The periplasmic nitrite reductase of Thauera selenatis may catalyze the reduction of selenite to elemental selenium

Thauera selenatis grows anaerobically with selenate, nitrate or nitrite as the terminal electron acceptor; use of selenite as an electron acceptor does not support growth. When grown with selenate, the product was selenite; very little of the selenite was further reduced to elemental selenium. When grown in the presence of both selenate and nitrate both electron acceptors were reduced concomitantly; selenite formed during selenate respiration was further reduced to elemental selenium. Mutants lacking the periplasmic nitrite reductase activity were unable to reduce either nitrite or selenite. Mutants possessing higher activity of nitrite reductase than the wild-type, reduced nitrite and selenite more rapidly than the wild-type. Apparently, the nitrite reductase (or a component of the nitrite respiratory system) is involved in catalyzing the reduction of selenite to elemental selenium while also reducing nitrite. While periplasmic cytochrome C ₅₅₁ may be a component of the nitrite respiratory system, the level of this cytochrome was essentially the same in mutant and wild-type cells grown under two different growth conditions (i.e. with either selenate or selenate plus nitrate as the terminal electron acceptors). The ability of certain other denitrifying and nitrate respiring bacteria to reduce selenite will also be described.     

Isolation, Growth, and Metabolism of an Obligately Anaerobic, Selenate-Respiring Bacterium, Strain SES-3

A gram-negative, strictly anaerobic, motile vibrio was isolated from a selenate-respiring enrichment culture. The isolate, designated strain SES-3, grew by coupling the oxidation of lactate to acetate plus CO₂ with the concomitant reduction of selenate to selenite or of nitrate to ammonium. No growth was observed on sulfate or selenite, but cell suspensions readily reduced selenite to elemental selenium (Se⁰). Hence, SES-3 can carry out a complete reduction of selenate to Se⁰. Washed cell suspensions of selenate-grown cells did not reduce nitrate, and nitrate-grown cells did not reduce selenate, indicating that these reductions are achieved by separate inducible enzyme systems. However, both nitrate-grown and selenate-grown cells have a constitutive ability to reduce selenite or nitrite. The oxidation of [¹⁴C]lactate to ¹⁴CO₂ coupled to the reduction of selenate or nitrate by cell suspensions was inhibited by CCCP (carbonyl cyanide m-chlorophenylhydrazone), cyanide, and azide. High concentrations of selenite (5 mM) were readily reduced to Se⁰ by selenate-grown cells, but selenite appeared to block the synthesis of pyruvate dehydrogenase. Tracer experiments with [⁷⁵Se]selenite indicated that cell suspensions could achieve a rapid and quantitative reduction of selenite to Se⁰. This reduction was totally inhibited by sulfite, partially inhibited by selenate or nitrite, but unaffected by sulfate or nitrate. Cell suspensions could reduce thiosulfate, but not sulfite, to sulfide. These results suggest that reduction of selenite to Se⁰ may proceed, in part, by some of the components of a dissimilatory system for sulfur oxyanions.     

Cell yield (YM) of Thauera selenatis grown anaerobically with acetate plus selenate or nitrate

Thauera selenatis was grown anaerobically in minimal medium with either selenate or nitrate as the terminal electron acceptor and acetate as the carbon source and electron donor. The molar cell protein yields, Y_M-protein (selenate) and Y_M-protein (nitrate), were found to be 7.8 g cell protein/mol selenite formed and 7.5 g cell protein/mol nitrite formed, respectively. These values represent Y_M values of 57 and 55 g (dry weight)/mol acetate when selenate or nitrate was the electron acceptor, respectively. Based upon a calculated Y_ATP value of 10.0 g (dry weight) cells/mol ATP, for growth on acetate in inorganic salts, growth with selenate as the terminal electron acceptor theoretically yielded 5.7 ATP/acetate oxidized, and 5.5 ATP when nitrate was the terminal electron acceptor. The results support the conclusion that energy is conserved via electron transport phosphorylation when selenate or nitrate reduction are the terminal electron acceptors during anaerobic growth with acetate.     

