Higher Nitrite Reductase Research Articles

The circularly permuted globin domain of androglobin exhibits atypical heme stabilization and nitric oxide interaction.

In the decade since the discovery of androglobin, a multi-domain hemoglobin of metazoans associated with ciliogenesis and spermatogenesis, there has been little advance in the knowledge of the biochemical and structural properties of this unusual member of the hemoglobin superfamily. Using a method for aligning remote homologues, coupled with molecular modelling and molecular dynamics, we have identified a novel structural alignment to other hemoglobins. This has led to the first stable recombinant expression and characterization of the circularly permuted globin domain. Exceptional for eukaryotic globins is that a tyrosine takes the place of the highly conserved phenylalanine in the CD1 position, a critical point in stabilizing the heme. A disulfide bond, similar to that found in neuroglobin, forms a closed loop around the heme pocket, taking the place of androglobin's missing CD loop and further supporting the heme pocket structure. Highly unusual in the globin superfamily is that the heme iron binds nitric oxide as a five-coordinate complex similar to other heme proteins that have nitric oxide storage functions. With rapid autoxidation and high nitrite reductase activity, the globin appears to be more tailored toward nitric oxide homeostasis or buffering. The use of our multi-template profile alignment method to yield the first biochemical characterisation of the circularly permuted globin domain of androglobin expands our knowledge of the fundamental functioning of this elusive protein and provides a pathway to better define the link between the biochemical traits of androglobin with proposed physiological functions.

Microbial induced calcium precipitation by Zobellella denitrificans sp. LX16 to simultaneously remove ammonia nitrogen, calcium, and chemical oxygen demand in reverse osmosis concentrates

In recent years, with the rapid development of industrial revolution and urbanization, the generation and treatment of a large number of salt-containing industrial wastewater has attracted wide attention. A novel salt-tolerant Zobellella denitrificans sp. LX16 with excellent nitrogen removal and biomineralization capabilities was isolated in this experiment. Kinetic experiments were conducted to determine the optimal condition. Under this condition, chemical oxygen demand (COD) can be entirely removed together with ammonia nitrogen, and the removal efficiency of calcium was 88.09%. Growth curves and nitrogen balance tests showed that strain LX16 not only had good HNAD and MICP capabilities, but also had high nitrite reductase and nitrate reductase activities during this process. Three-dimensional fluorescence results reflected that when external carbon sources were lacking or salinity was high, humic acid could effectively enhance the metabolic activity of heterotrophic nitrifying aerobic denitrifying microorganisms through extracellular electron transfer, and the substances produced in the metabolic process could promote biommineralization. Moreover, combined with SEM, SEM-EDS, XRD and FTIR analysis, it is concluded that the microbial surface can provide nucleation sites to form calcium salts, and with the increase of alkalinity to generate Ca5(PO4)3OH. The theoretical basis for the use of biological treatment in reverse osmosis wastewater have been proved by this experiment.

Corrigendum: Alleviation of Nitrogen and Sulfur Deficiency and Enhancement of Photosynthesis in Arabidopsis thaliana by Overexpression of Uroporphyrinogen III Methyltransferase (UPM1)

[This corrects the article DOI: 10.3389/fpls.2017.02265.].

Nitrite kinetics during anoxia: The role of abiotic reactions versus microbial reduction

Anoxic spells in soil induce denitrification, i.e. the sequential reduction NO3−→NO2−→NO→N2O→N2, catalysed by the four enzymes NAR, NIR, NOR and NOS, respectively. Transient accumulation of all intermediates is inevitable, but the concentrations depend on the regulation of gene expression and the physical/chemical properties of the soil. Nitrite is chemically unstable at low pH, decomposing via a conglomerate of abiotic reactions with metals and organic compounds which can result in production of NO, N2O, N2 and nitrosated organic compounds (R-NO). There is evidence that acidic soils accumulate less nitrite than neutral soils, but it is unclear if this is due to high abiotic decomposition rate (VADEC) or fast enzymatic reduction of nitrite (VNIR) at low pH. To investigate this, we monitored the kinetics of NO2−, NO, N2O and N2 during anoxic incubations of three organic soils with pHCaCl2 ranging from 3.4 to 7.2, taken from a long-term liming experiment. In parallel, we determined the rate of abiotic nitrite decay (VADEC) and its product stoichiometry (NO, N2O and R-NO) in gamma-irradiated soils. VADEC was clearly first-order with respect to HNO2 (kHNO2 = 1.4 h−1), N-gas production (NO, N2O and N2) accounted for only ∼50% of VADEC, the rest was ascribed to nitrosation (R-NO). During denitrification (live soil incubation), the nitrite concentrations reached 2–3 mM in the soils with pH 4.9 and 7.2, while the soil with pH 3.4 kept nitrite concentrations at 20–50 μM, except for a short spike reaching 160 μM after 40 h. Estimated rates of nitrite scavenging by the two competing sinks (NIR and ADEC) showed that NIR was the strongst nitrite sink in soil with pH 3.4 (VNIR > VADEC), while VNIR ≈ VADEC in the soil with pH 5.9. In the soil with pH 7.2, VADEC was insignificant. Thus, the regulation of denitrification (high VNIR relative to VNAR) played a crucial role in determining nitrite kinetics, hence the fate of nitrite in acid soils. High nitrite reductase activity effectively minimized abiotic nitrite decomposition and nitrosation of soil organic matter. The results shed light on regulation of denitrification in acid soils, and its implications for the fate of nitrogen during denitrification events.

