The discovery that mammalian cells have the ability to synthesize the free radical nitric oxide (NO) has stimulated an extraordinary impetus for scientific research in all the fields of biology and medicine. Since its early description as an endothelial-derived relaxing factor, NO has emerged as a fundamental signaling device regulating virtually every critical cellular function, as well as a potent mediator of cellular damage in a wide range of conditions. Recent evidence indicates that most of the cytotoxicity attributed to NO is rather due to peroxynitrite, produced from the diffusion-controlled reaction between NO and another free radical, the superoxide anion. Peroxynitrite interacts with lipids, DNA, and proteins via direct oxidative reactions or via indirect, radical-mediated mechanisms. These reactions trigger cellular responses ranging from subtle modulations of cell signaling to overwhelming oxidative injury, committing cells to necrosis or apoptosis. In vivo, peroxynitrite generation represents a crucial pathogenic mechanism in conditions such as stroke, myocardial infarction, chronic heart failure, diabetes, circulatory shock, chronic inflammatory diseases, cancer, and neurodegenerative disorders. Hence, novel pharmacological strategies aimed at removing peroxynitrite might represent powerful therapeutic tools in the future. Evidence supporting these novel roles of NO and peroxynitrite is presented in detail in this review.

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Nitric Oxide and Peroxynitrite in Health and Disease

Nitric oxide contrasts with most intercellular messengers because it diffuses rapidly and isotropically through most tissues with little reaction but cannot be transported through the vasculature due to rapid destruction by oxyhemoglobin. The rapid diffusion of nitric oxide between cells allows it to locally integrate the responses of blood vessels to turbulence, modulate synaptic plasticity in neurons, and control the oscillatory behavior of neuronal networks. Nitric oxide is not necessarily short lived and is intrinsically no more reactive than oxygen. The reactivity of nitric oxide per se has been greatly overestimated in vitro because no drain is provided to remove nitric oxide. Nitric oxide persists in solution for several minutes in micromolar concentrations before it reacts with oxygen to form much stronger oxidants like nitrogen dioxide. Nitric oxide is removed within seconds in vivo by diffusion over 100 microns through tissues to enter red blood cells and react with oxyhemoglobin. The direct toxicity of nitric oxide is modest but is greatly enhanced by reacting with superoxide to form peroxynitrite (ONOO-). Nitric oxide is the only biological molecule produced in high enough concentrations to out-compete superoxide dismutase for superoxide. Peroxynitrite reacts relatively slowly with most biological molecules, making peroxynitrite a selective oxidant. Peroxynitrite modifies tyrosine in proteins to create nitrotyrosines, leaving a footprint detectable in vivo. Nitration of structural proteins, including neurofilaments and actin, can disrupt filament assembly with major pathological consequences. Antibodies to nitrotyrosine have revealed nitration in human atherosclerosis, myocardial ischemia, septic and distressed lung, inflammatory bowel disease, and amyotrophic lateral sclerosis.

Nitric oxide, superoxide, and peroxynitrite: the good, the bad, and ugly.

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.

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Cell biology and molecular basis of denitrification.

Free radicals are common outcome of normal aerobic cellular metabolism. In-built antioxidant system of body plays its decisive role in prevention of any loss due to free radicals. However, imbalanced defense mechanism of antioxidants, overproduction or incorporation of free radicals from environment to living system leads to serious penalty leading to neuro-degeneration. Neural cells suffer functional or sensory loss in neurodegenerative diseases. Apart from several other environmental or genetic factors, oxidative stress (OS) leading to free radical attack on neural cells contributes calamitous role to neuro-degeneration. Though, oxygen is imperative for life, imbalanced metabolism and excess reactive oxygen species (ROS) generation end into a range of disorders such as Alzheimer's disease, Parkinson's disease, aging and many other neural disorders. Toxicity of free radicals contributes to proteins and DNA injury, inflammation, tissue damage and subsequent cellular apoptosis. Antioxidants are now being looked upon as persuasive therapeutic against solemn neuronal loss, as they have capability to combat by neutralizing free radicals. Diet is major source of antioxidants, as well as medicinal herbs are catching attention to be commercial source of antioxidants at present. Recognition of upstream and downstream antioxidant therapy to oxidative stress has been proved an effective tool in alteration of any neuronal damage as well as free radical scavenging. Antioxidants have a wide scope to sequester metal ions involved in neuronal plaque formation to prevent oxidative stress. In addition, antioxidant therapy is vital in scavenging free radicals and ROS preventing neuronal degeneration in post-oxidative stress scenario.

