrier protein when GSH depletion occurs in the cell. The dinitrosyl-diglutathionyl iron complex, which is formed by S-nitrosoglutathione decomposition in the presence of physiological concentrations of GSH and traces of ferrous ions, binds with extraordinary affinity to one active site of this dimeric enzyme (K(i) < 10(-12) m) and triggers negative cooperativity in the vacant subunit (K(i) = 10(-9) m). The complex bound to the enzyme is stable for hours, whereas in the free form and at low concentrations, its life time is only a few minutes. ESR and molecular modeling studies provide a reasonable explanation of this strong interaction, suggesting that Tyr(7) and enzyme-bound GSH could be involved in the coordination of the iron atom. All of the observed findings suggest that glutathione transferase P1-1, by means of an intersubunit communication, may act as a NO carrier under different cellular conditions while maintaining its well known detoxificating activity toward dangerous compounds.