Interspecific variation and plasticity in hemoglobin nitrite reductase activity and its correlation with oxygen affinity in vertebrates

Deoxygenated hemoglobin (Hb) is a nitrite reductase that reduces naturally occurring nitrite to nitric oxide (NO), supplying physiological relevant NO under hypoxic conditions. The nitrite reductase activity is modulated by the allosteric equilibrium between the R and T structures of Hb that also determines oxygen affinity. In the present study we investigated nitrite reductase activity and O2 affinity in Hbs from ten different vertebrate species under identical conditions to disclose interspecific variations and allow an extended test for a correlation between the rate constant for nitrite reduction and O2 affinity. We also tested plastic changes in Hb properties via addition of T-structure-stabilizing organic phosphates (ATP and GTP). The decay in deoxyHb during its reaction with nitrite was exponential-like in ectotherms (Atlantic hagfish, carp, crucian carp, brown trout, rainbow trout, cane toad, Indian python and red-eared slider turtle), while it was sigmoid in mammals (harbor porpoise and rabbit). Typically, hypoxia-tolerant species showed a faster reaction than intolerant species. Addition of ATP and GTP decreased O2 affinity and slowed the rate of nitrite reduction in a concentration-dependent manner. The initial second order rate constant of the deoxyHb-mediated nitrite reduction showed a strong curvilinear correlation with oxygen affinity among all ectothermic vertebrates, and the relationship also applied to plastic variations of Hb properties via organic phosphates. The relationship predicts high nitrite reductase activity in hypoxic tolerant species with high Hb-O2 affinity and reveals that the decrease in erythrocyte ATP and/or GTP during acclimation to hypoxia in ectotherms increases the erythrocyte NO generating capacity.

Comparative Evaluation of Biochemical Changes in Different Safflower Varieties (Carthamus tinctorius L.) under Water Deficit

Studies to determine drought induced biochemical changes in safflower and their utilization in identifying stress tolerant genotypes were conducted under water deficit (60% field capacity) conditions at Nuclear Institute for Agriculture and Biology, Faisalabad, Pakistan. Nitrate Reductase (NRA) and Nitrite Reductase (NiRA) activities, total soluble proteins, DNA contents, fresh and dry biomass of plant and plant yield were adversely affected by drought stress in all safflower genotypes. However, genotypes Thori 78 and PI-387820 showed less reduction in these attributes. Total free amino acids, reducing, non reducing sugars and total sugars increased in all genotypes under drought stress. Comparison among safflower genotypes indicated that V1 (with greater biomass, yield, high NiRA, proteins and DNA level) performed best under drought stress followed by V6 (with high NiRA, proteins and unsaturation/saturation level). V3 proved itself poorer upon the basis of growth and biochemical parameters. From the results it can be concluded that biochemical markers can be used to select drought tolerant safflower genotypes.

The oxidative denitrosylation mechanism and nitric oxide release from human fetal and adult hemoglobin, an experimentally based model simulation study

