Nitric Oxide Reductase Activity Research Articles

EPR spectroscopy of putative enzyme intermediates in the NO reductase and the auto-nitrosylation reaction of Desulfovibrio vulgaris hybrid cluster protein.

The hybrid cluster protein (Hcp) contains a unique 4Fe cluster that is a hybrid of μ-S and μ-O bridges. Escherichiacoli Hcp has recently been found to carry NO reductase activity as well as S-nitrosylation activity in NO-based signaling. In other species, the physiological activity has not been established. No reaction mechanism of any Hcp has been proposed. Here, we show that Desulfovibriovulgaris (Hildenborough) Hcp has nitric oxide reductase activity with benzyl viologen as electron donor. With EPR spectroscopy, we identify three unexpected putative reaction intermediates: both in reduced and oxidized Hcp, dinitrosyl iron complexes are formed. Also, the hybrid cluster in reduced Hcp, but not in oxidized Hcp, binds the product N2 O. Possible implications for a reaction mechanism are discussed.

Concomitant exercise training attenuates the cardioprotective effects of pharmacological therapy in a murine model of acute infectious myocarditis

When administered alone, preinfection exercise training and benznidazole-based chemotherapy induce cardioprotection in Chagas disease. However, the effect of concomitant exercise and benznidazole treatment is unknown. We investigated whether exercise and specific chemotherapy could interact to modulate parasitemia, inflammation, redox status and heart damage in a murine model of T. cruzi infection. Wistar rats were randomized into an uninfected control group (CNT) and four groups infected with T. cruzi: sedentary untreated (SUN) and treated (STR), and trained untreated (TUN) and treated (TTR). Running training was administered 5 days/week for 4 weeks. Treated animals concomitantly received 100 mg/kg/day benznidazole. Heart inflammation and reactive damage were not detected in CNT animals. Compared to SUN, TUN animals presented increased levels of parasitemia, myocarditis, nitric oxide, hydrogen peroxide, protein carbonyl, malondialdehyde, cytokines (IFN-γ, TNF-α, IL-4, IL-6, IL-10 and IL-17), catalase, superoxide dismutase and glutathione reductase activity, as well as reduced heart non-protein antioxidant levels (P < 0.05). TTR animals exhibited higher levels of parasitemia, myocarditis, hydrogen peroxide, malondialdehyde, IFN-γ, TNF-α and IL-6 than STR animals (P < 0.05), which showed the lowest levels of all analyzed parameters compared to the other groups (P < 0.05). Our findings indicate that exercise aggravates acute infection. When concomitantly administered with benznidazole, exercise training impaired parasitic control and chemotherapy-induced cardioprotection in T. cruzi-infected rats. Considering that exercise training and T. cruzi infection constitute independent metabolic challenges, the negative effects of concomitant treatment are potentially related to the overlapping oxidative and immunoinflammatory demands of exercise and the infection itself.

The Response of nor and nos Contributes to Staphylococcus aureus Virulence and Metabolism.

Staphylococcus aureus causes a wide spectrum of disease, with the site and severity of infection dependent on virulence traits encoded within genetically distinct clonal complexes (CCs) and bacterial responses to host innate immunity. The production of nitric oxide (NO) by activated phagocytes is a major host response to which S. aureus metabolically adapts through multiple strategies that are conserved in all CCs, including an S. aureus nitric oxide synthase (Nos). Previous genome analysis of CC30, a lineage associated with chronic endocardial and osteoarticular infections, revealed a putative NO reductase (Nor) not found in other CCs that potentially contributes to NO resistance and clinical outcome. Here, we demonstrate that Nor has true nitric oxide reductase activity, with nor expression enhanced by NO stress and anaerobic growth. Furthermore, we demonstrate that nor is regulated by MgrA and SrrAB, which modulate S. aureus virulence and hypoxic response. Transcriptome analysis of the S. aureus UAMS-1, UAMS-1 Δnor, and UAMS-1 Δnos strains under NO stress and anaerobic growth demonstrates that Nor contributes to nucleotide metabolism and Nos to glycolysis. We demonstrate that Nor and Nos contribute to enhanced survival in the presence of human human polymorphonuclear cells and have organ-specific seeding in a tail vein infection model. Nor contributes to abscess formation in an osteological implant model. We also demonstrate that Nor has a role in S. aureus metabolism and virulence. The regulation overlap between Nor and Nos points to an intriguing link between regulation of intracellular NO, metabolic adaptation, and persistence in the CC30 lineage.IMPORTANCEStaphylococcus aureus can cause disease at most body sites, and illness spans asymptomatic infection to death. The variety of clinical presentations is due to the diversity of strains, which are grouped into distinct clonal complexes (CCs) based on genetic differences. The ability of S. aureus CC30 to cause chronic infections relies on its ability to evade the oxidative/nitrosative defenses of the immune system and survive under different environmental conditions, including differences in oxygen and nitric oxide concentrations. The significance of this work is the exploration of unique genes involved in resisting NO stress and anoxia. A better understanding of the functions that control the response of S. aureus CC30 to NO and oxygen will guide the treatment of severe disease presentations.

