Nitroxyl Anion Research Articles

Analysis of the pathways of nitric oxide utilization in mitochondria

The regulatory role that mitochondria play in cell dysfunction and cell-death pathways involves the concept of a complex and multisite regulation of cellular respiration and energy production signaled by cellular and intercellular messengers. Hence, the role of nitric oxide, as a physiological regulator acting directly on the mitochondrial respiratory chain acquires further relevance. This article provides a survey of the major regulatory roles of nitric oxide on mitochondrial functions as an expression of two major metabolic pathways for nitric oxide consumption: a reductive pathway, involving mitochondrial ubiquinol and yielding nitroxyl anion and an oxidative pathway involving superoxide anion and yielding peroxynitrite. The modulation of the decay pathways for nitrogen-and oxygen-centered radicals is further analyzed as a function of the redox transitions of mitochondrial ubiquinol. The interplay among these redox processes and its implications for mitochondrial function is discussed in terms of the mitochondrial steady-state levels (and gradients) of nitric oxide and superoxide anion.

Attenuation of NMDA Receptor Activity and Neurotoxicity by Nitroxyl Anion, NO −

Recent evidence indicates that the NO-related species, nitroxyl anion (NO −), is produced in physiological systems by several redox metal–containing proteins, including hemoglobin, nitric oxide synthase (NOS), superoxide dismutase, and S-nitrosothiols (SNOs), which have recently been identified in brain. However, the chemical biology of NO − remains largely unknown. Here, we show that NO −—unlike NO ·, but reminiscent of NO + transfer (or S-nitrosylation)—reacts mainly with Cys-399 in the NR2A subunit of the N-methyl-D-aspartate (NMDA) receptor to curtail excessive Ca 2+ influx and thus provide neuroprotection from excitotoxic insults. This effect of NO − closely resembles that of NOS, which also downregulates NMDA receptor activity under similar conditions in culture.

Neuronal protection and destruction by NO.

Nitric oxide (NO)-related species include different redox states of the NO group, which have recently been reported to exist endogenously in biological tissues including the brain. The importance of these different NO-related species is that their distinct chemical reactivities can influence the life and death of neurons in response to various insults. In the case of NO+ equivalents (having one less electron than NO.), the mechanism of reaction often involves S-nitrosylation or transfer of the NO group to the sulfhydryl of a cysteine residue (or more properly to a thiolate anion) to form an RS-NO; further oxidation of critical thiols can possibly then form disulfide bonds from neighboring cysteine residues. We have mounted both physiological and chemical evidence that N-methyl-D-aspartate receptor (NMDAR) activity and caspase enzyme activity can be decreased by S-nitrosylation, as can other signaling molecules involved in neuronal apoptotic pathways, to afford neuroprotection. Over the past 5 years, beginning with our report on the NMDAR, evidence has accumulated that S-nitrosylation can regulate the biological activity of a great variety of proteins, in some ways akin to phosphorylation. Thus, this chemical reaction is gaining acceptance as a newly-recognized molecular switch to control protein function via reactive thiol groups, such as those encountered on the NMDAR and in the active site of caspases. One method of producing S-nitrosylation of the NMDAR and caspases is the administration of nitroglycerin, and nitroglycerin can be neuroprotective in acute focal ischemia/reperfusion models via mechanisms other than increasing cerebral blood flow. In contrast, NO* itself does not appear to react with thiol under physiological conditions. In fact, the favored reaction of NO* is with O2*- (superoxide anion) to form ONOO- (peroxynitrite), which can lead to neurotoxicity. A third NO-related species with one added electron compared to NO* is nitroxyl anion (NO-). NO- -unlike NO* but reminiscent of NO+ transfer - reacts with critical thiol groups of the NMDA receptor to curtail excessive Ca2+ influx and thus provide neuroprotection from excitotoxic insults.