Removing Selenate from Groundwater with a Vegetable Oil-Based Biobarrier

Vegetable oil–based permeable reactive biobarriers (PRBs) were evaluated as a method for remediating groundwater containing unacceptable amounts of selenate. PRBs formed by packing laboratory columns with sand coated with soybean oil were used. In an initial 24-week study a simulated groundwater containing 10 mg L⁻¹ selenate-Se was supplied to three soil columns and the selenate and selenite content of the effluent waters monitored. Two of the soil columns were effective at removing selenate and, during the final 21 weeks of the study, effluents from these columns contained almost no selenate or selenite. Almost all (95%) of the selenate removed was recovered as immobilized selenium sequestered in the solid matrix of the column. For unknown reasons, the third column failed to reduce selenate. A second study looked at the ability of PRBs to remove selenate when nitrate was present. As was done in the first study, three columns were evaluated but this time the water supplied to the columns contained 20 mg L⁻¹ nitrate-N and 10 mg L⁻¹ selenate-Se. Nitrate quickly disappeared from the effluents of these columns and during the final 23 weeks of the study, the nitrate content of the effluent water averaged less than 0.03 μg ml⁻¹ nitrate-N. Selenate was also removed by these columns but at a slower rate than observed with nitrate. In the final 6 weeks of the study, about 95% of the selenate applied to the columns was removed. In situ PRBs containing soybean oil might be used to remediate groundwater contaminated with both selenate and nitrate.     

Induction of nitrite/nitrate synthesis in murine macrophages by BCG infection, lymphokines, or interferon-gamma

Macrophage synthesis of nitrite and nitrate after activation by BCG infection or by treatment in vitro with both T cell-derived (lymphokines (LK) or recombinant murine interferon-gamma (IFN-gamma] and bacterial (lipopolysaccharide (LPS) and heat-killed bacillus Calmette-Guerin (hk BCG] agents was studied by using macrophages from C3H/He and C3H/HeJ mice. Spleen and peritoneal macrophages isolated from BCG-infected donors that were producing nitrate continued to synthesize nitrite and nitrate in culture. LPS treatment in vitro (25 or 50 micrograms/ml) additionally increased this nitrite/nitrate synthesis. Thioglycolate-elicited macrophages from non-infected C3H/HeJ mice treated with LK also produced nitrite/nitrate, and concurrent LPS (0.1 to 50 micrograms/ml) treatment resulted in enhanced synthesis. Recombinant IFN-gamma also stimulated nitrite/nitrate synthesis by C3H/He and CeH/HeJ macrophages as did LPS (C3H/He only) and hk BCG. When given concurrently with either LPS or hk BCG, IFN-gamma enhanced C3H/He and C3H/HeJ macrophage nitrite/nitrate synthesis over that produced by macrophages treated with either LPS or hk BCG alone. Macrophages activated in vitro exhibited a 4 to 12 hr lag time before engaging in nitrite/nitrate synthesis, which then proceeded for 36 to 42 hr at linear rates. Daily medium renewal did not alter the synthesis kinetics but increased the total amount of nitrite/nitrate produced. Nitrate and nitrite were stable under the conditions of culture and when added did not influence additional macrophage synthesis. Taken together, these results indicate that T cell lymphokines and IFN-gamma are powerful modulators of macrophage nitrite/nitrate synthesis during BCG infection and in vitro, and nitrite/nitrate synthesis appears to be common property of both primed and fully activated macrophage populations.     

