Role of Carbonyl Compounds for N-Nitrosamine Formation during Nitrosation: Kinetics and Mechanisms.

N-Nitrosamines are potential human carcinogens frequently detected in natural and engineered aquatic systems. This study sheds light on the role of carbonyl compounds in the formation of N-nitrosamines by nitrosation of five secondary amines via different pathways. The results showed that compared to a control system, the presence of formaldehyde enhances the formation of N-nitrosamines by a factor of 5-152 at pH 7, depending on the structure of the secondary amines. Acetaldehyde showed a slight enhancement effect on N-nitrosamine formation, while acetone and benzaldehyde did not promote nitrosation reactions. For neutral and basic conditions, the iminium ion was the dominant intermediate for N-nitrosamine formation, while carbinolamine became the major contributor under acidic conditions. Negative free energy changes (<-19 kcal mol-1) and relatively low activation energies (<18 kcal mol-1) of the reactions of secondary amines with N2O3, iminium ions with nitrite and carbinolamines with N2O3 from quantum chemical computations further support the proposed reaction pathways. This highlights the roles of the iminium ion and carbinolamine in the formation of N-nitrosamines during nitrosation in the presence of carbonyl compounds, especially in the context of industrial wastewater.

Redox Reactivity of Nonsymbiotic Phytoglobins towards Nitrite.

Nonsymbiotic phytoglobins (nsHbs) are a diverse superfamily of hemoproteins grouped into three different classes (1, 2, and 3) based on their sequences. Class 1 Hb are expressed under hypoxia, osmotic stress, and/or nitric oxide exposure, while class 2 Hb are induced by cold stress and cytokinins. Both are mainly six-coordinated. The deoxygenated forms of the class 1 and 2 nsHbs from A. thaliana (AtHb1 and AtHb2) are able to reduce nitrite to nitric oxide via a mechanism analogous to other known globins. NsHbs provide a viable pH-dependent pathway for NO generation during severe hypoxia via nitrite reductase-like activity with higher rate constants compared to mammalian globins. These high kinetic parameters, along with the relatively high concentrations of nitrite present during hypoxia, suggest that plant hemoglobins could indeed serve as anaerobic nitrite reductases in vivo. The third class of nsHb, also known as truncated hemoglobins, have a compact 2/2 structure and are pentacoordinated, and their exact physiological role remains mostly unknown. To date, no reports are available on the nitrite reductase activity of the truncated AtHb3. In the present work, three representative nsHbs of the plant model Arabidopsis thaliana are presented, and their nitrite reductase-like activity and involvement in nitrosative stress is discussed. The reaction kinetics and mechanism of nitrite reduction by nsHbs (deoxy and oxy form) at different pHs were studied by means of UV-Vis spectrophotometry, along with EPR spectroscopy. The reduction of nitrite requires an electron supply, and it is favored in acidic conditions. This reaction is critically affected by molecular oxygen, since oxyAtHb will catalyze nitric oxide deoxygenation. The process displays unique autocatalytic kinetics with metAtHb and nitrate as end-products for AtHb1 and AtHb2 but not for the truncated one, in contrast with mammalian globins.

C-Nitrosation, C-Nitration, and Coupling of Flavonoids with N-Acetyltryptophan Limit This Amine N-Nitrosation in a Simulated Cured and Cooked Meat.

Nitrite is a common additive in cured meat formulation that provides microbiological safety, lipid oxidation management, and typical organoleptic properties. However, it is associated with the formation of carcinogenic N-nitrosamines. In this context, the antinitrosating capacity of selected flavonoids and ascorbate was evaluated in a simulated cooked and cured meat under formulation and digestion conditions. N-Acetyltryptophan was used as a secondary amine target. (-)-Epicatechin, rutin, and quercetin were all able to limit the formation of N-acetyl-N-nitrosotryptophan (NO-AcTrp) at pH 2.5 and pH 5 although (-)-epicatechin was 2 to 3-fold more efficient. Kinetics for the newly identified compounds allowed us to unravel common mechanistic pathways, which are flavonoid oxidation by nitrite followed by C-nitration and an original covalent coupling between NO-AcTrp and flavonoids or their nitro and nitroso counterparts. C-nitrosation of the A-ring was evidenced only for (-)-epicatechin. These major findings suggest that flavonoids could help to manage N-nitrosamine formation during cured meat processing, storage, and digestion.

