Ecology of Thioploca spp.: nitrate and sulfur storage in relation to chemical microgradients and influence of Thioploca spp. on the sedimentary nitrogen cycle.

Microsensors, including a recently developed NO3(-) biosensor, were applied to measure O(2) and NO3(-) profiles in marine sediments from the upwelling area off central Chile and to investigate the influence of Thioploca spp. on the sedimentary nitrogen metabolism. The studies were performed in undisturbed sediment cores incubated in a small laboratory flume to simulate the environmental conditions of low O(2), high NO3(-), and bottom water current. On addition of NO3(-) and NO2(-), Thioploca spp. exhibited positive chemotaxis and stretched out of the sediment into the flume water. In a core densely populated with Thioploca, the penetration depth of NO3(-) was only 0.5 mm and a sharp maximum of NO3(-) uptake was observed 0.5 mm above the sediment surface. In sediments with only few Thioploca spp., NO3(-) was detectable down to a depth of 2 mm and the maximum consumption rates were observed within the sediment. No chemotaxis toward nitrous oxide (N2O) was observed, which is consistent with the observation that Thioploca does not denitrify but reduces intracellular NO3(-) to NH(4)(+). Measurements of the intracellular NO3(-) and S(0) pools in Thioploca filaments from various depths in the sediment gave insights into possible differences in the migration behavior between the different species. Living filaments containing significant amounts of intracellular NO3(-) were found to a depth of at least 13 cm, providing final proof for the vertical shuttling of Thioploca spp. and nitrate transport into the sediment.     

Anaerobic ammonium oxidation measured in sediments along the Thames estuary, United Kingdom

Until recently, denitrification was thought to be the only significant pathway for N(2) formation and, in turn, the removal of nitrogen in aquatic sediments. The discovery of anaerobic ammonium oxidation in the laboratory suggested that alternative metabolisms might be present in the environment. By using a combination of (15)N-labeled NH(4)(+), NO(3)(-), and NO(2)(-) (and (14)N analogues), production of (29)N(2) and (30)N(2) was measured in anaerobic sediment slurries from six sites along the Thames estuary. The production of (29)N(2) in the presence of (15)NH(4)(+) and either (14)NO(3)(-) or (14)NO(2)(-) confirmed the presence of anaerobic ammonium oxidation, with the stoichiometry of the reaction indicating that the oxidation was coupled to the reduction of NO(2)(-). Anaerobic ammonium oxidation proceeded at equal rates via either the direct reduction of NO(2)(-) or indirect reduction, following the initial reduction of NO(3)(-). Whether NO(2)(-) was directly present at 800 micro M or it accumulated at 3 to 20 micro M (from the reduction of NO(3)(-)), the rate of (29)N(2) formation was not affected, which suggested that anaerobic ammonium oxidation was saturated at low concentrations of NO(2)(-). We observed a shift in the significance of anaerobic ammonium oxidation to N(2) formation relative to denitrification, from 8% near the head of the estuary to less than 1% at the coast. The relative importance of anaerobic ammonium oxidation was positively correlated (P < 0.05) with sediment organic content. This report of anaerobic ammonium oxidation in organically enriched estuarine sediments, though in contrast to a recent report on continental shelf sediments, confirms the presence of this novel metabolism in another aquatic sediment system.     

Denitrification, dissimilatory reduction of nitrate to ammonium, and nitrification in a bioturbated estuarine sediment as measured with N and microsensor techniques

Nitrogen and oxygen transformations were studied in a bioturbated (reworked by animals) estuarine sediment (Norsminde Fjord, Denmark) by using a combination of N isotope (NO(3)), specific inhibitor (C(2)H(2)), and microsensor (N(2)O and O(2)) techniques in a continuous-flow core system. The estuarine water was NO(3) rich (125 to 600 muM), and NO(3) was consistently taken up by the sediment on the four occasions studied. Total NO(3) uptake (3.6 to 34.0 mmol of N m day) corresponded closely to N(2) production (denitrification) during the experimental steady state, which indicated that dissimilatory, as well as assimilatory, NO(3) reduction to NH(4) was insignificant. When C(2)H(2) was applied in the flow system, denitrification measured as N(2)O production was often less (58 to 100%) than the NO(3) uptake because of incomplete inhibition of N(2)O reduction. The NO(3) formed by nitrification and not immediately denitrified but released to the overlying water, uncoupled nitrification, was calculated both from NO(3) dilution and from changes in NO(3) uptake before and after C(2)H(2) addition. These two approaches gave similar results, with rates ranging between 0 and 8.1 mmol of N m day on the four occasions. Attempts to measure total nitrification activity by the difference between NH(4) fluxes before and after C(2)H(2) addition failed because of non-steady-state NH(4) fluxes. The vertical distribution of denitrification and oxygen consumption was studied by use of N(2)O and O(2) microelectrodes. The N(2)O profiles measured during the experimental steady state were often irregularly shaped, and the buildup of N(2)O after C(2)H(2) was added was much too fast to be described by a simple diffusion model. Only bioturbation by a dense population of infauna could explain these observations. This was corroborated by the relationship between diffusive and total fluxes, which showed that only 19 to 36 and 29 to 62% of the total O(2) uptake and denitrification, respectively, were due to diffusion-reaction processes at the regular sediment surface, excluding animal burrows.     

