▪ Abstract At the interface between the innate and adaptive immune systems lies the high-output isoform of nitric oxide synthase (NOS2 or iNOS). This remarkable molecular machine requires at least 17 binding reactions to assemble a functional dimer. Sustained catalysis results from the ability of NOS2 to attach calmodulin without dependence on elevated Ca2+. Expression of NOS2 in macrophages is controlled by cytokines and microbial products, primarily by transcriptional induction. NOS2 has been documented in macrophages from human, horse, cow, goat, sheep, rat, mouse, and chicken. Human NOS2 is most readily observed in monocytes or macrophages from patients with infectious or inflammatory diseases. Sustained production of NO endows macrophages with cytostatic or cytotoxic activity against viruses, bacteria, fungi, protozoa, helminths, and tumor cells. The antimicrobial and cytotoxic actions of NO are enhanced by other macrophage products such as acid, glutathione, cysteine, hydrogen peroxide, or superoxid...

Nitric oxide and macrophage function

Denitrification is a distinct means of energy conservation, making use of N oxides as terminal electron acceptors for cellular bioenergetics under anaerobic, microaerophilic, and occasionally aerobic conditions. The process is an essential branch of the global N cycle, reversing dinitrogen fixation, and is associated with chemolithotrophic, phototrophic, diazotrophic, or organotrophic metabolism but generally not with obligately anaerobic life. Discovered more than a century ago and believed to be exclusively a bacterial trait, denitrification has now been found in halophilic and hyperthermophilic archaea and in the mitochondria of fungi, raising evolutionarily intriguing vistas. Important advances in the biochemical characterization of denitrification and the underlying genetics have been achieved with Pseudomonas stutzeri, Pseudomonas aeruginosa, Paracoccus denitrificans, Ralstonia eutropha, and Rhodobacter sphaeroides. Pseudomonads represent one of the largest assemblies of the denitrifying bacteria within a single genus, favoring their use as model organisms. Around 50 genes are required within a single bacterium to encode the core structures of the denitrification apparatus. Much of the denitrification process of gram-negative bacteria has been found confined to the periplasm, whereas the topology and enzymology of the gram-positive bacteria are less well established. The activation and enzymatic transformation of N oxides is based on the redox chemistry of Fe, Cu, and Mo. Biochemical breakthroughs have included the X-ray structures of the two types of respiratory nitrite reductases and the isolation of the novel enzymes nitric oxide reductase and nitrous oxide reductase, as well as their structural characterization by indirect spectroscopic means. This revealed unexpected relationships among denitrification enzymes and respiratory oxygen reductases. Denitrification is intimately related to fundamental cellular processes that include primary and secondary transport, protein translocation, cytochrome c biogenesis, anaerobic gene regulation, metalloprotein assembly, and the biosynthesis of the cofactors molybdopterin and heme D1. An important class of regulators for the anaerobic expression of the denitrification apparatus are transcription factors of the greater FNR family. Nitrate and nitric oxide, in addition to being respiratory substrates, have been identified as signaling molecules for the induction of distinct N oxide-metabolizing enzymes.

/pdf/cell-biology-and-molecular-basis-of-denitrification-red4l5iffz.pdf

Cell biology and molecular basis of denitrification.

Redox signaling: nitrosylation and related target interactions of nitric oxide.

Regulation of biosynthesis of nitric oxide.