Closing Dichloramine Decomposition Nitrogen and Oxygen Mass Balances: Relative Importance of End-Products from the Reactive Nitrogen Species Pathway.

In drinking water chloramination, monochloramine autodecomposition occurs in the presence of excess free ammonia through dichloramine, the decay of which was implicated in N-nitrosodimethylamine (NDMA) formation by (i) dichloramine hydrolysis to nitroxyl which reacts with itself to nitrous oxide (N2O), (ii) nitroxyl reaction with dissolved oxygen (DO) to peroxynitrite or mono/dichloramine to nitrogen gas (N2), and (iii) peroxynitrite reaction with total dimethylamine (TOTDMA) to NDMA or decomposition to nitrite/nitrate. Here, the yields of nitrogen and oxygen-containing end-products were quantified at pH 9 from NHCl2 decomposition at 200, 400, or 800 µeq Cl2·L-1 with and without 10 µM-N TOTDMA under ambient DO (∼500 µM-O) and, to limit peroxynitrite formation, low DO (≤40 µM-O). Without TOTDMA, the sum of free ammonia, monochloramine, dichloramine, N2, N2O, nitrite, and nitrate indicated nitrogen recoveries ±95% confidence intervals were not significantly different under ambient (90 ± 6%) and low (93 ± 7%) DO. With TOTDMA, nitrogen recoveries were less under ambient (82 ± 5%) than low (97 ± 7%) DO. Oxygen recoveries under ambient DO were 88-97%, and the so-called unidentified product of dichloramine decomposition formed at about three-fold greater concentration under ambient compared to low DO, like NDMA, consistent with a DO limitation. Unidentified product formation stemmed from peroxynitrite decomposition products reacting with mono/dichloramine. For a 2:2:1 nitrogen/oxygen/chlorine atom ratio and its estimated molar absorptivity, unidentified product inclusion with uncertainty may close oxygen recoveries and increase nitrogen recoveries to 98% (ambient DO) and 100% (low DO).

Simultaneous aerobic nitrogen and phosphate removal capability of novel salt-tolerant strain, Pseudomonas mendocina A4: Characterization, mechanism and application potential.

A salt-tolerant strain, Pseudomonas mendocina A4, was isolated from brackish-water ponds showing simultaneous heterotrophic nitrification-aerobic denitrification and phosphorus removal capability. The optimal conditions for nitrogen and phosphate removal of strain A4 were pH 7-8, carbon/nitrogen ratio 10, phosphorus/nitrogen ratio 0.2, temperature 30 °C, and salinity range of 0-5 % using sodium succinate as the carbon source. The nitrogen and phosphate removal efficiencies were 96-100 % and 88-96 % within 24 h, respectively. The nitrogen and phosphate removal processes were matched with the modified Gompertz model, and the underlying mechanisms were confirmed by the activities of key metabolic enzymes. Under 10 % salinity, the immobilization technology was employed to enhance the nitrogen and phosphate removal efficiencies of strain A4, achieving 87 % and 76 %, respectively. These findings highlight the potential application of strain A4 in both freshwater and marine culture wastewater treatment.

Simultaneous inorganic nitrogen and phosphate removal by aerobic-heterotrophic fungus Fusarium keratoplasticum FSP1: Performance, pathway and application.