Correlation between anammox activity and microscale distribution of nitrite in a subtropical mangrove sediment

The distribution of anaerobic ammonium oxidation (anammox) in nature has been addressed by only a few environmental studies, and our understanding of how anammox bacteria compete for substrates in natural environments is therefore limited. In this study, we measure the potential anammox rates in sediment from four locations in a subtropical tidal river system. Porewater profiles of NO(x)(-) (NO2- plus NO3-) and NO2- were measured with microscale biosensors, and the availability of NO2- was compared with the potential for anammox activity. The potential rate of anammox increased with increasing distance from the mouth of the river and correlated strongly with the production of nitrite in the sediment and with the average concentration or total pool of nitrite in the suboxic sediment layer. Nitrite accumulated both from nitrification and from NO(x)(-) reduction, though NO(x)(-) reduction was shown to have the greatest impact on the availability of nitrite in the suboxic sediment layer. This finding suggests that denitrification, though using NO2- as a substrate, also provides a substrate for the anammox process, which has been suggested in previous studies where microscale NO2- profiles were not measured.     

Impact of nitrate on the structure and function of bacterial biofilm communities in pipelines used for injection of seawater into oil fields

We studied the impact of NO(3)(-) on the bacterial community composition, diversity, and function in in situ industrial, anaerobic biofilms by combining microsensor profiling, (15)N and (35)S labeling, and 16S rRNA gene-based fingerprinting. Biofilms were grown on carbon steel coupons within a system designed to treat seawater for injection into an oil field for pressurized oil recovery. NO(3)(-) was added to the seawater in an attempt to prevent bacterial H(2)S generation and microbially influenced corrosion in the field. Microprofiling of nitrogen compounds and redox potential inside the biofilms showed that the zone of highest metabolic activity was located close to the metal surface, correlating with a high bacterial abundance in this zone. Upon addition, NO(3)(-) was mainly reduced to NO(2)(-). In biofilms grown in the absence of NO(3)(-), redox potentials of <-450 mV at the metal surface suggested the release of Fe(2+). NO(3)(-) addition to previously untreated biofilms induced a decline (65%) in bacterial species richness, with Methylophaga- and Colwellia-related sequences having the highest number of obtained clones in the clone library. In contrast, no changes in community composition and potential NO(3)(-) reduction occurred upon subsequent withdrawal of NO(3)(-). Active sulfate reduction was below detection levels in all biofilms, but S isotope fractionation analysis of sulfide deposits suggested that it must have occurred either at low rates or episodically. Scanning electron microscopy revealed that pitting corrosion occurred on all coupons, independent of the treatment. However, uniform corrosion was clearly mitigated by NO(3)(-) addition.     

Effects of nitrate and nitrite on dissimilatory iron reduction by Shewanella putrefaciens 200.

The inhibitory effects of nitrate (NO3-) and nitrite (NO2-) on dissimilatory iron (FE3+) reduction were examined in a series of electron acceptor competition experiments using Shewanella putrefaciens 200 as a model iron-reducing microorganism. S. putrefaciens 200 was found to express low-rate nitrate reductase, nitrite reductase, and ferrireductase activity after growth under highly aerobic conditions and greatly elevated rates of each reductase activity after growth under microaerobic conditions. The effects of NO3- and NO2- on the Fe3+ reduction activity of both aerobically and microaerobically grown cells appeared to follow a consistent pattern; in the presence of Fe3+ and either NO3- or NO2-, dissimilatory Fe3+ and nitrogen oxide reduction occurred simultaneously. Nitrogen oxide reduction was not affected by the presence of Fe3+, suggesting that S. putrefaciens 200 expressed a set of at least three physiologically distinct terminal reductases that served as electron donors to NO3-, NO2-, and Fe3+. However, Fe3+ reduction was partially inhibited by the presence of either NO3- or NO2-. An in situ ferrozine assay was used to distinguish the biological and chemical components of the observed inhibitory effects. Rate data indicated that neither NO3- nor NO2- acted as a chemical oxidant of bacterially produced Fe2+. In addition, the decrease in Fe3+ reduction activity observed in the presence of both NO3- and NO2- was identical to the decrease observed in the presence of NO2- alone. These results suggest that bacterially produced NO2- is responsible for inhibiting electron transport to Fe3+.     