Cytotoxicity and Site-specific DNA Damage Induced by Nitroxyl Anion (NO−) in the Presence of Hydrogen Peroxide: IMPLICATIONS FOR VARIOUS PATHOPHYSIOLOGICAL CONDITIONS

Nitroxyl anion (NO(-)), the one-electron reduction product of nitric oxide (NO(.)), is formed under various physiological conditions. We have used four different assays (DNA strand breakage, 8-oxo-deoxyguanosine formation in calf thymus DNA, malondialdehyde generation from 2'-deoxyribose, and analysis of site-specific DNA damage using (32)P-5'-end-labeled DNA fragments of the human p53 tumor suppressor gene and the c-Ha-ras-1 protooncogene) to study the effects of NO(-) generated from Angeli's salt on DNA damage. It was found that strong oxidants are generated from NO(-), especially in the presence of H(2)O(2) plus Fe(III)-EDTA or Cu(II). NO(.) released from diethylamine-NONOate had no such effect. Distinct effects of hydroxyl radical (HO(.)) scavengers and patterns of site-specific DNA cleavage caused by Angeli's salt alone or by Angeli's salt, H(2)O(2) plus metal ion suggest that NO(-) acts as a reductant to catalyze the formation of the HO(.) from H(2)O(2) plus Fe(III) and formation of Cu(I)-peroxide complexes with a reactivity similar to HO(.) from H(2)O(2) and Cu(II). Angeli's salt and H(2)O(2) exerted synergistically cytotoxic effects to MCF-7 cells, determined by lactate dehydrogenase release assay. Thus NO(-) may play an important role in the etiology of various pathophysiological conditions such as inflammation and neurodegenerative diseases, especially when H(2)O(2) and transition metallic ions are present.

Induction of DNA strand breakage and base oxidation by nitroxyl anion through hydroxyl radical production

Nitroxyl anion (NO −), the one-electron reduction product of nitric oxide (NO ), has been reported to be formed under various physiological conditions and to be cytotoxic, although the mechanism responsible for the toxic effects has not been identified. We have studied the effects of NO − generated from Angeli’s salt (sodium trioxodinitrate) or Piloty’s acid ( N-hydoxybenzenesulfonamide) on DNA strand breakage and DNA base oxidation in vitro. Induction of strand breakage was dose- and time-dependent upon incubation of plasmid pBR322 with Angeli’s salt or Piloty’s acid. Similarly, 8-oxo-2′-deoxyguanosine and malondialdehyde were formed when calf-thymus DNA or 2′-deoxyribose, respectively, were incubated with Angeli’s salt. Electron acceptors (ferricyanide, 4-hydroxy-TEMPO), that convert NO − to NO , inhibited the reactions, indicating that NO −, but not NO , is responsible for the reactions. Furthermore, the reactions were also inhibited by the presence of hydroxyl radical (HO ) scavengers, antioxidants, metal chelators and superoxide dismutase and catalase, implying involvement of free HO . These results suggest that NO − is a possible endogenous source of HO , that may be formed either directly from the reaction product of NO − with NO (N 2O 2 •−) or indirectly through H 2O 2 formation. Thus NO − may play an important role as a cause of diverse pathophysiological conditions such as inflammation and neurodegenerative diseases.

Analysis of 3-Nitrotyrosine in Biological Fluids and Protein Hydrolyzates by High-Performance Liquid Chromatography Using a Postseparation, On-line Reduction Column and Electrochemical Detection: Results with Various Nitrating Agents

Nitric oxide reacts rapidly with superoxide to form the strong nitrating agent peroxynitrite, which is responsible for much of the tissue damage associated with diverse pathophysiological conditions such as inflammation. The occurrence of free or protein-bound nitrotyrosine (NTYR) has been considered as evidence forin vivoformation of peroxynitrite. However, various agents can nitrate tyrosine, and their relative significancein vivohas not been determined due to lack of a sensitive method to analyze NTYR in tissue proteins and biological fluids. We have developed a new HPLC-electrochemical detection method to analyze NTYR in protein hydrolyzates or biological fluids. The sample is injected directly into a reversed-phase HPLC column and NTYR is subsequently reduced by a platinum column to 3-aminotyrosine, which is quantified with an electrochemical detector. The method is simple, selective, and sensitive (detection limit, 0.1 pmol per 20-μl injection). We have applied this method to comparein vitrothe ability of various nitrating agents to form NTYR in bovine serum albumin and human plasma. Yields of NTYR formed in human plasma proteins incubated with 1 or 10 mM nitrating agent decreased in the following order: synthetic peroxynitrite > 3-morpholino-sydonimine, a generator of both NO and superoxide > Angeli's salt, which forms nitroxyl anion (NO−) > spermine-NONOate, which releases NO > sodium nitrite plus hypochlorite, which forms the nitrating agent nitryl chloride (NO2Cl). A simple purification method using a C18 Sep-Pak cartridge is also described for analysis of free NTYR in human plasma.

NO donor and biological properties of different benzofuroxans.