Generation of unbound nitric oxide (NO) via the oxidative denitrosylation (ODN) mechanism is proposed to involve the simultaneous reaction of nitrite with oxy and deoxy hemoglobin (Hb(O2) (k1) and Hb (k13)) to yield respectively, *NO2 and Hb(+2)(NO). These two reaction pathways are coupled when *NO2 reacts with Hb(+2)(NO) to yield Hb(+3)(NO) (k22), a species that releases NO rapidly. Here, I have constructed an experimentally based molecular model of the ODN mechanism (k1-k31), focusing on the high nitrite reductase activity of R-state hemoglobin. This model was used to test the hypothesis that human fetal hemoglobin (HbF) can generate unbound NO faster and to a greater extent than HbA, consequent to a 25-fold larger value of k1, which was determined in an earlier study. The results show that despite the use of identical values for k22, there was a 44-fold larger apparent rate of reaction of *NO2 with HbF(NO) compared to HbA(NO), for reactions simulated at 410 μM nitrite and 100 μM hemoglobin (heme basis), 50% oxygen saturation at pH 7.4 and 37°C. This faster reaction was associated with the generation of about 11 μM peak unbound NO. In contrast, HbA failed to generate unbound NO rapidly under the same conditions. However, raising the concentration of nitrite into the millimolar range did generate unbound NO in the HbA simulation, in agreement with the experimental literature, and that result was associated with acceleration in the rate of reaction of NO2 with HbA(NO). Unbound NO could be generated at 410 μM nitrite in the HbA simulation by lowering the pH. This too was associated with an acceleration in the rate of reaction of NO2 with HbA(NO). Furthermore, generation of unbound NO could be assigned to the pH-dependent increase in k1, independent of the associated increase in k(13). Finally, selective exchange of the HbA value of k1 for the HbF value, keeping all other constants and conditions unchanged, generated kinetic patterns for the various species of the "k1-modified" HbA simulation, which were virtually indistinguishable from those seen in the HbF simulation. Taken together, these findings show that rapid and extensive generation of unbound NO within the ODN mechanism is controlled by the value of k1. The faster and more extensive generation of unbound NO by HbF at micromolar nitrite concentration suggests a possible second function for HbF in sickle cell disease, namely enhanced vasodilation. The failure of 410 μM nitrite to generate unbound NO in the HbA simulation at pH 7.4, contrasts with evidence in the literature showing that exposure of intact red cells to 100 to 200 μM nitrite in PBS, promoted NO release into the gas phase. I point out that this difference in outcome may be due to the higher activity of HbA when bound to the cytoplasmic domain of the red cell membrane anion transport protein SLC4A1 (band 3) and to the demonstrated capacity of band 3 to transport nitrite.

Nitrite reductase activities of myoglobins from five whale species with distinct diving capacities

The O2 carrier myoglobin (Mb) is expressed at high concentrations in the skeletal muscle of whales where it represents a major O2 store during diving. In addition to its well-known roles in O2 storage and delivery, Mb when deoxygenated can reduce endogenous nitrite to nitric oxide (NO), which in turn may downregulate respiration under hypoxia and protect against tissue damage upon reoxygenation. Although these functional properties of Mb are expected to contribute to the diving capacity of whales, very little is known about the variation in Mb function among whale species with different diving abilities. In this study, we have therefore measured O2 affinities and nitrite reductase activities of purified Mbs from five whale species; three toothed whales (harbor porpoise, sperm whale, bottlenose whale) and two baleen whales (minke whale and humpback whale) with distinct diving capacities and known average dive durations. We show that differences in O2 affinities and nitrite reductase activities are present among whale Mbs, but that these do not correlate with the average dive duration of the selected species. However, a higher Mb O2 affinity found in toothed whales is significantly correlated to a higher nitrite reductase activity, an adaptation that may increase NO production per unit time in the hypoxic muscle. In addition, the higher Mb concentrations present in the extreme diving whales compared with the more moderate divers may enhance the rate of NO production from nitrite during hypoxia and this can increase the beneficial effects of NO during and after long dives.

Effect of ruminal administration ofEscherichia coliwild type or a genetically modified strain with enhanced high nitrite reductase activity on methane emission and nitrate toxicity in nitrate-infused sheep

The effects of two kinds of Escherichia coli (E. coli) strain, wild-type E. coli W3110 and E. coli nir-Ptac, which has enhanced NO(2) reduction activity, on oral CH(4) emission and NO(3) toxicity in NO(3)-treated sheep were assessed in a respiratory hood system in a 4 x 6 Youden square design. NO(3) (1.3 g NaNO(3)/kg(0.75) body weight) and/or E. coli strains were delivered into the rumen through a fistula as a single dose 30 min after the morning meal. Escherichia coli cells were inoculated for sheep to provide an initial E. coli cell density of optical density at 660 nm of 2, which corresponded to 2 x 10(10) cells/ml. The six treatments consisted of saline, E. coli W3110, E. coli nir-Ptac, NO(3), NO(3) plus E. coli W3110, and NO(3) plus E. coli nir-Ptac. CH(4) emission from sheep was reduced by the inoculation of E. coli W3110 or E. coli nir-Ptac by 6 % and 12 %, respectively. NO(3) markedly inhibited CH(4) emission from sheep. Compared with sheep given NO(3) alone, the inoculation of E. coli W3110 to NO(3)-infused sheep lessened ruminal and plasma toxic NO(2) accumulation and blood methaemoglobin production, while keeping ruminal methanogenesis low. Ruminal and plasma toxic NO(2) accumulation and blood methaemoglobin production in sheep were unaffected by the inoculation of E. coli nir-Ptac. These results suggest that ruminal methanogenesis may be reduced by the inoculation of E. coli W3110 or E. coli nir-Ptac. The inoculation of E. coli W3110 may abate NO(3) toxicity when NO(3) is used to inhibit CH(4) emission from ruminants.