Small-scale distribution of interstitial nitrite in freshwater sediment microcosms: the role of nitrate and oxygen availability,and sediment permeability

The spatial distribution of interstitial NO2(-) concentrations was studied in NO3(-)-exposed freshwater sediment microcosms, using pore water extractions as well as ion-selective microsensors. Porewater extractions revealed ecotoxicologically critical NO2(-) concentrations in hypoxic and anoxic sediment layers in which significant NO3(-) consumption took place. In contrast, the use of ion-selective microsensors demonstrated the high capacity of the thin oxic surface layer of the sediments to consume NO2(-) and to produce NO3(-). Two modes of NO3(-) supply to the sediments were compared: In treatments with NO3(-) supply to the overlying water, a subsurface maximum of NO2(-) concentration was observed, coinciding with the site of maximum NO3(-) consumption. When NO3(-) was perfused up through the sediment cores, however, NO2(-) accumulated throughout the entire sediment column. Such spatially extensive NO2(-) accumulations were only observed in sediments poor in organic matter with a relatively high permeability. By manipulating the O2 content of the overlying water, the release of NO2(-) from the sediments could be influenced: In treatments with air-saturated overlying water, the sediments did not release detectable amounts of NO2(-) into the water phase. When kept hypoxic (25% air saturation) instead, significant NO2(-) accumulations were recorded in the overlying water. These findings suggest that in treatments with air-saturated overlying water, NO2(-) that was produced in deeper sediment layers (denitrifying conditions) was completely consumed at the oxic sediment surface (nitrifying conditions) before it could reach the overlying water.     

Effect of nitrite and nitrate on in situ sulfide production in an activated sludge immobilized agar gel film as determined by use of microelectrodes

Microelectrode, fluorescence in situ hybridization (FISH), and denaturing gradient gel electrophoresis (DGGE) analyses were used to investigate the effect of nitrite and nitrate on in situ sulfide production in an activated sludge immobilized agar gel film. Microelectrode measurements of O(2), H(2)S, NO(3)(-), NO(2)(-), and pH revealed that the addition of NO(2)(-) and NO(3)(-) forced sulfate reduction zones deeper in the agar gel and significantly reduced the in situ sulfide production levels. The sulfate reduction zone was consequently separated from O(2) and NO(2)(-) or NO(3)(-) respiration zones with increasing the concentrations of NO(2)(-) and NO(3)(-). These NO(2)(-) and NO(3)(-) treatments had only a transient effect on sulfide production. The in situ sulfide production quickly recovered to the previous levels when NO(2)(-) and NO(3)(-) were removed. The PCR-DGGE and FISH analyses revealed that 2-day-continuous addition of 500 microM NO(3)(-) did not change the metabolically active sulfate-reducing bacterial (SRB) community. On the basis of these data, it could be concluded that the addition of NO(2)(-) and NO(3)(-) did not kill SRB, but induced the interspecies competition for common carbon source (i.e., acetate) between nitrate-reducing heterotrophic bacteria and SRB and enhanced the oxidation of the produced sulfide, which were main possible causes of the suppression of in situ sulfide production in the agar gel.     