Energy recovery in the form of N2O by denitrifying bacteria

Nitrous oxide (N2O) is a highly energetic oxidant. This work documents a novel process that recovers N2O by inhibiting the activity of the nitrous-oxide reductase (N2OR) in denitrifying bacteria. N2O recovery was demonstrated in batch experiments in which NO was the only electron-acceptor, with Fe(II)EDTA being the chelating agent for NO. Increasing NO-Fe(II)EDTA led to greater accumulation of N2O over N2 in batch studies (up to 70% of total gas produced), and N2O accumulation correlated to loss of nitrous-oxide reductase (N2OR) activity and conformational changes to cytochrome c, the immediate electron donor for N2OR. Stable and high production of N2O was also documented in long-term experiments with NO-Fe(II)EDTA as the only acceptor for denitrifying biomass. Mechanistically, the conformational change of cytochrome c was the dominant cause for the loss of electron-transfer capacity, which led to a decrease of N2OR activity and the accumulation of N2O. And this is the first time to recover energy source from flue gas in the form of N2O. Recovery of N2O by suppressing N2OR widens the options for energy recovery from nitrogen-based wastes.

Impacts of long term copper exposure on abundance of nitrogen cycling genes and denitrification activity in pasture soils

Soils are the main source of nitrous oxide (N2O) emissions, with 60–70% of global N2O production due to the enhanced denitrification activity with elevated nitrogen (N) availability associated with agricultural intensification. The amendment of agricultural soils with biosolids, the residual product of wastewater treatment, has been used for several decades as an inexpensive source of organic matter and N. Biosolids also contain elevated levels of trace elements such as copper (Cu), which influence the activity of the bacterial enzyme nitrous oxide reductase (N2OR). We extracted DNA from a pasture soil 20 years after application of biosolid and Cu salts and used an environmental genomics approach (GeoChip) to evaluate broad changes in the abundance and structure of families of N-cycling genes. We then focussed on abundances of individual bacterial genes involved in sequential N-reduction (nirS, nirK, and nosZ). There was no relationship between total soil Cu concentrations and the different N-cycling gene families (GeoChip; P > 0.1). However, increasing Zn was associated with decreased nitrification (amoA) and dissimilatory N-reduction (napA and nrfA) gene-family abundances (P < 0.1). When focused on individual bacterial denification pathway genes (qPCR), nosZ (nitrous oxide reductase) was found to be sensitive to Cu concentrations. Moreover, the activity of the Cu-dependent enzyme N2OR is potentially affected by historical Cu concentrations found in these pasture soils. The results highlight the sensitivity of a few key functional taxa (nosZ type denitrifying bacteria) to long-term Cu exposure in soil, and how these effects can translate into alteration of wider N-cycling processes such as those affecting net N2O emissions.

Nitric Oxide Reductase Activity in Heme-Nonheme Binuclear Engineered Myoglobins through a One-Electron Reduction Cycle.

FeBMbs are structural and functional models of native bacterial nitric oxide reductases (NORs) generated through engineering of myoglobin. These biosynthetic models replicate the heme-nonheme diiron site of NORs and allow substitutions of metal centers and heme cofactors. Here, we provide evidence for multiple NOR turnover in monoformyl-heme-containing FeBMb1 proteins loaded with FeII, CoII, or ZnII metal ions at the FeB site (FeII/CoII/ZnII-FeBMb1(MF-heme)). FTIR detection of the ν(NNO) band of N2O at 2231 cm-1 provides a direct quantitative measurement of the product in solution. A maximum number of turnover is observed with FeII-FeBMb1(MF-heme), but the NOR activity is retained when the FeB site is loaded with ZnII. These data support the viability of a one-electron semireduced pathway for the reduction of NO at binuclear centers in reducing conditions.