To investigate the effect of benzofusion on NO donor properties and related biological activities of the furoxan system. The biological properties considered were the ability to increase the cytosolic levels of cGMP in C6 cells and vasodilation. NO donor properties were investigated either in the presence or the absence of cysteine by using the Griess reaction, chemiluminescence, and gas chromatography. Increase of cytosolic cGMP levels were evaluated by radioimmunoassay. Vasodilating activity was assessed on rat aorta strips precontracted with noradrenaline, in the presence and the absence of oxyhemoglobin (HbO2) and methylene blue (MB), respectively. Benzofuroxan and its methyl and cyano derivatives were unable to release NO under the experimental conditions. Generally these compounds displayed feeble vasodilating properties and were able to weakly stimulate soluble guanylate cyclase (sGC). By contrast, benzodifuroxan and benzotrifuroxan were able to produce both NO* and its reduced form NO- , the nitroxyl anion. They displayed potent vasodilating properties and were able to increase cytosolic levels of cGMP in a concentration-dependent manner. The simple benzofuroxans considered here are devoid of the capability to release NO, they weakly stimulate sGC as well as manifest feeble vasodilating properties by a mechanism that does not involve a thiol-induced NO production. By contrast, benzodifuroxan and benzotrifuroxan behave as typical NO donor furoxans.

Antioxidant and pro-oxidant actions of flavonoids: effects on DNA damage induced by nitric oxide, peroxynitrite and nitroxyl anion

Antioxidant and pro-oxidant activities of flavonoids have been reported. We have studied the effects of 18 flavonoids and related phenolic compounds on DNA damage induced by nitric oxide (NO), peroxynitrite, and nitroxyl anion (NO −). Similarly to our previous findings with catecholamines and catechol-estrogens, DNA single-strand breakage was induced synergistically when pBR322 plasmid was incubated in the presence of an NO-releasing compound (diethylamine NONOate) and a flavonoid having an ortho-trihydroxyl group in either the B ring (e.g., epigallocatechin gallate) or the A ring (e.g., quercetagetin). Either NO or any of the above flavonoids alone did not induce strand breakage significantly. However, most of the tested flavonoids inhibited the peroxynitrite-mediated formation of 8-nitroguanine in calf-thymus DNA, measured by a new HPLC-electrochemical detection method, as well as the peroxynitrite-induced strand breakage. NO − generated from Angeli’s salt caused DNA strand breakage, which was also inhibited by flavonoids but at only high concentrations. On the basis of these findings, we propose that NO − and/or peroxynitrite could be responsible for DNA strand breakage induced by NO and a flavonoid having an ortho-trihydroxyl group. Our results indicate that flavonoids have antioxidant properties, but some act as pro-oxidants in the presence of NO.

Reactions catalyzed by tetrahydrobiopterin-free nitric oxide synthase.

Murine macrophage nitric oxide synthase (NOS) was expressed in E. coli and purified in the presence (holoNOS) or absence (H4B-free NOS) of (6R)-tetrahydro-L-biopterin (H4B). Isolation of active enzyme required the coexpression of calmodulin. Recombinant holoNOS displayed similar spectral characteristics and activity as the enzyme isolated from murine macrophages. H4B-free NOS exhibited a Soret band at approximately 420 nm and, by analytical gel filtration, consisted of a mixture of monomers and dimers. H4B-free NOS catalyzed the oxidation of NG-hydroxy-L-arginine (NHA) with either hydrogen peroxide (H2O2) or NADPH and O2 as substrates. No product formation from arginine was observed under either condition. The amino acid products of NHA oxidation in both the H2O2 and NADPH/O2 reactions were determined to be citrulline and Ndelta-cyanoornithine (CN-orn). Nitrite and nitrate were also formed. Chemiluminescent analysis did not detect the formation of nitric oxide (*NO) in the NADPH/O2 reaction. The initial inorganic product of the NADPH/O2 reaction is proposed to be the nitroxyl anion (NO-) based on the formation of a ferrous nitrosyl complex using the heme domain of soluble guanylate cyclase as a trap, and the formation of a ferrous nitrosyl complex of H4B-free NOS during turnover of NHA and NADPH. NO- is unstable and, under the conditions of the reaction, is oxidized to nitrite and nitrate. At 25 degreesC, the H2O2-supported reaction had a specific activity of 120 +/- 14 nmol min-1 mg-1 and the NADPH-supported reaction had a specific activity of 31 +/- 6 nmol min-1 mg-1 with a KM,app for NHA of 129 +/- 9 microM. HoloNOS catalyzed the H2O2-supported reaction with a specific activity of 815 +/- 30 nmol min-1 mg-1 and the NADPH-dependent reaction to produce *NO and citrulline at 171 +/- 20 nmol min-1 mg-1 with a KM, app for NHA in the NADPH reaction of 36.9 +/- 0.3 microM.