Bacterium-based NO2- biosensor for environmental applications

A sensitive NO2- biosensor that is based on bacterial reduction of NO2- to N2O and subsequent detection of the N2O by a built-in electrochemical N2O sensor was developed. Four different denitrifying organisms lacking NO3- reductase activity were assessed for use in the biosensor. The relevant physiological aspects examined included denitrifying characteristics, growth rate, NO2- tolerance, and temperature and salinity effects on the growth rate. Two organisms were successfully used in the biosensor. The preferred organism was Stenotrophomonas nitritireducens, which is an organism with a denitrifying pathway deficient in both NO3- and N2O reductases. Alternatively Alcaligenes faecalis could be used when acetylene was added to inhibit its N2O reductase. The macroscale biosensors constructed exhibited a linear NO2- response at concentrations up to 1 to 2 mM. The detection limit was around 1 microM NO2-, and the 90% response time was 0.5 to 3 min. The sensor signal was specific for NO2-, and interference was observed only with NH2OH, NO, N2O, and H2S. The sensor signal was affected by changes in temperature and salinity, and calibration had to be performed in a system with a temperature and an ionic strength comparable to those of the medium analyzed. A broad range of water bodies could be analyzed with the biosensor, including freshwater systems, marine systems, and oxic-anoxic wastewaters. The NO2- biosensor was successfully used for long-term online monitoring in wastewater. Microscale versions of the NO2- biosensor were constructed and used to measure NO2- profiles in marine sediment.     

Respiration-linked proton flux in Wolinella succinogenes during reduction of N-oxides

Formate uncoupled proton translocation in formate-grown Wolinella succinogenes cells supplied with N-oxides as terminal electron acceptors. In suspensions containing KSCN (but not valinomycin), H2 supported proton translocation when NO3-, NO2-, and NO were provided as oxidants. H+/N-oxide ratios were 4.77 for NO3-, 2.49 for NO2-, and 1.75 for NO. KSCN inhibits N2O reduction thus precluding use of N2O as oxidant. Repeated exposure of cells to NO inhibited their ability to translocated protons with NO as oxidant but only slightly diminished and did not eliminate their capacity for NO3(-)- or NO2(-)-dependent proton flux. Substituting reduced benzyl viologen for H2 and measuring proton uptake provided results consistent with an extramembranal location for the N- oxide reductases. The uncoupler, carbonyl cyanide m-chlorophenylhydrazone, collapsed proton gradients, permitted uptake of 2 mol H+/mol NO3- or NO2-, but unaccountably inhibited NO3- reduction by 50% while leaving H+ uptake stoichiometry of the cells unaffected.     

Effect of nitrate on methane production and fermentation by slurries of human faecal bacteria

Most probable number counts showed that denitrifying species were the numerically predominant NO3- reducing bacteria in the faeces of five methanogenic individuals [about 10(10) bacteria (g dry wt faeces)-1]. In faecal slurries, however, denitrification was a relatively minor route of NO3- dissimilation, since only about 3% of the NO3- was converted to gaseous products, with NO3- being mainly reduced to NO2- and NH4+. When KNO2 was added to the slurries, denitrification became quantitatively more significant with approximately 23% of the NO2- being lost as gaseous products. The addition of KNO3 (10 mM) to slurries containing either starch or casein significantly decreased H2 and CH4 production. The effect of NO3- on methanogenesis was twofold: firstly, H2 accumulation decreased due to diversion of electrons towards NO3-/NO2- reduction, and as a result of H2 being used as an electron donor for NO3- reduction, resulting in the removal of the methanogenic substrate; secondly, there was direct inhibition of methane-producing bacteria by NO3- and NO2-. In starch-containing slurries, acetate: butyrate molar ratios were increased when NO3- was added but this effect was not observed when casein replaced starch. These results show that the ability of NO3-/NO2- to act as an electron sink can significantly influence the major products of the human colonic fermentation.     

Inhibition by sulfide of nitric and nitrous oxide reduction by denitrifying Pseudomonas fluorescens.

The influence of low redox potentials and H2S on NO and N2O reduction by resting cells of denitrifying Pseudomonas fluorescens was studied. Hydrogen sulfide and Ti(III) were added to achieve redox potentials near -200 mV. The control without reductant had a redox potential near +200 mV. Production of 13NO, [13N]N2O, and [13N]N2 from 13NO3- and 13NO2- was followed. Total gas production was similar for all three treatments. The accumulation of 13NO was most significant in the presence of sulfide. A parallel control with autoclaved cells indicated that the 13NO production was largely biological. The sulfide inhibition was more dramatic at the level of N2O reduction; [13N]N2O became the major product instead of [13N]N2, the dominant product when either no reductant or Ti(III) was present. The results indicate that the specific action of sulfide rather than the low redox potential caused a partial inhibition of NO reduction and a strong inhibition of N2O reduction in denitrifying cells.     

Hydrogenase activity in Azospirillum brasilense is inhibited by nitrite, nitric oxide, carbon monoxide, and acetylene.