Fungi with multiple contaminant removal function have rarely been studied. Here, a novel fungal strain Fusarium keratoplasticum FSP1, which was isolated from halophilic granular sludge, is reported for first time to perform simultaneous nitrogen and phosphate removal. The strain showed wide adaptability under C/N ratios of 30-35, salinities of 0 %-3 % (m/v), and pH of 7.5-9.5. The maximum removal rates of ammonium, nitrate and nitrite were 4.43, 4.01 and 2.97 mg N/L/h. The nitrogen balance, enzyme activity and substrate conversion experiments demonstrated a single strain FSP1 can assimilate inorganic nitrogen and convert inorganic nitrogen to gaseous nitrogen through heterotrophic nitrification or aerobic denitrification. About 39 %-42 % of the degraded phosphorus was in the extracellular polymeric substances (EPS). Orthophosphate was the main phosphorus species in the cell, whereas phosphate monoester and diester were in the EPS. The novel strain FSP1 is a potential candidate for wastewater treatment.

Advancements in Preprocessing and Analysis of Nitrite and Nitrate since 2010 in Biological Samples: A Review.

As a substance present in organisms, nitrite is a metabolite of nitric oxide and can also be ingested. Nitrate is the metabolite of nitrite. Therefore, it is necessary to measure it quickly, easily and accurately to evaluate the health status of humans. Although there have been several reviews on analytical methods for non-biological samples, there have been no reviews focused on both sample preparation and analytical methods for biological samples. First, rapid and accurate nitrite measurement has significant effects on human health. Second, the detection of nitrite in biological samples is problematic due to its very low concentration and matrix interferences. Therefore, the pretreatment plus measuring methods for nitrite and nitrate obtained from biological samples since 2010 are summarized in the present review, and their prospects for the future are proposed. The treatment methods include liquid-liquid microextraction, various derivatization reactions, liquid-liquid extraction, protein precipitation, solid phase extraction, and cloud point extraction. Analytical methods include spectroscopic methods, paper-based analytical devices, ion chromatography, liquid chromatography, gas chromatography-mass spectrometry, electrochemical methods, liquid chromatography-mass spectrometry and capillary electrophoresis. Derivatization reagents with rapid quantitative reactions and advanced extraction methods with high enrichment efficiency are also included. Nitrate and nitrate should be determined at the same time by the same analytical method. In addition, much exploration has been performed on formulating fast testing through microfluidic technology. In this review, the newest developments in nitrite and nitrate processing are a focus in addition to novel techniques employed in such analyses.

Granulation of partial denitrification sludge: Advances in mechanism understanding, technologies development and perspectives.

The high-rate and stably efficient nitrite generation is vital and still challenges the wide application of partial denitrification (PD) and anammox technology. Increasing attention has been drawn to the granulation of PD biomass. However, the knowledge of PD granular sludge is still limited in terms of granules characterization and mechanisms of biomass aggregation for high nitrite accumulation. This work reviewed the performance and granulation of PD biomass for high nitrite accumulation via nitrate reduction, including the system start-up, influential factors, granular characteristics, hypothetical mechanism, challenges and perspectives in future application. The physiochemical characterization and key influential factors were summarized in view of nitrite production, morphology analysis, extracellular polymer substance structure, as well as microbial mechanisms. The PD granules exhibit potential advantages of a high biomass density, good settleability, high hydraulic loading rates, and strong shock resistance. A novel granular sludge-based PD combined with anammox process was proposed to enhance the capability of nitrogen removal. In the future, PD granules utilizing different electron donors is a promising way to broaden the application of anammox technology in both municipal and industrial wastewater treatment.

Bringing Nitric Oxide to the Molybdenum World-A Personal Perspective.

Molybdenum-containing enzymes of the xanthine oxidase (XO) family are well known to catalyse oxygen atom transfer reactions, with the great majority of the characterised enzymes catalysing the insertion of an oxygen atom into the substrate. Although some family members are known to catalyse the "reverse" reaction, the capability to abstract an oxygen atom from the substrate molecule is not generally recognised for these enzymes. Hence, it was with surprise and scepticism that the "molybdenum community" noticed the reports on the mammalian XO capability to catalyse the oxygen atom abstraction of nitrite to form nitric oxide (NO). The lack of precedent for a molybdenum- (or tungsten) containing nitrite reductase on the nitrogen biogeochemical cycle contributed also to the scepticism. It took several kinetic, spectroscopic and mechanistic studies on enzymes of the XO family and also of sulfite oxidase and DMSO reductase families to finally have wide recognition of the molybdoenzymes' ability to form NO from nitrite. Herein, integrated in a collection of "personal views" edited by Professor Ralf Mendel, is an overview of my personal journey on the XO and aldehyde oxidase-catalysed nitrite reduction to NO. The main research findings and the path followed to establish XO and AO as competent nitrite reductases are reviewed. The evidence suggesting that these enzymes are probable players of the mammalian NO metabolism is also discussed.