Inhibition of microbial H<Subscript>2</Subscript>S production in an oil reservoir model column by nitrate injection

The effect of nitrate addition on microbial H2S production in a seawater-flooded oil reservoir model column with crude oil as carbon and energy source was investigated. Injection of 0.5 mM nitrate for 2.5-3.5 months led to complete elimination of H2S (initially 0.45-0.67 mM). The major decline in H2S level coincided with the first complete nitrate consumption and production of nitrite. When nitrate was excluded, H2S production resumed after approximately 2.5 months and reached previous levels after approximately 5 months. Using a fluorescent antibody technique, three populations each of sulfate-reducing bacteria (SRB) and nitrate-reducing bacteria (NRB) were monitored. SRB dominated the anoxic zone prior to nitrate addition, comprising 64-93% of the total bacterial population. The monitored NRB constituted less than 6% and no increase was observed during nitrate addition (indicating that other, unidentified, NRB populations were present). After 1-3 months without significant H2S production (3.5-5.5 months with nitrate), the SRB population collapsed, the fraction being reduced to 9-25%. The dominant SRB strain in the column, which constituted on average 94% of the monitored SRB population, was partly/completely inhibited by 50/75 microM nitrite in batch culture tests. Similar nitrite concentrations (50-150 microM) were detected in the column when the H2S level declined, indicating that nitrite inhibition was the main cause of H2S elimination. The results from this study indicate that nitrate/nitrite can be used to prevent detrimental SRB activity in oil reservoirs.     

Failure of an enriched nitrite-DPAO population to use nitrate as an electron acceptor

The metabolism of denitrifying polyphosphate accumulating organisms (DPAO) is not completely known. Recent reports suggest the existence of two types of DPAOs: those that can use nitrate and nitrite as electron acceptors (nitrate-DPAO) and those that can only use nitrite (nitrite-DPAO). Then, the survival of nitrite-DPAO in nitrate reducing environments is due to the existence of flanking denitrifying species, which reduce nitrate to nitrite. This works aims at a better understanding of the nitrite-DPAO population. For this aim, a nitrite-DPAO population was previously selected in a SBR using nitrite as electron acceptor. Then, nitrate utilisation by nitrite-DPAO was studied within a short-term period (4 days) and within a long-term period (50 days) with simultaneous nitrite and nitrate additions. The results obtained clearly indicate that nitrite-DPAO fail to use nitrate as electron acceptor even after 50 days of periodic dosing of nitrate and agree with the dual DPAO theory. Moreover, this failure casts doubts on the feasibility of nitrite based EBPR systems (i.e. partial nitrification + nitrite-DPAO) because these systems will not be able to denitrify an occasional nitrate inlet, which will remain in the effluent.     

Nitrite reduction by a mixed culture under conditions relevant to shortcut biological nitrogen removal

Dissimilative reduction of nitrite by nitrite-acclimated cellswas investigated in a batch reactor under various environmental conditions that can beencountered in shortcut biological nitrogen removal (SBNR: ammonia to nitrite andnitrite to nitrogen gas). The maximum specific nitrite reduction rate was as much as 4.3 times faster than the rate of nitrate reduction when individually tested, but the reaction was inhibited in the presence of nitrate when the initial nitrate concentration was greater than approximately 25 mg-N/l or the initialNO ₃ ^- N/NO ₂ ^- N ratio was larger than 0.5. Nitrite reduction was also inhibited by nitrite itself when theconcentration was higher than that to which the cells had been acclimated. Therefore, it was desirable to avoid excessively high nitrite and nitrate concentrations in a denitrification reactor. Nitrite reduction, however, was not affected by an alkaline pH (in the range of 7–9) or a high concentration of FA (in the range of 16–39 mg/l), which can be common in SBNR processes. The chemical oxygen demand (COD) requirement for nitrite reduction was approximately 22–38% lower than that for nitrate reduction, demonstrating that the SBNR process can be economical. The specific consumption,measured as the ratio of COD consumed to nitrogen removed, was affected by the availability of COD and the physiological state of the cells. The ratio increased when the cells grew rapidly and were storing carbon and electrons.     