Nitrite, NO, CO, and C2H2 inhibited O2-dependent H2 uptake (H3H oxidation) in denitrifying Azospirillum brasilense Sp7 grown anaerobically on N2O or NO3-. The apparent Ki values for inhibition of O2-dependent H2 uptake were 20 microM for NO2-, 0.4 microM for NO, 28 microM for CO, and 88 microM for C2H2. These inhibitors also affected methylene blue-dependent H2 uptake, presumably by acting directly on the hydrogenase. Nitrite and NO inhibited H2 uptake irreversibly, whereas inhibition due to CO was easily reversed by repeatedly evacuating and backfilling with N2. The C2H2 inhibition was not readily reversed, partly due to difficulty in removing the last traces of this gas from solution. The NO2- inhibition of malate-dependent respiration was readily reversed by repeatedly washing the cells, in contrast to the effect of NO2- on H2-dependent respiration. These results suggest that the low hydrogenase activities observed in NO3(-)-grown cultures of A. brasilense may be due to the irreversible inhibition of hydrogenase by NO2- and NO produced by NO3- reduction.     

Hydrogen Sulfide Promotes Root Organogenesis in Ipomoea batatas, Salix matsudana and Glycine max

In this report, we demonstrate that sodium hydrosulfide (NaHS), a hydrogen sulfide (H2S) donor, promoted adventitious root formation mediated by auxin and nitric oxide (NO). Application of the H2S donor to seedling cuttings of sweet potato (Ipomoea batatas L.) promoted the number and length of adventltious roots in a dose-dependent manner. It was also verified that H2S or HS- rather than other sulfur-containing components derived from NariS could be attributed to the stimulation of adventitious root formation. A rapid Increase In endogenous H2S, indole acetic acid (IAA) and NO were sequentially observed in shoot tips of sweet potato seedlings treated with HallS. Further investigation showed that HzS-mediated root formation was alleviated by N-l-naphthylphthalamic acid (NPA), an IAA transport inhibitor, and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), an NO scavenger. Similar phenomena in H2S donor-dependent root organogenesis were observed in both excised willow (Sallx matsudana var. tortuosa Vilm) shoots and soybean (Glycine max L.) seedlings. These results indicated that the process of H2S-induced adventitious root formation was likely mediated by IAA and NO, and that H2S acts upstream of IAA and NO signal transduction pathways.     

Phylogeny and distribution of nitrate-storing Beggiatoa spp. in coastal marine sediments

Filamentous sulphide-oxidizing Beggiatoa spp. often occur in large numbers in the coastal seabed without forming visible mats on the sediment surface. We studied the diversity, population structure and the nitrate-storing capability of such bacteria in the Danish Limfjorden and the German Wadden Sea. Their distribution was compared to the vertical gradients of O2, NO3- and H2S as measured by microsensors. The main Beggiatoa spp. populations occurred in a 0.5-3 cm thick intermediate zone, below the depth of oxygen and nitrate penetration but above the zone of free sulphide. The Beggiatoa spp. filaments were found to store nitrate, presumably in liquid vacuoles up to a concentration of 370 mM NO3-, similar to the related large marine sulphur bacteria, Thioploca and Thiomargarita. The observations indicate that marine Beggiatoa spp. can live anaerobically and conserve energy by coupling sulphide oxidation with the reduction of nitrate to dinitrogen and/or ammonia. Calculations of the diffusive nitrate flux and the potential sulphide oxidation by Beggiatoa spp. show that the bacteria may play a critical role for the sulphur cycling and the nitrogen balance in these coastal environments. 16S rDNA sequence analysis shows a large diversity of these uncultured, nitrate-storing Beggiatoa spp. Smaller (9-17 micro m wide) and larger (33-40 micro m wide) Beggiatoa spp. represent novel phylogenetic clusters distinct from previously sequenced, large marine Beggiatoa spp. and Thioploca spp. Fluorescence in situ hybridization (FISH) of the natural Beggiatoa spp. populations showed that filament width is a conservative character of each phylogenetic species but a given filament width may represent multiple phylogenetic species in a mixed population.     