Versatile MXene composite probe-mediated homogeneous electrochemiluminescence biosensor with integrated signal transduction and near-infrared modulation strategy for concanavalin A detection.

Based on the highly specific interaction between concanavalin A (Con A) and glucose (Glu), a competitive electrochemiluminescence (ECL) biosensor was constructed for ultrasensitive detection of Con A. Nanocomposites with excellent electrocatalytic and photothermal properties were obtained by covalently bonding zinc oxide quantum dots (ZnO QDs) to vanadium carbide MXene (V2C MXene) surfaces. The modification of ZnO QDs hinders the aggregation of V2C MXene and increases the catalytic activity of oxygen reduction reaction, thus amplifying the luminol cathodic emission. In addition, the excellent photothermal performance of the V2C MXene-ZnO QDs can convert light energy into heat energy under the irradiation of 808 nm near infrared laser, thus increasing the temperature of the reaction system and accelerating the electron transfer process to realize the synergistic amplified homogeneous ECL system. This innovative work not only enriches the fundamental research on multifunctional MXene nanomaterials for biosensing, but also provides an effective strategy for ECL signal amplification.

Biomimetic catalysis of nitrite reductase enzyme using copper complexes in chemical and electrochemical reduction of nitrite.

Copper nitrite reductase mimetics were synthesized using three new tridentate ligands sharing the same N,N,N motif of coordination. The ligands were based on L-proline modifications, attaching a pyridine and a triazole to the pyrrolidine ring, and differ by a pendant group (R = phenyl, n-butyl and n-propan-1-ol). All complexes coordinate nitrite, as evidenced by cyclic voltammetry, UV-Vis, FTIR and electron paramagnetic resonance (EPR) spectroscopies. The coordination mode of nitrite was assigned by FTIR and EPR as κ2O chelate mode. Upon acidification, EPR experiments indicated a shift from chelate to monodentate κO mode, and 15N NMR experiments of a Zn2+ analogue, suggested that the related Cu(II) nitrous acid complex may be reasonably stable in solution, but in equilibrium with free HONO under non catalytic conditions. Reduction of nitrite to NO was performed both chemically and electrocatalytically, observing the highest catalytic activities for the complex with n-propan-1-ol as pendant group. These results support the hypothesis that a hydrogen bond moiety in the secondary coordination sphere may aid the protonation step.

Efficient nitrogen removal by a novel extreme strain, Pseudomonas reactans WL20-3 under dual stresses of low temperature and high alkalinity: Characterization, mechanism, and application.

Although many studies report the resistance of heterotrophic nitrification-aerobic denitrification (HN-AD) strains to single environmental stress, there is no research on its resistance to dual stresses of low temperature and high alkalinity. A novel bacterium Pseudomonas reactants WL20-3 isolated in this study showed removal efficiencies of 100%, 100%, and 97.76% for ammonium, nitrate, and nitrite, respectively, at 4 °C and pH 11.0. Transcriptome analysis revealed that the resistance of strain WL20-3 to dual stresses was attributed not only to the regulation of genes in the nitrogen metabolic pathway, but also to genes in other pathways such as the ribosome, oxidative phosphorylation, amino acid metabolism, and ABC transporters. Additionally, WL20-3 removed 83.98% of ammonium from actual wastewater at 4 °C and pH 11.0. This study isolated a novel strain WL20-3 with superior nitrogen removal under dual stresses and provided a molecular understanding of its tolerance mechanism to low temperature and high alkalinity.

Morphological Effects of Au Nanoparticles on Electrochemical Sensing Platforms for Nitrite Detection.