Oxidative stress and neurodegenerative diseases: a review of upstream and downstream antioxidant therapeutic options.

Reactive oxygen species (ROS) are generated as by-products of cellular metabolism, primarily in the mitochondria. When cellular production of ROS overwhelms its antioxidant capacity, damage to cellular macromolecules such as lipids, protein, and DNA may ensue. Such a state of “oxidative stress” is thought to contribute to the pathogenesis of a number of human diseases including those of the lung. Recent studies have also implicated ROS that are generated by specialized plasma membrane oxidases in normal physiological signaling by growth factors and cytokines. In this review, we examine the evidence for ligand-induced generation of ROS, its cellular sources, and the signaling pathways that are activated. Emerging concepts on the mechanisms of signal transduction by ROS that involve alterations in cellular redox state and oxidative modifications of proteins are also discussed.

Reactive oxygen species in cell signaling

The rate constant for the reaction of NO with ·O2− was determined to be (6.7 ± 0.9) × 109 1 mol−1 s−1, considerably higher than previously reported. Rate measurements were made from pH 5.6 to 12.5 ...

The reaction of no with superoxide

The reaction of nitric oxide with organic peroxyl radicals.

Formation and reactions of the ·SH/·S- and HSSH·-/HSS·2- radicals in aqueous solutions have been studied by excimer laser flash photolysis and by pulse radiolysis. Acidic H2S solutions can be photolyzed with 193 nm laser pulses and produce a transient species with λmax at 240 nm, ascribed to the ·SH/·S- radical. Solutions of SH- can be photolyzed also with 248 nm laser pulses to produce the ·SH/·S- radical. The same radical is formed by oxidation of SH- ions with SO4·- and CO3·- radicals. At pH > 5, ·SH/·S- reacts with SH- (kf = 4 × 109 L mol-1 s-1, kr = 5 × 105 s-1) to form HSSH·-/HSS·2-, with λmax at 380 nm. Both ·SH/·S- and HSSH·-/HSS·2- react rapidly with O2; the former produces SO2·- (k = 5 × 109 L mol-1 s-1), and the latter produces O2·- (k = 4 × 108 L mol-1 s-1). Both radicals react with olefinic compounds. The monomeric radical oxidizes Fe(CN)64-, SO32-, ClO2-, and chlorpromazine. The dimeric radical is a weaker oxidant toward ferrocyanide but reduces N-methylpyridinium compounds. The reduction po...

Reduction Potential of the Sulfhydryl Radical: Pulse Radiolysis and Laser Flash Photolysis Studies of the Formation and Reactions of ·SH and HSSH·-in Aqueous Solutions

The kinetics of the acqueous-phase reactions of the free radicals ·OH, ·Cl, and SO· with the halogenated acetates, CH2FCOO−, CHF2COO−, CF3COO−, and with CH2ClCOO−, CHCl2COO−, CCl3COO− were investigated. Generally, the reactivity decreases with increasing halogen substitution and is in the order k(·OH) > k(SO·) > k(·Cl), but there is no general relation between the effect on reactivity of chlorine and fluorine substitution. © 1995 John Wiley & Sons, Inc.

Rate constants for some reactions of free radicals with haloacetates in aqueous solution

Rate constants have been measured for the reactions of the sulfate radical, SO4˙−, with alkanes, alkenes, alcohols, ethers, and amines in 95% acetonitrile solution. The rate constants were in the range of 106 L mol−1 s−1 for the abstraction reactions and 107−109 L mol−1 s−1 for the addition and electron transfer reactions. These values are 20 to 80 times lower than those measured in aqueous solutions. Furthermore, the rate constants for the reactions of SO4˙− with the primary alcohols increase with the number of carbon atoms and then level off, in contrast to the behavior observed in aqueous solution, where the rate constant increases more sharply for the larger alcohols. © 1993 John Wiley & Sons, Inc.

S. Padmaja

Papers

The reaction of no with superoxide

The reaction of nitric oxide with organic peroxyl radicals.

Reduction Potential of the Sulfhydryl Radical: Pulse Radiolysis and Laser Flash Photolysis Studies of the Formation and Reactions of ·SH and HSSH·-in Aqueous Solutions

Rate constants for some reactions of free radicals with haloacetates in aqueous solution

Rate constants for reactions of SO4˙− radicals in acetonitrile