Impact of hydraulic retention time and current on the microbial community and denitrification genes in a continuous‐flow biofilm electrode reactor

AbstractBACKGROUNDA biofilm‐electrode reactor (BER) was developed on the basis of hydrogenotrophic denitrification. The BER can be strengthened by adding organic matter, and the nitrate removal will be enhanced by a combination of heterotrophic and autotrophic denitrification. Denitrification in a biological system can be affected by operating parameters, and the microbial community and functional genes can be changed at the same time. In this study, the effect of hydraulic retention time (HRT) and current on the denitrification of a low chemical oxygen demand to nitrogen ratio (COD/N), saline wastewater in a continuous‐flow BER was evaluated and compared to a conventional biofilm reactor (BR).RESULTSThe highest extent of nitrate removal was obtained at the optimum HRT (8 h) and current (10 mA). Low levels of ammonia (0.5–4 mg L−1), produced through dissimilatory nitrate reduction to ammonia (DNRA), were observed. Quantitative real‐time polymerase chain reaction analysis revealed that the proportion of nirS‐type denitrifiers (3.75–8.30%) exceeded nirK‐type denitrifiers (0.24–0.90%) in both BR and BER. The genera of T78, Paracoccus and Azoarcus were identified as the dominant denitrifying bacteria in the BER. The cooperative activity of nitrate, nitrite and nitrous oxide reductase is necessary to reduce nitrate to dinitrogen in the denitrification process. The highest ratio of nosZ/nir genes in the bacterial community was 0.25 when the applied current was 10 mA.CONCLUSIONCompared to other denitrification reactors, the BER is more effective to treat low‐COD/N, saline wastewater with short HRT and low current. © 2018 Society of Chemical Industry

Acute response of soil denitrification and N2O emissions to chlorothalonil: A comprehensive molecular mechanism

The fungicide chlorothalonil (CHT) has been widely used in the tea orchard due to its high-efficiency and sterilization. It has been reported that repeated application of CHT inhibits soil nitrification process. However, the acute impact of CHT on soil denitrification and associated N2O emissions is unclear. This study evaluated nitrate (NO3−) removal, denitrifying gene abundance and denitrifying enzyme activity of tea orchard soil after a 72-h-exposure to CHT. It was found that increasing CHT from 5 to 25 mg kg−1 suppressed the NO3− removal efficiency from 74.6% to 54.1%, but increased N2O emissions from 23.1% to 94.8%. Following treatment with 25 mg kg−1 of CHT, the abundances of the nirK, nirS and nosZ genes were reduced by 31.6%, 22.1%, and 50.7%, respectively. Alternatively, the declines of the electron transport system activity (ETSA) value and adenosine triphosphate (ATP) content suggested that CHT had an inhibitory effect on microbial metabolism. Enzyme activity studies further revealed that the decrease of nitrate reductase (NAR), nitrite reductase (NIR) and nitric oxide reductase (NOR) activities was the main reason for the suppression of denitrification by CHT. Furthermore, positive correlations were observed between denitrifying reductase activity and the intracellular metabolism, indicating that the decrease in microbial metabolism should also be responsible for the inhibitory effect of CHT on the denitrifying process. Overall, it was found that the acute exposure of soil to CHT could inhibit the denitrification process and significantly increase N2O emissions, which might result in destruction of the soil nitrogen cycle and exacerbation of global warming.

Greenhouse gas emission from the soybean rhizosphere is mitigated by promoting nitrous oxide reductase activity in bradyrhizobia

Greenhouse gas emission from the soybean rhizosphere is mitigated by promoting nitrous oxide reductase activity in bradyrhizobia

Diversity and complexity of flavodiiron NO/O2 reductases.

Flavodiiron proteins (FDPs) are a family of enzymes endowed with nitric oxide (NO) or oxygen reductase activities, forming the innocuous nitrous oxide (N2O) or water molecules, respectively. FDPs are widespread in the three life kingdoms, and have a modular nature, being each monomer minimally constituted by a metallo-β-lactamase-like domain containing a catalytic diiron centre, followed by a flavodoxin one, with a flavin mononucleotide. Since their discovery, additional domains have been found in FDPs, attached to the C-terminus, and containing either extra metal (iron) centers or extra flavin binding modules. Following an extensive analysis of genomic databases, we identified novel domain compositions, and proposed a new classification of FDPs in eight classes based on the nature and number of extra domains.

Valence Localization at a Bio-inspired Mixed-Valent {Cu2 S}2+ Motif upon Solvation in Acetonitrile: Effect on Nitrous Oxide Reductase (N2 Or) Activity.