Au nanoparticles were synthesized in a soft template of pseudo-polyanions composed of polyvinylpyrrolidone (PVP) and sodium dodecyl sulfate (SDS) by the in situ reduction of chloroauric acid (HAuCl4) with PVP. The particle sizes and morphologies of the Au nanoparticles were regulated with concentrations of PVP or SDS at room temperature. Distinguished from the Au nanoparticles with various shapes, Au nanoflowers (AuNFs) with rich protrusion on the surface were obtained at the low final concentration of SDS and PVP. The typical AuNF synthesized in the PVP (50 g·L-1)-SDS (5 mmol·L-1)-HAuCl4 (0.25 mmol·L-1) solution exhibited a face-centered cubic structure dominated by a {111} crystal plane with an average equivalent particle size of 197 nm and an average protrusion height of 19 nm. Au nanoparticles with four different shapes, nanodendritic, nanoflower, 2D nanoflower, and nanoplate, were synthesized and used to modify the bare glassy carbon electrode (GCE) to obtain Au/GCEs, which were assigned as AuND/GCE, AuNF/GCE, 2D-AuNF/GCE, and AuNP/GCE, respectively. Electrochemical sensing platforms for nitrite detection were constructed by these Au/GCEs, which presented different detection sensitivity for nitrites. The results of cyclic voltammetry (CV) demonstrated that the AuNF/GCE exhibited the best detection sensitivity for nitrites, and the surface area of the AuNF/GCE was 1.838 times of the bare GCE, providing a linear c(NO2-) detection range of 0.01-5.00 µmol·L-1 with a limit of detection of 0.01 µmol·L-1. In addition, the AuNF/GCE exhibited good reproducibility, stability, and high anti-interference, providing potential for application in electrochemical sensing platforms.

Self-supported polypyrrole flexible electrodes for electrochemical reduction of nitrite.

The efficiency of an electrochemical oxidation/reduction process strongly depends on the working electrode's surface area to volume ratio. By making electrodes flexible and employing different configurations such as roll-to-roll membrane, the surface area to volume ratio can be enhanced, therefore improving the overall efficiency of electrochemical processes. Conductive polymers emerge as a new framework to enable alternative electrochemical water treatment cell configurations. Self-standing polypyrrole flexible electrodes were synthesized by electropolymerization and evaluated on the treatment of an oxyanion pollutant: nitrite. Mechanical characterization through stress-strain curves and bending tests demonstrated high electrode resilience that sustained over 1000 bending cycles without impacting mechanical integrity or electrocatalytic responses. The electrocatalytic response towards nitrite reduction was assessed under linear scan voltammetry (LSV) and removal performance evaluated under potentiostatic conditions reaching 79% abatement of initial concentrations of nitrite of 15 mg/L [NO2--N]. Self-standing flexible electrodes appear as a novel framework to enable modular compact water treatment unit designs that maximize the electrode area/volume ratio and substitute expensive platinum group metal (PGMs) electrocatalysts.

GC-MS Studies on Nitric Oxide Autoxidation and S-Nitrosothiol Hydrolysis to Nitrite in pH-Neutral Aqueous Buffers: Definite Results Using 15N and 18O Isotopes.