Selenium reduction by a denitrifying consortium

A denitrifying bacterial consortium obtained from the Pullman, Washington wastewater treatment facility was enriched under denitrifying conditions and its ability to reduce selenite and selenate was studied. Replicate experiments at two different experimental conditions were performed. All experiments were performed under electron-acceptor limiting conditions, with acetate as the carbon source and nitrate the electron acceptor. In the first set of experiments, selenite was present, whereas, in the second set, selenate was added. A significant lag period of approximately 150 h was necessary before selenite or selenate reduction was observed. During this lag period, nitrate and nitrite use was observed. Once selenite or selenate reduction had started, nitrate and nitrite reduction was concomitant with selenium species reduction. Trace amounts of selenite were detected during the selenate reduction study. Analysis of the data indicates that, once selenium species reduction was induced, the rate of reduction was proportional to the selenium species concentration and to the biomass concentration. Furthermore, at similar biomass and contaminant concentrations, selenite reduction is approximately four times faster than selenate reduction. Copyright 1999 John Wiley & Sons, Inc.     

沼泽红假单胞菌(Rhodopseudomonas palustris)CQV97对无机三态氮共存水体中氮素的去除效率及其影响因素

研究水体环境因素(温度、光照和pH)、小分子有机碳和有机氮化合物对一株具有高效脱氮潜力的沼泽红假单胞菌(Rhodopseudomonas palustris)CQV97在无机三态氮共存体系中脱除无机三态氮的影响规律。结果显示,该菌株在20~40℃,500~5 000lux,pH 6.0~9.0环境条件下,能够脱除高浓度无机三态氮(其中亚硝氮不低于40mg·L-1),表明该菌株具有较强的适应复杂环境的能力;以乙酸钠/乙醇为唯一碳源时,该菌株能有效地去除无机三态氮,而以葡萄糖为唯一碳源时,能有效去除硝氮,但不能去除氨氮,亚硝氮明显积累,表明环境中小分子有机碳源影响菌体对无机三态氮的去除能力;体系中添加高浓度(120mg·L-1)蛋白胨或尿素时,由于有机氮降解的释氨作用,菌体对氨氮的去除能力受到明显抑制,氨氮积累明显,13d时氨氮去除率仅分别为16%(蛋白胨)和6%(尿素),但硝氮和亚硝氮的去除能力并没有受到明显影响。异位处理实际水体结果表明,菌株可使水体中氨氮含量明显降低、硝氮和亚硝氮被完全去除。综上,沼泽红假单胞菌CQV97菌株环境适应能力强,具有高效脱除水体无机三态氮的应用潜力,这为进一步开发高效脱氮微生物制剂及其合理使用奠定了基础。     

The effect of nitrogen refeeding on starved cells of Platymonas striata Butcher

A rapid uptake of nitrogen was observed in nitrogen-starved cells of Platymonas striata after refeeding with ammonium or nitrate ions. This was followed by a net loss of nitrogen per cell. Cells initially grown in and then starved in a regime of continuous light showed greater increases in average cell nitrogen on refeeding with ammonium or nitrate ions than did cells initially grown in and then starved in a regime of alternating light and darkness. A particulate subcellular location was observed for nitrate reductase (EC 1.6.6.1) in broken cell suspensions prepared by sonication. Nitrite reductase (EC 1.6.6.4) was located in the soluble fraction of these cell suspensions. Broken cell preparations displayed a lowered nitrate reductase activity as compared with the particulate component of these preparations. This was shown not to be due to heat-stable inhibitors present in the soluble phase of the cell. It appeared to be an artefact produced by the high nitrite reductase activity of the broken cell preparations, which removed much of the nitrite as it was formed. Nitrogen starvation of nitrate-grown cultures produced cellular increases in nitrate reductase and nitrite reductase activities which were further increased after the addition of nitrate. The results are discussed.Abbreviations ASP2 complete culture medium - ASP2 INF medium lacking in inorganic nitrogen - ASP2 NF medium lacking all nitrogen - NAR nitrate reductase - NIR nitrite reductase - EDTA Ethylenediaminetetracetic acid - PVP Polyvinylpyrollidone, M.W. 44,000     