Reactions of nitric oxide with mitochondrial cytochrome c: a novel mechanism for the formation of nitroxyl anion and peroxynitrite.

The aerobic reactions of nitric oxide with cytochrome c were analysed. Nitric oxide (NO) reacts with ferrocytochrome c at a rate of 200 M-1 s-1 to form ferricytochrome c and nitroxyl anion (NO-). Ferricytochrome c was detected by optical spectroscopy; NO- was detected by trapping with metmyoglobin (Mb3+) to form the EPR-detectable Mb-nitrosyl complex, and by the formation of dimers in yeast ferrocytochrome c via cross-linking of the free cysteine residue. The NO- formed subsequently reacted with oxygen to form peroxynitrite, as measured by the oxidation of dihydrorhodamine 123. NO binds to ferricytochrome c to form the ferricytochrome c-NO complex. The on-rate for this reaction is 1.3+/-0.4x10(3) M-1.s-1, and the off-rate is 0.087+/-0.054 s-1. The dissociation constant (Kd) of the complex is 22+/-7 microM. These reactions of NO with cytochrome c are likely to be relevant to mitochondrial metabolism of NO. Ferricytochrome c can act as a reversible sink for excess NO in the mitochondria. The reduction of NO to NO- by ferrocytochrome c may play a role in the irreversible inhibition of mitochondrial oxygen consumption by peroxynitrite. It is generally assumed that peroxynitrite would be formed in mitochondria via the reaction of NO with superoxide. The finding that NO- is formed from the reaction of NO and ferrocytochrome c provides a means of producing peroxynitrite in the absence of superoxide, via the reaction of NO- with oxygen.

Intracellular but not extracellular conversion of nitroxyl anion into nitric oxide leads to stimulation of human neutrophil migration.

Considerable controversy exists in the literature with regard to the nature of the agent mediating the biological effects of nitroxyl (NO-) donors. Here it is demonstrated that Angeli's salt (AS), a generator of NO-, enhanced human neutrophil migration. Under aerobic conditions, AS was converted to peroxynitrite to a small extent. However, using methionine, a scavenger of peroxynitrite, it was shown that peroxynitrite was not involved in AS-induced migration. AS equally enhanced human neutrophil migration under aerobic and anaerobic conditions, which strongly suggests that extracellular conversion of NO- to .NO by oxygen was not required. Furthermore, metHb and L-cysteine, which react more readily with NO- than with .NO, inhibited AS-induced migration, whereas the response towards gaseous .NO remained unaffected. AS induced an increase in the intracellular level of cGMP, although the curves for migration and cGMP level appeared to be slightly different in their concentration dependence. An inhibitor of soluble guanylate cyclase and antagonists of cGMP-dependent protein kinase had a more pronounced inhibitory effect on .NO-induced migration than on AS-induced migration. This suggests that the cGMP signalling cascade is partially, but not solely, responsible for AS-induced migration. As it has been demonstrated that soluble guanylate cyclase can only be activated by .NO, and not by NO-, these data indicate that NO- is at least partly converted intracellularly to .NO.

Reactions between nitric oxide and haemoglobin under physiological conditions

The tenet of high-affinity nitric oxide (NO) binding to a haemoglobin (Hb) has shaped our view of haem proteins and of small diffusible signaling molecules. Specifically, NO binds rapidly to haem iron in Hb (k approximately 10[7] M[-1] s[-1]) and once bound, the NO activity is largely irretrievable (Kd approximately 10[-5] s[-1]); the binding is purportedly so tight as to be unaffected by O2 or CO. However, these general principles do not consider the allosteric state of Hb or the nature of the allosteric effector, and they mostly derive from the functional behaviour of fully nitrosylated Hb, whereas Hb is only partially nitrosylated in vivo. Here we show that oxygen drives the conversion of nitrosylhaemoglobin in the 'tense' T (or partially nitrosylated, deoxy) structure to S-nitrosohaemoglobin in the 'relaxed' R (or ligand-bound, oxy) structure. In the absence of oxygen, nitroxyl anion (NO-) is liberated in a reaction producing methaemoglobin. The yields of both S-nitrosohaemoglobin and methaemoglobin are dependent on the NO/Hb ratio. These newly discovered reactions elucidate mechanisms underlying NO function in the respiratory cycle, and provide insight into the aetiology of S-nitrosothiols, methaemoglobin and its related valency hybrids. Mechanistic reexamination of NO interactions with other haem proteins containing allosteric-site thiols may be warranted.