We demonstrate, based on experimental and theoretical evidence, that the isolated [2(CH3 CN)2 ]2+ complex prepared in CH3 CN and containing a mixed-valent {Cu2II,I S} core evolves towards a new [2(CH3 CN)3 ]2+ species upon solvation in CH3 CN. Unlike its type III structural analogue [2(H2 O)(OTf)]+ active toward N2 O reduction, this new type I compound is inactive. This outcome opens new perspectives for a rational for N2 O activation using bio-inspired Cu/S complexes, especially on the role of the valence localization/delocalization and the Cu-Cu bond on the reactivity.

Dimethyl pyrazol-based nitrification inhibitors effect on nitrifying and denitrifying bacteria to mitigate N2O emission

Nitrous oxide (N2O) emissions have been increasing as a result of intensive nitrogen (N) fertilisation. Soil nitrification and denitrification are the main sources of N2O, and the use of ammonium-based fertilisers combined with nitrification inhibitors (NIs) could be useful in mitigating N2O emissions from agricultural systems. In this work we looked at the N2O mitigation capacity of two dimethylpyrazol-based NIs, 3,4-dimethylpyrazole phosphate (DMPP) and 2-(N-3,4-dimethyl-1H-pyrazol-1-yl) succinic acid isomeric mixture (DMPSA), on soil nitrifying and denitrifying microbial populations under two contrasting soil water contents (40% and 80% soil water filled pore space; WFPS). Our results show that DMPP and DMPSA are equally efficient at reducing N2O emissions under 40% WFPS conditions by inhibiting bacterial ammonia oxidation. In contrast, at 80% WFPS DMPSA was less efficient than DMPP at reducing N2O emissions. Interestingly, at 80% WFPS, where lowered oxygen availability limits nitrification, both DMPP and DMPSA not only inhibited nitrification but also stimulated N2O reduction to molecular nitrogen (N2) via nitrous oxide reductase activity (Nos activity). Therefore, in this work we observed that DMP-based NIs stimulated the reduction of N2O to N2 by nitrous oxide reductase during the denitrification process.

A new fluorescence biosensor for nitric oxide detection based on cytochrome P450 55B1

Nitric oxide (NO) is an important biological signaling molecule, which plays an important role in the regulation of cell function of the nervous, vascular and immune systems. Nitric oxide also is involved in the parthenogenesis of Parkinson’s disease and tumor angiogenesis. Thus, the detection of nitric oxide is essential to understand its physiological and pathological process. Cytochrome P450 55B1 from Chlamydomonas reinhardtii is known to function as nitric oxide reductase (NOR). Here we describe the development of a new fluorescence biosensor for NO based on the NOR activity of P450 55B1. In this paper, we constructed pET28a-55b1 vector and expressed P450 55B1 in Escherichia coli successfully. We studied the interaction between P450 55B1 and NO by spectrometric method. The result show that the fluorescence intensity of iron porphyrin of P450 55B1 increase gradually with the addition of NO. When the concentration of NO is below 22.5μM, the fluorescence intensity increase linearly with the concentration of NO from a baseline value of ΔF=0.0675+0.15 CNO (in μM), which is slightly affected by potentially interfering chemicals. NO release from the rat liver homogenate stimulated by l-arginine was determined by the fluorescence NO biosensor. The results demonstrated that our strategy can be used to detect the concentration of NO in biologically samples.

Transcriptional and metabolic regulation of denitrification in Paracoccus denitrificans allows low but significant activity of nitrous oxide reductase under oxic conditions

Oxygen is known to repress denitrification at the transcriptional and metabolic levels. It has been a common notion that nitrous oxide reductase (N2 OR) is the most sensitive enzyme among the four N-oxide reductases involved in denitrification, potentially leading to increased N2 O production under suboxic or fluctuating oxygen conditions. We present detailed gas kinetics and transcription patterns from batch culture experiments with Paracoccus denitrificans, allowing in vivo estimation of e(-) -flow to O2 and N2 O under various O2 regimes. Transcription of nosZ took place concomitantly with that of narG under suboxic conditions, whereas transcription of nirS and norB was inhibited until O2 levels approached 0 μM in the liquid. Catalytically functional N2 OR was synthesized and active in aerobically raised cells transferred to vials with 7 vol% O2 in headspace, but N2 O reduction rates were 10 times higher when anaerobic pre-cultures were subjected to the same conditions. Upon oxygen exposure, there was an incomplete and transient inactivation of N2 OR that could be ascribed to its lower ability to compete for electrons compared with terminal oxidases. The demonstrated reduction of N2 O at high O2 partial pressure and low N2 O concentrations by a bacterium not known as a typical aerobic denitrifier may provide one clue to the understanding of why some soils appear to act as sinks rather than sources for atmospheric N2 O.