Effect of Escherichia coli wild type or its derivative with high nitrite reductase activity on in vitro ruminal methanogenesis and nitrate/nitrite reduction1

The effects of two kinds of Escherichia coli strains, wild-type E. coli W3110 or E. coli nir-Ptac, which has enhanced nitrite reduction activity, on in vitro CH4 production and nitrate and nitrite reduction in cultures of mixed ruminal microorganisms was investigated using continuous incubation systems. Escherichia coli nir-Ptac, a derivative of wild-type E. coli W3110, was constructed by replacing self promoter of nir BD operon encoding subunits of nitrite reductase in E. coli W3110 by tac promoter to make the expression of nir BD higher and constitutive. The nitrite reductase activity of E. coli nir-Ptac was approximately twice as high as E. coli W3110. The culture media consisted of 400 mL of strained ruminal fluid taken from two nonlactating Holstein cows receiving a basal diet of orchardgrass hay at maintenance level (55 g of DM/kg of BW0.75 daily), and 400 mL of autoclaved artificial saliva. Treatments were arranged in two separate 3 x 3 factorials consisting of nitrate (NaNO3; 0, 5, or 10 mM) without E. coli or inoculated with E. coli W3110 or E. coli nir-Ptac, or nitrite (NaNO2; 0, 1 or 2 mM) without E. coli or inoculated with E. coli W3110 or E. coli nir-Ptac. The control culture contained no chemical or microbial additives. Escherichia coli cells were inoculated into in vitro mixed ruminal cultures at approximately 2 x 10(8) to 10(9) cells/mL. Methane production by ruminal microorganisms was decreased markedly (P < 0.001) by the addition of nitrate and nitrite, and by the inoculation of cultures with E. coli W3110 or E. coli nir-Ptac (P < 0.01). With mixed nitrite-containing cultures, E. coli nir-Ptac inhibited (P < 0.001) in vitro nitrite accumulation and CH4 production more than E. coli W3110, which may be due to the tac promoter-enhanced nitrite reductase activity of E. coli nir-Ptac accelerating electrons to be consumed for nitrite reduction rather than CH4 biosynthesis. In conclusion, anaerobic cultures of E. coli W3110 or E. coli nir-Ptac may decrease CH4 production in the rumen. The inoculation of E. coli W3110 or, especially, E. coli nir-Ptac to mixed ruminal microorganisms may decrease nitrite toxicity when ruminants consume high-nitrate-containing forages and when nitrite is applied to abate ruminal CH4 production.

Nitrite reductase activity of sulphate-reducing bacteria prevents their inhibition by nitrate-reducing, sulphide-oxidizing bacteria.

Sulphate-reducing bacteria (SRB) can be inhibited by nitrate-reducing, sulphide-oxidizing bacteria (NR-SOB), despite the fact that these two groups are interdependent in many anaerobic environments. Practical applications of this inhibition include the reduction of sulphide concentrations in oil fields by nitrate injection. The NR-SOB Thiomicrospira sp. strain CVO was found to oxidize up to 15 mM sulphide, considerably more than three other NR-SOB strains that were tested. Sulphide oxidation increased the environmental redox potential (Eh) from -400 to +100 mV and gave 0.6 nitrite per nitrate reduced. Within the genus Desulfovibrio, strains Lac3 and Lac6 were inhibited by strain CVO and nitrate for the duration of the experiment, whereas inhibition of strains Lac15 and D. vulgaris Hildenborough was transient. The latter had very high nitrite reductase (Nrf) activity. Southern blotting with D. vulgaris nrf genes as a probe indicated the absence of homologous nrf genes from strains Lac3 and Lac6 and their presence in strain Lac15. With respect to SRB from other genera, inhibition of the known nitrite reducer Desulfobulbus propionicus by strain CVO and nitrate was transient, whereas inhibition of Desulfobacterium autotrophicum and Desulfobacter postgatei was long-lasting. The results indicate that inhibition of SRB by NR-SOB is caused by nitrite production. Nrf-containing SRB can overcome this inhibition by further reducing nitrite to ammonia, preventing a stalling of the favourable metabolic interactions between these two bacterial groups. Nrf, which is widely distributed in SRB, can thus be regarded as a resistance factor that prevents the inhibition of dissimilatory sulphate reduction by nitrite.