Reassessment of the in vivo Assay for Nitrate Reductase in Leaves

The in vivo assay procedure for nitrate reductase and its dependence on the concentration of nitrate and other ions were examined. It was found that high ion concentrations led to an increased release of nitrite to the reaction media which could be interpreted as a stimulated nitrate reductase activity. This phenomenon is not an osmotic effect, since equivalent concentrations of mannitol did not lead to identical results. The effect of ions on the enhanced nitrite production was attributed to changes in cell membrane permeability rather than to a supply of substrate. This conclusion is based on several findings: (a) in in vitro assays, the rate of nitrite production was not affected by ion concentrations: (b) the stimulation of nitrite production was obtained by various ions and not only by nitrate; (c) pretreatment of alfalfa leaves with nitrate did not increase the NO₂^? release rate to the external solution; and (d) nitrate and nitrite export from leaf discs to the solution was stimulated even in discs which were enzymatically inactive. Calcium ions in the presence of KNO₃ inhibited the enhanced nitrite production, probably due to alteration of membrane stability. The effect of ions on the rate of nitrite production was reversible and the high rate of nitrite production was reduced to the control rate when discs were transferred to a solution of low concentration.     

Simultaneous Reduction of Nitrate and Selenate by Cell Suspensions of Selenium-Respiring Bacteria

Washed-cell suspensions of Sulfurospirillum barnesii reduced selenate [Se(VI)] when cells were cultured with nitrate, thiosulfate, arsenate, or fumarate as the electron acceptor. When the concentration of the electron donor was limiting, Se(VI) reduction in whole cells was approximately fourfold greater in Se(VI)-grown cells than was observed in nitrate-grown cells; correspondingly, nitrate reduction was ~11-fold higher in nitrate-grown cells than in Se(VI)-grown cells. However, a simultaneous reduction of nitrate and Se(VI) was observed in both cases. At nonlimiting electron donor concentrations, nitrate-grown cells suspended with equimolar nitrate and selenate achieved a complete reductive removal of nitrogen and selenium oxyanions, with the bulk of nitrate reduction preceding that of selenate reduction. Chloramphenicol did not inhibit these reductions. The Se(VI)-respiring haloalkaliphile Bacillus arsenicoselenatis gave similar results, but its Se(VI) reductase was not constitutive in nitrate-grown cells. No reduction of Se(VI) was noted for Bacillus selenitireducens, which respires selenite. The results of kinetic experiments with cell membrane preparations of S. barnesii suggest the presence of constitutive selenate and nitrate reduction, as well as an inducible, high-affinity nitrate reductase in nitrate-grown cells which also has a low affinity for selenate. The simultaneous reduction of micromolar Se(VI) in the presence of millimolar nitrate indicates that these organisms may have a functional use in bioremediating nitrate-rich, seleniferous agricultural wastewaters. Results with ⁷⁵Se-selenate tracer show that these organisms can lower ambient Se(VI) concentrations to levels in compliance with new regulations proposed for release of selenium oxyanions into the environment.     

High-resolution microanalysis of nitrite and nitrate in neuronal tissues by capillary electrophoresis with conductivity detection

Nitrites and nitrates are widely used reporters of endogenous activity of nitric oxide synthases (NOS), an important group of enzymes producing the gaseous signal molecule nitric oxide (NO). However, due to the great chemical heterogeneity of neuronal tissues, standard analytical protocols for evaluation of neuronal nitrite/nitrate concentrations are inefficient. We optimized a high-performance capillary zone electrophoresis (CZE) technique to analyze nitrite/nitrate concentrations in submicroliter samples from mammalian neuronal tissues. The measurements were made using a PrinCE 476 computerized capillary electrophoresis system with a Crystal 1000 contact conductivity detector. Isotachophoretic stacking injection of 1000- to 10000-fold diluted samples, which had been pretreated with a custom-designed solid-phase microextraction (SPME) cartridge, was employed to assay micromolar and nanomolar nitrite and nitrate levels in the presence of the high millimolar chloride concentrations characteristic of many biological samples. In the presented technique, a 10-microl volume of diluted ganglionic sample was used for chloride removal and sample cleanup. The method yields high analytical performance, including good reproducibility, resolution, and accuracy. The limits of detection relative to undiluted sample matrix were 8.9 nM (0.41 ppb) and 3.54 nM (0.22 ppb) for nitrite and nitrate, respectively. In addition, this technique resolves other anions that are present in neuronal tissues at sub-nanomolar concentrations and can be broadly applied for high-throughput anionic profiling. In rat dorsal root ganglia, endogenous levels of nitrate (231+/-29 microM; n=6) and nitrite (24-96 microM) were found. These concentrations exceeded those previously found in neuronal tissue homogenates using different techniques.     