Nitrite (O=N-O-, NO2-) and nitrate (O=N(O)-O-, NO3-) are ubiquitous in nature. In aerated aqueous solutions, nitrite is considered the major autoxidation product of nitric oxide (●NO). ●NO is an environmental gas but is also endogenously produced from the amino acid L-arginine by the catalytic action of ●NO synthases. It is considered that the autoxidation of ●NO in aqueous solutions and in O2-containing gas phase proceeds via different neutral (e.g., O=N-O-N=O) and radical (e.g., ONOO●) intermediates. In aqueous buffers, endogenous S-nitrosothiols (thionitrites, RSNO) from thiols (RSH) such as L-cysteine (i.e., S-nitroso-L-cysteine, CysSNO) and cysteine-containing peptides such as glutathione (GSH) (i.e., S-nitrosoglutathione, GSNO) may be formed during the autoxidation of ●NO in the presence of thiols and dioxygen (e.g., GSH + O=N-O-N=O → GSNO + O=N-O- + H+; pKaHONO, 3.24). The reaction products of thionitrites in aerated aqueous solutions may be different from those of ●NO. This work describes in vitro GC-MS studies on the reactions of unlabeled (14NO2-) and labeled nitrite (15NO2-) and RSNO (RS15NO, RS15N18O) performed in pH-neutral aqueous buffers of phosphate or tris(hydroxyethylamine) prepared in unlabeled (H216O) or labeled H2O (H218O). Unlabeled and stable-isotope-labeled nitrite and nitrate species were measured by gas chromatography-mass spectrometry (GC-MS) after derivatization with pentafluorobenzyl bromide and negative-ion chemical ionization. The study provides strong indication for the formation of O=N-O-N=O as an intermediate of ●NO autoxidation in pH-neutral aqueous buffers. In high molar excess, HgCl2 accelerates and increases RSNO hydrolysis to nitrite, thereby incorporating 18O from H218O into the SNO group. In aqueous buffers prepared in H218O, synthetic peroxynitrite (ONOO-) decomposes to nitrite without 18O incorporation, indicating water-independent decomposition of peroxynitrite to nitrite. Use of RS15NO and H218O in combination with GC-MS allows generation of definite results and elucidation of reaction mechanisms of oxidation of ●NO and hydrolysis of RSNO.

Reducing O2 sensitivity in electrochemical nitric oxide releasing catheters: An O2-tolerant copper(II)-ligand nitrite reduction catalyst and a glucose oxidase catheter coating.

Electrocatalytic nitric oxide (NO) generation from nitrite (NO2-) within a single lumen of a dual-lumen catheter using CuII-ligand (CuII-L) mediators have been successful at demonstrating NO's potent antimicrobial and antithrombotic properties to reduce bacterial counts and mitigate clotting under low oxygen conditions (e.g., venous blood). Under more aerobic conditions, the O2 sensitivity of the Cu(II)-ligand catalysts and the reaction of O2 (highly soluble in the catheter material) with the NO diffusing through the outer walls of the catheters results in a large decreases in NO fluxes from the surfaces of the catheters, reducing the utility of this approach. Herein, we describe a new more O2-tolerant CuII-L catalyst, [Cu(BEPA-EtSO3)(OTf)], as well as a potentially useful immobilized glucose oxidase enzyme-coating approach that greatly reduces the NO reactivity with oxygen as the NO partitions and diffuses through the catheter material. Results from this work demonstrate that very effective NO fluxes (>1*10-10 mol min-1 cm-2) from a single-lumen silicone rubber catheter can be achieved in the presence of up to 10% O2 saturated solutions.

Multi-parameter control-based operation strategy for mainstream deammonification in an integrated anaerobic biofilm reactor-step feed MBR.

The mainstream deammonification of municipal wastewater has been recognized as one of the greatest challenges in wastewater engineering. The conventional activated sludge process has disadvantages of high energy input and sludge production. To tackle this situation, an innovative A-B process, where an anaerobic biofilm reactor (AnBR) functioned as the A stage for energy recovery, and a step-feed membrane bioreactor (MBR) functioned as the B stage for mainstream deammonification, was constructed for carbon-neutral wastewater treatment. For addressing the challenge associated with selective retention of ammonia-oxidizing bacteria (AOB) over nitrite oxidizing bacteria (NOB), a multi-parameter control-based operation strategy was developed with synergistic control of influent COD redistribution, dissolved oxygen (DO) concentration and sludge retention time (SRT) in the innovative AnBR - step-feed MBR system. Results showed that more than 85% of wastewater COD could be removed with the direct production of methane gas in the AnBR. A relatively stable partial nitritation, which is a prerequisite of anammox, was achieved with the successful suppression of NOB, leading to 98% of ammonium-N and 73% of total nitrogen removed. Anammox bacteria could well survive and enrich in the integrated system, and the contribution of anammox to the total nitrogen removal was more than 70% at optimal conditions. Reactions network involved in the nitrogen transformation in the integrated system was further constructed through the mass balance and microbial community structure analyses. Consequently, this study demonstrated a practically feasible process configuration with high operation and control flexibility towards stable mainstream deammonification of municipal wastewater.