Direct measurement of nitric oxide generation from nitric oxide synthase.

Although nitric oxide synthase (NOS) is widely considered as the major source of NO in biological cells and tissues, direct evidence demonstrating NO formation from the purified enzyme has been lacking. It was recently reported that NOS does not synthesize NO, but rather generates nitroxyl anion (NO-) that is subsequently converted to NO by superoxide dismutase (SOD). To determine if NOS synthesizes NO, electron paramagnetic resonance (EPR) spectroscopy was applied to directly measure NO formation from purified neuronal NOS. In the presence of the NO trap Fe2+-N-methyl-D-glucamine dithiocarbamate, NO gives rise to characteristic EPR signals with g = 2.04 and aN = 12.7 G, whereas NO- is undetectable. In the presence of L-arginine (L-Arg) and cofactors, NOS generated prominent NO signals. This NO generation did not require SOD, and it was blocked by the specific NOS inhibitor N-nitro-L-arginine methyl ester. Isotope-labeling experiments with L-[15N]Arg further demonstrated that NOS-catalyzed NO arose from the guanidino nitrogen of L-Arg. Measurement of the time course of NO formation demonstrated that it paralleled that of L-citrulline. The conditions used in the prior study were shown to result in potent superoxide generation, and this may explain the failure to measure NO formation in the absence of SOD. These experiments provide unequivocal evidence that NOS does directly synthesize NO from L-Arg.

Differential Effects of Nitric Oxide Species on Pc12 Cell Survival

Background: Nitric oxide (NO•) is known to be both neurotoxic and neuroprotective. There are three redox-related species: neutral NO•, the nitroxyl anion (NO-) and the nitrosonium cation(NO+).

The role of glutathione in the transport and catabolism of nitric oxide

Nitric oxide acts as a neuronal and vascular messenger implying diffusion through intracellular environments containing 5–10 mM glutathione. Nitric oxide reacts with glutathione under aerobic conditions generating S-nitrosoglutathione (GSNO). GSNO reacts with glutathione ( k = 8.3 × 10 −3 M −1 · s −1) to generate nitrous oxide and glutathione disulfide (GSSG). Anaerobically, glutathione reacts with nitric oxide generating nitrous oxide and GSSG ( k = 4.8 × 10 −4 s −1 at 5 mM GSH). In both aerobic and anaerobic situations the nitroxyl anion may be an intermediate in the synthesis of nitrous oxide and, under aerobic conditions, nitroxyl anion may generate peroxynitrite. We present a hypothesis for the intracellular interaction between nitric oxide and glutathione.

21] Nitric oxide and metal-catalyzed reactions

This chapter discusses the nitric oxide (NO) and metal-catalyzed reactions. The main biological functions of NO. include the stimulation of the soluble guanylate cyclase and elevation of cyclic guanosine monophosphate (cGMP) in various cells. NO activates soluble guanylate cyclase through an interaction with iron in the heme of the enzyme. NO can undergo numerous reactions; it can act as either a weak oxidizing compound or as a reducing agent, forming NO+ (nitrosonium cation) or NO– (nitroxyl anion). Some of the complexes are diamagnetic and others paramagnetic, which can be detected by electron paramagnetic resonance (EPR). Paramagnetic species are formed from the reaction of NO with ferrous ion in aqueous solution containing one or more additional coordinating anionic ligands. Application of the methods described in the chapter enables to determine the antioxidative effects imposed by NO. on ferrous complexes. The overall effects of NO. as activator, modulator, or inhibitor seem to be dictated by its site of biosynthesis, its concentration, and the presence and concentration of transition metals.