Control of nitrate and nitrite assimilation by carbohydrate reserves,adenosine nucleotides and pyridine nucleotides in leaves of Zea mays L. under dark conditions

The rate of in-vivo nitrate reduction by leaf segments of Zea mays L. was found to decline during the second hour of dark anaerobic treatment. On transfer to oxygen the capacity to reduce nitrate under dark conditions was restored. These observations led to the proposal that nitrate reductase is a regulatory enzyme with ADP acting as a negative effector. The effect of ADP on the invitro activity of nitrate reductase and the changes in the in-vivo adenylate pool under dark-N₂ and dark-O₂ were investigated. It was found that ADP inhibited the activity of partially purified nitrate reductase. Similarly, the in-vivo anaerobic inhibition of nitrate reduction was associated with a build-up of ADP in the leaf tissue. Under anaerobic conditions nitrite accumulated and on transfer to oxygen the accumulated nitrite was reduced. To explain this phenomenon the following hypothesis was proposed and tested. Under anaerobic conditions the supply of reducing equivalents for nitrite reduction in the plastid becomes restricted and nitrite accumulates as a consequence. On transfer to oxygen this restriction is removed and nitrite disappears. This capacity to reduce accumulated nitrite was found to be dependent on the carbohydrate status of the leaf tissue.     

NO3 −/NO2 − assimilation in halophilic archaea: physiological analysis, nasA and nasD expressions

The haloarchaeon Haloferax mediterranei is able to assimilate nitrate or nitrite using the assimilatory nitrate pathway. An assimilatory nitrate reductase (Nas) and an assimilatory nitrite reductase (NiR) catalyze the first and second reactions, respectively. The genes involved in this process are transcribed as two messengers, one polycistronic (nasABC; nasA encodes Nas) and one monocistronic (nasD; codes for NiR). Here we report the Hfx mediterranei growth as well as the Nas and NiR activities in presence of high nitrate, nitrite and salt concentrations, using different approaches such as physiological experiments and enzymatic activities assays. The nasA and nasD expression profiles are also analysed by real-time quantitative PCR. The results presented reveal that the assimilatory nitrate/nitrite pathway in Hfx mediterranei takes place even if the salt concentration is higher than those usually present in the environments where this microorganism inhabits. This haloarchaeon grows in presence of 2 M nitrate or 50 mM nitrite, which are the highest nitrate and nitrite concentrations described from a prokaryotic microorganism. Therefore, it could be attractive for bioremediation applications in sewage plants where high salt, nitrate and nitrite concentrations are detected in wastewaters and brines.     

Selenite uptake and incorporation by Selenomonas ruminatium

Selenite uptake and incorporation in Selenomonas ruminantium was constitutive with an inducible component. It was distinct from sulphate or selenate transport, since sulphate and selenate did not inhbit uptake, nor could sulphate or selenate uptake be demonstrated. Selenite uptake had an apparent K _m of 1.28 mM and a V _max of 148 ng Se min^-1 mg^-1 protein. Uptake was sensitive to inhibition by 2,4-dinitrophenol (DNP), carbonyl cyanide m-chlorophenyl hydrazone (CCCP), azide, iodoacetic acid (IAA) and N-ethylmaleimide (NEM), but not chloropromazine (CPZ), N,N-dicyclohexyl-carbodiimide (DCCD), quinine, arsenate, or fluoride. Treatment of cells accumulating ⁷⁵[Se]-Selenite with 2,4,DNP inhibited uptake, but did not cause efflux. Transport of selenite was inhibited by sulphite and nitrite, but not by nitrate, phosphate, sulphate of selenate. ⁷⁵[Se]-Selenite was incorporated into selenocystine, selenoethionine, selenohomocysteine, and selenomethionine and was also reduced to red elemental selenium.     