Bacterial Mobilization of Nutrients From Biochar-Amended Soils.

Soil amendments with biochar to improve soil fertility and increase soil carbon stocks have received some high-level attention. Physical and chemical analyses of amended soils and biochars from various feedstocks are reported, alongside some evaluations of plant growth promotion capabilities. Fewer studies investigated the soil microbiota and their potential to increase cycling and mobilization of nutrients in biochar-amended soils. This review is discussing the latest findings in the bacterial contribution to cycling and mobilizing nitrogen, phosphorus, and sulfur in biochar-amended soils and potential contributions to plant growth promotion. Depending on feedstock, pyrolysis, soil type, and plant cover, changes in the bacterial community structure were observed for a majority of the studies using amplicon sequencing or genetic fingerprinting methods. Prokaryotic nitrification largely depends on the availability of ammonium and can vary considerably under soil biochar amendment. However, denitrification to di-nitrogen and in particular, nitrous oxide reductase activity is commonly enhanced, resulting in reduced nitrous oxide emissions. Likewise, bacterial fixation of di-nitrogen appears to be regularly enhanced. A paucity of studies suggests that bacterial mobilization of phosphorus and sulfur is enhanced as well. However, most studies only tested for extracellular sulfatase and phosphatase activity. Further research is needed to reveal details of the bacterial nutrient mobilizing capabilities and this is in particular the case for the mobilization of phosphorus and sulfur.

Impact of internal recycle ratio on nitrous oxide generation from anaerobic/anoxic/oxic biological nitrogen removal process

To evaluate the effects of internal recycle ratio on nitrous oxide (N2O) generation, we set up three laboratory-scale anaerobic–anoxic–oxic (A2O) wastewater treatment processes, with internal recycle ratios of 100%, 200%, and 300%, respectively. Total nitrogen (TN) removal was markedly enhanced from 45.8% to 61.9%, as the internal recycle ratio increased from 100% to 300%. N2O generation was increased from 3.47 to 9.81×10−2mg/L during the treatment process, with the anoxic section showing the largest N2O increment from denitrification. This phenomenon is attributed to the increased amount of nitrate (NO3−-N) substrate available for denitrification, due to the increased volume of internal recycle liquid, as well as the increased amount of oxygen, which could restrain the activity of nitrous oxide reductase brought to the anoxic section. Nitrous oxide reductase was more sensitive to oxygen than nitrate and nitrite reductases. Microorganism analysis indicated that the population of nosZ gene-containing bacteria was only slightly affected by the recycle ratio. However, the number of nosZ gene copies decreased as the internal recycle ratio increased from 100% to 300%; this result reveals noticeable decreases in the denitrification capacity of the system for reducing N2O to N2.

Can pH amendments in grazed pastures help reduce N2O emissions from denitrification? – The effects of liming and urine addition on the completion of denitrification in fluvial and volcanic soils

Soil pH plays a critical role in determining the overall rate of several important processes in the agricultural nitrogen cycle. During denitrification, the activity of nitrous oxide reductase (N2O-R) is reduced at low pH. This effect has led to suggestions that soil pH adjustment via liming to enhance the activity of this enzyme might be a viable agricultural greenhouse gas mitigation strategy by enhancing the reduction of N2O in the soil to climatically inert N2. We assessed the effect of liming on the apparent activity of N2O-R by measuring the denitrification end products, N2O and N2, in a series of short-term anaerobic incubations. We compared a weakly-buffered fluvial soil and a well-buffered, volcanic soil under different incubation temperatures and in the presence or absence of a ∼600 kg ha−1 cow urine-N amendment. Our results indicated that the liming effect was heavily modulated by soil type, temperature, and urine amendment. Liming (at rates of 1.5 and 3.0 t ha−1 for the volcanic soil and at rates of 5 and 10 t ha−1 for the fluvial soil) caused pH increases of between 0.43 and 1.25 pH units. The highest reductions in N2O in the fluvial soil occurred when the 1.5 t ha−1 rate was used in the fluvial soils under urine addition and at the higher temperature. The combined flux of N2O + N2 did not change with liming. However, an interaction of soil type and urine amendment caused large differences in the partitioning of the denitrification end-products between N2O and N2 – an effect that overwhelmed the relatively modest effects of liming. When the soils were amended with urine-N, the resulting denitrification gases from the volcanic soil were mostly in the form of N2O (60–77%), whereas in the fluvial soil the denitrification products were mostly in the form of N2 and a much smaller portion were in the form of N2O (11–45%). Nevertheless, we found liming-induced enhancements of N2O-R of 15–20% (P < 0.05) in urine-amended, fluvial soil. We suggest some possible mechanisms that would explain such large differences in the N2O/(N2O + N2) product ratio.