Interaction of Nitric Oxide with Photoexcited Rose Bengal: Evidence for One-Electron Reduction of Nitric Oxide to Nitroxyl Anion

The interaction of nitric oxide (·NO) with Rose Bengal (RB) in the presence of electron donors was investigated. Upon illumination of a mixture of RB and·NO with visible light, an enhancement in the rate of·NO consumption was observed that increased with increasing RB concentration. In the presence of electron donors (NADH, glutathione, or ascorbate), the rates of·NO depletion increased further. NADH enhanced·NO depletion to a greater extent than either glutathione or ascorbate. Photoactivated RB under anaerobic conditions reacts with NADH to form the RB anion radical (RB·−), which has a characteristic visible absorption band centered at 418 nm. Rose Bengal anion radical disproportionates to give RB and a colorless reduced form of RB, RBH−. The net result of this process is the photobleaching of RB. The presence of·NO during irradiation of RB and NADH introduced a lag time into the kinetics of RB photobleaching. The length of this lag time was proportional to the concentration of·NO. A similar lag time, which was also dependent on the·NO concentration, was observed in the kinetics of formation of RB·−. The three-line electron spin resonance (ESR) spectrum of RB·−, with an intensity ratio 1:2:1, was obtained during irradiation of RB and NADH under anaerobic conditions.·NO introduced a concentration-dependent lag time into the kinetics of the appearance of this ESR signal. We propose that·NO oxidizes RB·−to regenerate RB and thus inhibit photobleaching until·NO is consumed. This reaction predicts the formation of NO−, the one-electron reduced form of·NO. Nitrous oxide, a characteristic dimerization product of NO−, was detected by gas chromatography. This evidence indicates the occurrence of a Type I mechanism between photoactivated RB and·NO.

Electrochemical study of the nitroxyl radical derivatives built in the π‐conjugated poly(phenylenevinylene) skeleton

AbstractThe electrochemical oxidation and reduction of the stable neutral nitroxyl radical gave the oxoaminium salt and hydroxylamine or nitroxyl anion, respectively. The electrochemical behavior of the N‐butylnitroxyl radicals or N‐butylhydroxylamines built in to various phenylenevinylene species were discussed in connection with its developed π‐conjugated structure based on cyclic voltammetric measurements. The aromatic nitroxyl radical showed two pairs of oxidation–reduction waves, but the acidic proton (hydroxylamine) changed its cathodic peak to a broad one with a shift to the anodic side. The π‐conjugated poly(phenylenevinylene) backbone and mobile proton of the hydroxylamine unstabilized the nitroxyl radical by retaining the energy gap.

Interactions of Nitric Oxide and Redox-related species with biological targets

The toxicity of NO and the redox-related species NO+( the nitrosonium cation, supplied as the nitroprusside anion) and NO–( the nitroxyl anion, supplied by decomposition of the monoprotonated trioxodinitrate anion or Piloty's acid, N-hydroxybenzenesulfonamide) to the food spoilage organism Clostridium sporogenes, together with that of nitrite, Roussin's black salt and SIN-1, was assessed. Distinct toxic effects were shown by each of the three species. The nitroprusside anion inhibits growth, with Ki(50)= 20 µmol l–1, and hydrogen production to similar extents. At higher concentration of nitroprusside, the cell suspension is clarified, due either to clumping or lysis. Electron microscopy shows that sodium nitroprusside at 10 µmol l–1 causes blistering of cells and, at 20 µmol l–1, lysis of cells. This results from nitrosation of thiol groups in the cell wall. Analysis for thiols shows a decrease of about 90% of the thiol groups in the cell wall for nitroprusside-treated cells, while electron paramagnetic resonance (EPR) spectrometry confirms the production of reduced nitroprusside species resulting from reaction with the thiol groups. EPR also shows the formation of mononuclear dinitrosyliron–sulfur complexes with g= 2.03, which is probably due to the NO released during the reactions of the reduced nitroprusside. Nitric oxide and the nitroxyl ion do not cause lysis of cells but both cause clumping at sufficiently high concentrations. The effects of NO were shown equally on both growth and dihydrogen production, but the nitroxyl anion differs from the other two species in showing greater toxicity towards dihydrogen production than to growth, reflecting the high affinity of this species towards iron–sulfur clusters.

Biochemistry of nitric oxide and its redox-activated forms.

Nitric oxide (NO.), a potentially toxic molecule, has been implicated in a wide range of biological functions. Details of its biochemistry, however, remain poorly understood. The broader chemistry of nitrogen monoxide (NO) involves a redox array of species with distinctive properties and reactivities: NO+ (nitrosonium), NO., and NO- (nitroxyl anion). The integration of this chemistry with current perspectives of NO biology illuminates many aspects of NO biochemistry, including the enzymatic mechanism of synthesis, the mode of transport and targeting in biological systems, the means by which its toxicity is mitigated, and the function-regulating interaction with target proteins.