Resolution of Distinct Membrane-Bound Enzymes from Enterobacter cloacae SLD1a-1 That Are Responsible for Selective Reduction of Nitrate and Selenate Oxyanions

Enterobacter cloacae SLD1a-1 is capable of reductive detoxification of selenate to elemental selenium under aerobic growth conditions. The initial reductive step is the two-electron reduction of selenate to selenite and is catalyzed by a molybdenum-dependent enzyme demonstrated previously to be located in the cytoplasmic membrane, with its active site facing the periplasmic compartment (C. A. Watts, H. Ridley, K. L. Condie, J. T. Leaver, D. J. Richardson, and C. S. Butler, FEMS Microbiol. Lett. 228:273-279, 2003). This study describes the purification of two distinct membrane-bound enzymes that reduce either nitrate or selenate oxyanions. The nitrate reductase is typical of the NAR-type family, with α and β subunits of 140 kDa and 58 kDa, respectively. It is expressed predominantly under anaerobic conditions in the presence of nitrate, and while it readily reduces chlorate, it displays no selenate reductase activity in vitro. The selenate reductase is expressed under aerobic conditions and expressed poorly during anaerobic growth on nitrate. The enzyme is a heterotrimeric (αβγ) complex with an apparent molecular mass of ~600 kDa. The individual subunit sizes are ~100 kDa (α), ~55 kDa (β), and ~36 kDa (γ), with a predicted overall subunit composition of α₃β₃γ₃. The selenate reductase contains molybdenum, heme, and nonheme iron as prosthetic constituents. Electronic absorption spectroscopy reveals the presence of a b-type cytochrome in the active complex. The apparent K_m for selenate was determined to be ~2 mM, with an observed V_max of 500 nmol SeO₄²⁻ min⁻¹ mg⁻¹ (k_cat, ~5.0 s⁻¹). The enzyme also displays activity towards chlorate and bromate but has no nitrate reductase activity. These studies report the first purification and characterization of a membrane-bound selenate reductase.     

Diverse effects of formate on the assimilatory metabolism of nitrate and nitrite inRhizobium

Two strains ofRhizobium, cowpeaRhizobium 32H1 andRhizobium japonicum CB 1809, showed a marked stimulation in growth on addition of formate to the minimal medium containing nitrate as the sole source of nitrogen. The amount of accumulated nitrite and specific nitrate reductase activity was much higher in cultures supplemented with formate than in the control medium. In contrast, growth, consumption of nitrite and specific nitrite reductase activity in minimal medium + nitrite was greatly reduced by the addition of formate. A chlorate resistant mutant (Chl-16) was isolated spontaneously which contained a nitrite reductase which was not inhibited by formate. The results suggest that formate serves as an electron donor for nitrate reductase and inhibits nitrite assimilation inRhizobium     

Oxygen exchange between nitrate molecules during nitrite oxidation by Nitrobacter

During oxidation of nitrite, cells of Nitrobacter winogradskyi are shown to catalyze the active exchange of oxygen atoms between exogenous nitrate molecules (production of 15N16/18O3- during incubation of 14N16/18O3-, 15N16O3-, and 15N16O2- in H216O). Little, if any, exchange of oxygens between nitrate and water also occurs (production of 15N16/18O3- during incubation of 15N16O3- and 14N16O2- in H218O). 15N species of nitrate were assayed by 18O-isotope shift in 15N NMR. Taking into account the O-exchange reactions which occur during nitrite oxidation, H2O is seen to be the source of O in nitrate produced by oxidation of nitrite by N. winogradskyi. The data do not establish whether the nitrate-nitrate O exchange is catalyzed by nitrite oxidase (H2O + HNO2----HNO3 + 2H+ + 2e-) or nitrate reductase (HNO3 + 2H+ + 2e-----HNO2 + H2O) or both enzymes in consort. The nitrate-nitrate exchange reaction suggests the existence of an oxygen derivative of a H2O-utilizing oxidoreductase.