Dissimilatory hexaheme c nitrite reductase of ?Spirillum? strain 5175: purification and properties

When grown with nitrate as terminal electron acceptor both the soluble (periplasm, cytoplasm) and the membrane fraction of “Spirillum” strain 5175 exhibited high nitrite reductase activity. The nitrite reductase obtained from the soluble fraction was purified 76-fold to electrophoretical homogeneity. The enzyme reduced nitrite to ammonia with a specific activity of 723 μmol NO inf2 sup- × (mg protein × min)-1. The molecular mass was 58±1 kDa by SDS-PAGE compared to 59±2 kDa determined by size exclusion chromatography under nondenaturing conditions. The enzyme (as isolated) contained 5.97±0.15 heme c molecules/Mr 58 kDa. The absorption spectrum was typical for c-type cytochrome with maxima at 280, 408, 532 and 610 nm (oxidized) and at 420, 523 and 553 nm (dithionite-reduced). The enzyme (as isolated) exhibited a complex set of high-spin and lowspin ferric heme resonances with g-values at 9.82, 3,85, 3.31, 2.95, 2.30 and 1.49 in agreement with data reported for electron paramagnetic resonance spectra of nitrite reductases from Desulfovibrio desulfuricans, Wolinella succinogenes and Escherichia coli.

Diverse effects of formate on the dissimilatory metabolism of nitrate in Pseudomonas denitrificans ATCC 13867: Growth, nitrite accumulation in culture, cellular activities of nitrate and nitrite reductases

The growth of Pseudomonas denitrificans ATCC 13867 under denitrifying conditions was significantly stimulated by adding an appropriate amount of formate (2.5 mM or above) to the growth medium. The accumulation of nitrite in the culture was markedly depressed so long as formate remained in the culture above a certain level. Cellular activities of enzymes participating in denitrification also changed. The cells grown in the presence of formate exhibited a lower nitrate reductase activity and, in contrast, a higher nitrite reductase activity than the cells grown without added formate.

Absorption of atmospheric NO2by plants and soils

Abstract NO. uptake by leaves and reduction systems of nitrite produced were investigated using 3-week-old kidney bean, sunflower, and com plants, which were grown in vermiculite beds supplied with tap water. The plants were fumigated with 4 ppm NO2 for 6 hr in an artificially-lit chamber (30 klux), and the following things were observed. Fumigation in the light at daytime caused severe injury (wilting) in kidney bean leaves, and slight injury in sunflower leaves, but com leaves were tolerant to this treatment. A high concentration of nitrite rapidly accumulated in the kidney bean leaves, but the nitrite reductase activity was only increased to a small extent. In sunflower leaves a high concentration of nitrite was accumulated, and the nitrite reductase activity also increased rapidly. Fumigation in the dark at nighttime caused low concentration of nitrite to accumulate in kidney bean and sunflower leaves, and it was leveled down at the later fumigation time. The nitrite reductase activity of the two plants was increased with a slow rate continuously during the NO2 fumigation period. On the other hand, in the com leaves, nitrite was not detected in the daytime, but only a low concentration of nitrite was detected in the nighttime. A high activity of nitrite reductase was detected compared with the other two plants. Application of nitrate through the culture solution reduced nitrite accumulation in the light but not in the dark. These results indicate that acute NO. injury is mainly related to nitrite accumulation. Therefore, low NO2 absorption and high nitrite reductase activity (including its rapid induction) may reduce injury in leaves. When kidney bean and sunftower plants were fumigated with 4 ppm NO2 at different times of the day, a high concentration of nitrite accumulated only in the daytime, and not in the nighttime even if they were fumigated in the light. Absorption of 15NO3 at night by sunflower leaves was around 14% that at daytime. These indicate that a diurnal change of NO2 uptake is operating.

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.

Nitrate reduction in citrus tree leaves

A nitrate-reducing enzyme system is active in citrus tree leaf fragments. The system is inactivated in disrupted and damaged cells of leaf macerate. The measurement of nitrite as an indication of nitrate reductase activity is some times misleading because of its rapid disappearance due to high nitrite reductase activity.