Adsorption of nitrate and nitrite anion by modified maize stalks from aqueous solutions.

The newly developed aminated maize stalk (AMS) was prepared by a chemical process using charred maize stalk (CMS). The AMS was used for the removal of nitrate and nitrite ions from aqueous media. The effects of initial anion concentration, contact time, and pH were studied by the batch method. The prepared adsorbent was characterized by FT-IR, XRD, FE-SEM , and elemental analysis. The concentration of the nitrate and nitrite solution before and after was determined with the help of a UV-Vis spectrophotometer. The maximum adsorption capacities were found to be 294.11 mg/g for nitrate and 232.55 mg/g for nitrite, respectively, at pH 5 for both ions attaining equilibrium within 60 min. The BET surface area of AMS was found to be 25.3 m2/g with a pore volume of 0.02cc/g. The pseudo-second-order kinetics model fit well, and the adsorption data supported the Langmuir isotherm. The findings revealed that AMS has a high capability for removing nitrate (NO3-) and nitrite (NO2-) ions from their aqueous solutions.

Molecular and kinetic properties of copper nitrite reductase from Sinorhizobium meliloti 2011 upon substituting the interfacial histidine ligand coordinated to the type 2 copper active site for glycine.

A copper-containing nitrite reductase catalyzes the reduction of nitrite to nitric oxide in the denitrifier Sinorhizobium meliloti 2011 (SmNirK), a microorganism used as bioinoculant in alfalfa seeds. Wild type SmNirK is a homotrimer that contains two copper centers per monomer, one of type 1 (T1) and other of type 2 (T2). T2 is at the interface of two monomers in a distorted square pyramidal coordination bonded to a water molecule and three histidine side chains, H171 and H136 from one monomer and H342 from the other. We report the molecular, catalytic, and spectroscopic properties of the SmNirK variant H342G, in which the interfacial H342 T2 ligand is substituted for glycine. The molecular properties of H342G are similar to those of wild type SmNirK. Fluorescence-based thermal shift assays and FTIR studies showed that the structural effect of the mutation is only marginal. However, the kinetic reaction with the physiological electron donor was significantly affected, which showed a âˆ¼ 100-fold lower turnover number compared to the wild type enzyme. UV-Vis, EPR and FTIR studies complemented with computational calculations indicated that the drop in enzyme activity are mainly due to the void generated in the protein substrate channel by the point mutation. The main structural changes involve the filling of the void with water molecules, the direct coordination to T2 copper ion of the second sphere aspartic acid ligand, a key residue in catalysis and nitrite sensing in NirK, and to the loss of the 3 N-O coordination of T2.

Solid State Kinetics of Nitrosation Using Native Sources of Nitrite.

While many reactive species are known to cause N-nitrosation, trace nitrite (NO2-), which may be present in several excipients, is a source of nitrosating agents in pharmaceutical formulations. In this study we have found that the salt form of NO2- can influence the favored nitrosation conditions and final amount of nitrosamine being formed. Using native levels of NO2-, most likely present as ammonium nitrite (NH4NO2), in microcrystalline cellulose, we have determined the kinetics of nitrosamine formation in solid state with dimethylamine substrate present in metformin, used as model compound. It was found that the competing degradation of NH4NO2 into N2 and H2O limited the amount of nitrosamine formation to a great extent. Empirically modelling the kinetic data predicted reaching at maximum 1.6% conversion over a hypothetical 3-year shelf-life. These results also showed that using other sources of NO2- as spiking reagents, such as NaNO2, may lead to unrealistic worst-case situations when the main form of NO2- in the drug product (DP) under evaluation may be NH4NO2. As well, measuring NO2- in freshly manufactured excipients containing NO2- potentially as NH4NO2 may lead to biased high NO2- content, which is not representative of the actual amounts present at the time of DP manufacture.