Dioxygen and nitric oxide scavenging by Treponema denticola flavodiiron protein: a mechanistic paradigm for catalysis.

Flavodiiron proteins (FDPs) contain a unique active site consisting of a non-heme diiron carboxylate site proximal to a flavin mononucleotide (FMN). FDPs serve as the terminal components for reductive scavenging of dioxygen (to water) or nitric oxide (to nitrous oxide), which combats oxidative or nitrosative stress in many bacteria. Characterizations of FDPs from spirochetes or from any oral microbes have not been previously reported. Here, we report characterization of an FDP from the anaerobic spirochete, Treponema (T.) denticola, which is associated with chronic periodontitis. The isolated T. denticola FDP exhibited efficient four-electron dioxygen reductase activity and lower but significant anaerobic nitric oxide reductase activity. A mutant T. denticola strain containing the inactivated FDP-encoding gene was significantly more air-sensitive than the wild-type strain. Single turnover reactions of the four-electron-reduced FDP (FMNH2-Fe(II)Fe(II)) (FDPred) with O2 monitored on the milliseconds to seconds time scale indicated initial rapid formation of a spectral feature consistent with a cis-μ-1,2-peroxo-diferric intermediate, which triggered two-electron oxidation of FMNH2. Reaction of FDPred with NO showed apparent cooperativity between binding of the first and second NO to the diferrous site. The resulting diferrous dinitrosyl complex triggered two-electron oxidation of the FMNH2. Our cumulative results on this and other FDPs indicate that smooth two-electron FMNH2 oxidation triggered by the FDPred/substrate complex and overall four-electron oxidation of FDPred to FDPox constitutes a mechanistic paradigm for both dioxygen and nitric oxide reductase activities of FDPs. Four-electron reductive O2 scavenging by FDPs could contribute to oxidative stress protection in many other oral bacteria.

Chromium speciation and biochemical changes vary in relation to plant ploidy

Uptake of trivalent chromium (Cr(III)-chloride), Cr speciation and consequences for the metabolism in chamomile plants with two ploidy levels have been studied. Depletion of fresh biomass, tissue water content and soluble proteins in response to high (120μM) Cr(III) was ploidy-independent. Cr mainly accumulated in the roots (only negligibly in the shoots) and total root Cr amount was higher in tetraploid ones including the proof with specific fluorescent indicator (naphthalimide–rhodamine) of Cr(III). Quantification of Cr(VI) detected its higher content in tetraploid roots (up to 4.2% from total Cr), indicating partial oxidation of applied Cr(III). Higher H2O2 presence but lower activities of peroxidases were observed in tetraploid roots while nitric oxide, superoxide dismutase and glutathione reductase activities did not differ extensively. Soluble phenols, lignin, non-protein thiols, individual thiols (glutathione and phytochelatin 2) and ascorbic acid responded to high Cr(III) similarly in both cultivars while decrease of minerals was more pronounced in tetraploid ones. It seems that Cr(III)-induced oxidative stress arises from high root Cr uptake and Cr(VI) presence and is related to depletion of thiols. Assay of Krebs cycle acids confirmed rather depletion under 120μM Cr(III) in both cultivars but increase in citric acid may indicate its involvement in root Cr chelation. Subsequent comparison of Cr(III)-chloride and Cr(III)-nitrate showed similar influence on Cr accumulation and majority of biochemical responses while different impact on phytochelatin 2 amount was the most distinct feature.

Probing the nitrite and nitric oxide reductase activity of cbb3 oxidase: resonance Raman detection of a six-coordinate ferrous heme-nitrosyl species in the binuclear b3/CuB center.

In this work we report the first spectroscopic evidence demonstrating that cbb3 oxidase catalyzes the reduction of nitrite to nitrous oxide under reducing anaerobic conditions. The reaction proceeds through the formation of a ferrous six-coordinate heme b3-nitrosyl species that has been characterized by resonance Raman spectroscopy.