Inhibition of NO3- and NO2- reduction by microbial Fe(III) reduction: evidence of a reaction between NO2- and cell surface-bound Fe2+

A recent study (D. C. Cooper, F. W. Picardal, A. Schimmelmann, and A. J. Coby, Appl. Environ. Microbiol. 69:3517-3525, 2003) has shown that NO(3)(-) and NO(2)(-) (NO(x)(-)) reduction by Shewanella putrefaciens 200 is inhibited in the presence of goethite. The hypothetical mechanism offered to explain this finding involved the formation of a Fe(III) (hydr)oxide coating on the cell via the surface-catalyzed, abiotic reaction between Fe(2+) and NO(2)(-). This coating could then inhibit reduction of NO(x)(-) by physically blocking transport into the cell. Although the data in the previous study were consistent with such an explanation, the hypothesis was largely speculative. In the current work, this hypothesis was tested and its environmental significance explored through a number of experiments. The inhibition of approximately 3 mM NO(3)(-) reduction was observed during reduction of a variety of Fe(III) (hydr)oxides, including goethite, hematite, and an iron-bearing, natural sediment. Inhibition of oxygen and fumarate reduction was observed following treatment of cells with Fe(2+) and NO(2)(-), demonstrating that utilization of other soluble electron acceptors could also be inhibited. Previous adsorption of Fe(2+) onto Paracoccus denitrificans inhibited NO(x)(-) reduction, showing that Fe(II) can reduce rates of soluble electron acceptor utilization by non-iron-reducing bacteria. NO(2)(-) was chemically reduced to N(2)O by goethite or cell-sorbed Fe(2+), but not at appreciable rates by aqueous Fe(2+). Transmission and scanning electron microscopy showed an electron-dense, Fe-enriched coating on cells treated with Fe(2+) and NO(2)(-). The formation and effects of such coatings underscore the complexity of the biogeochemical reactions that occur in the subsurface.     

Effect of sulfur compounds on biological reduction of nitric oxide in aqueous Fe(II)EDTA2- solutions.

Biological reduction of nitric oxide (NO) in aqueous solutions of EDTA chelated Fe(II) is one of the main steps in the BioDeNOx process, a novel bioprocess for the removal of nitrogen oxides (NOx) from polluted gas streams. Since NOx contaminated gases usually also contain sulfurous pollutants, the possible interferences of these sulfur compounds with the BioDeNOx process need to be identified. Therefore, the effect of the sulfur compounds Na2SO4, Na2SO3, and H2S on the biological NO reduction in aqueous solutions of Fe(II)EDTA2- (25 mM, pH 7.2, 55 degrees C) was studied in batch experiments. Sulfate and sulfite were found to not affect the reduction rate of Fe(II)EDTA2- complexed NO under the conditions tested. Sulfide, either dosed externally or formed during the batch incubation out of endogenous sulfur sources or the supplied sulfate or sulfite, influences the production and consumption of the intermediate nitrous oxide (N2O) during Fe(II)EDTA2- bound NO reduction. At low concentrations (0.2 g VSS/l) of denitrifying sludge, 0.2 mM free sulfide completely inhibited the nitrosyl-complex reduction. At higher biomass concentrations (1.3-2.3 g VSS/l), sulfide (from 15 microM to 0.8 mM) induced an incomplete NO denitrification with N2O accumulation. The reduction rates of NO to N2O were enhanced by anaerobic sludge, presumably because it kept FeEDTA in the reduced state.     

Copper,zinc superoxide dismutase as a univalent NO(-) oxidoreductase and as a dichlorofluorescin peroxidase.

Nitroxyl (NO(-)) may be produced by nitric-oxide synthase and by the reduction of NO by reduced Cu,Zn-SOD. The ability of NO(-) to cause oxidations and of SOD to inhibit such oxidations was therefore explored. The decomposition of Angeli's salt (AS) produces NO(-) and that in turn caused the aerobic oxidation of NADPH, directly or indirectly. O(2) was produced concomitant with the aerobic oxidation of NADPH by AS, as evidenced by the SOD-inhibitable reduction of cytochrome c. Both Cu,Zn-SOD and Mn-SOD inhibited the aerobic oxidation of NADPH by AS, but the amounts required were approximately 100-fold greater than those needed to inhibit the reduction of cytochrome c. This inhibition was not due to a nonspecific protein effect or to an effect of those large amounts of the SODs on the rate of decomposition of AS. NO(-) caused the reduction of the Cu(II) of Cu,Zn-SOD, and in the presence of O(2), SOD could catalyze the oxidation of NO(-) to NO. The reverse reaction, i.e. the reduction of NO to NO(-) by Cu(I),Zn-SOD, followed by the reaction of NO(-) with O(2) would yield ONOO(-) and that could explain the oxidation of dichlorofluorescin (DCF) by Cu(I),Zn-SOD plus NO. Cu,Zn-SOD plus H(2)O(2) caused the HCO(3)(-)-dependent oxidation of DCF, casting doubt on the validity of using DCF oxidation as a reliable measure of intracellular H(2)O(2) production.