Nitric oxide and superoxide, which are produced by several cell types, rapidly combine to form peroxynitrite. This reaction can result in nitric oxide scavenging, and thus mitigation of the biological effects of superoxide. Also, superoxide can trap and hence modulate the effects of nitric oxide; superoxide dismutase, by controlling superoxide levels, therefore can influence the reaction pathways open to nitric oxide. The production of peroxynitrite, however, causes its own sequelae of events: Although neither .NO nor superoxide is a strong oxidant, peroxynitrite is a potent and versatile oxidant that can attack a wide range of biological targets. The peroxynitrite anion is relatively stable, but its acid, peroxynitrous acid (HOONO), rearranges to form nitrate with a half-life of approximately 1 s at pH 7, 37 degrees C. HOONO exists as a Boltzmann distribution of rotamers; at 5-37 degrees C HOONO has an apparent acidity constant, pKa,app, of 6.8. Oxidation reactions of HOONO can involve two-electron processes (such as an SN2 displacement) or a one-electron transfer (ET) reaction in which the substrate is oxidized by one electron and peroxynitrite is reduced. These oxidation reactions could involve one of two mechanisms. The first mechanism is homolysis of HOONO to give HO. and .NO2, which initially are held together in a solvent cage. This caged pair of radicals (the "geminate" pair) can either diffuse apart, giving free radicals that can perform oxidations, or react together either to form nitrate or to reform HOONO (a process called cage return). A large amount of cage return can explain the small entropy of activation (Arrhenius A-factor) observed for the decomposition of HOONO. A cage mechanism also can explain the residual yield of nitrate that appears to be formed even in the presence of high concentrations of all of the scavengers studied to date, since scavengers capture only free HO. and .NO2 and not caged radicals. If the cage mechanism is correct, the rate of disappearance of peroxynitrite be slower in solvents of higher viscosity, and we do not find this to be the case. The second mechanism is that an activated isomer of peroxynitrous acid, HOONO*, can be formed in a steady state. The HOONO* mechanism can explain the inability of hydroxyl radical scavengers to completely block either nitrate formation or the oxidation of substrates such as methionine, since HOONO* would be less reactive, and therefore more selective, than the hydroxyl radical itself.(ABSTRACT TRUNCATED AT 400 WORDS)

https://www.physiology.org/doi/pdf/10.1152/ajplung.1995.268.5.L699

The chemistry of peroxynitrite: a product from the reaction of nitric oxide with superoxide

Activation of Escherichia coli oxidative stress regulon genes (sodA, zwf, fumC, nfo, etc.) is mediated by a two-stage regulatory system: the redox-sensitive SoxR protein transcriptionally activates the soxS gene, whose product then stimulates transcription of the regulon genes. Previous experiments showed that limited 3' truncation of soxR gene causes constitutive soxRS expression. DNA sequence analysis of the soxR genes from the soxRS-constitutive strains isolated originally (Greenberg et al. (1990) Proc. Natl. Acad. Sci. USA 87, 6181-6185) revealed that three alleles encode amino acid substitutions or a chain termination clustered near the C-terminus of SoxR. Two other single-amino-acid substitutions in constitutive alleles mapped to the helix-turn-helix motif and to a region of unknown function in the center of the polypeptide, respectively. No constitutive mutation was found within the region encoding the cysteines of the SoxR FeS center, in the soxR or soxS promoters, or in the soxS structural gene. Since an in-frame deletion of just nine SoxR residues (136-144; full-length SoxR = 154 residues) gave rise to a powerful constitutive allele, it appears that a small segment of the SoxR C-terminus maintains the protein in the inactive state. Conservely, an intact C-terminus is evidently not required for gene activation by SoxR.

A cluster of constitutive mutations affecting the C-terminus of the Redox-sensitive SoxR transcriptional activator

Nitric oxide (NO.) is produced as a cytotoxic free radical through enzymatic oxidation of L-arginine in activated macrophages. Pure NO. gas was previously found to induce the Escherichia coli soxRS oxidative stress regulon, which is readily monitored by using a soxS'::lac fusion. The soxRS system includes antioxidant defenses, such as a superoxide dismutase and a DNA repair enzyme for oxidative damage, and protects E. coli from the cytotoxicity of NO.-generating macrophages. Previous experiments involved exposing E. coli to a bolus of NO. rather than the steadily generated gas expected of activated macrophages. We show here detectable induction of soxS transcription by NO. delivered at rates as low as 25 microM/h. Maximal induction was observed at 25 microM NO. per h under anaerobic conditions but at 125 microM/h aerobically. After incubation with murine macrophages, soxS expression was induced in the phagocytosed bacteria up to approximately 30-fold after an 8-h exposure. This in vivo induction was almost completely eliminated by the NO. synthase inhibitor NG-monomethyl-L-arginine. The inhibitor increased the survival of a delta soxRS strain but not that of wild-type E. coli after phagocytosis, which suggests that induction of the soxRS regulon by NO. can counteract most of the cytotoxic effects of NO. production by the macrophages. We show that the soxRS-regulated enzyme glucose-6-phosphate dehydrogenase is an important element of the defense against macrophages.

https://iai.asm.org/content/iai/63/3/794.full.pdf

Roles of nitric oxide in inducible resistance of Escherichia coli to activated murine macrophages.

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A cluster of constitutive mutations affecting the C-terminus of the Redox-sensitive SoxR transcriptional activator

Roles of nitric oxide in inducible resistance of Escherichia coli to activated murine macrophages.