Inorganic nitric oxide metabolites participating in NO-dependent modifications of biopolymers

Biogenous nitric(II) oxide (NO), the higher nitrogen oxides (NO2, isomeric N2O3 and N2O4, ONOO-, etc.) that are NO-derived in vivo, and the products of their transformations are active compounds capable of reactions with biopolymers and low-molecular metabolites. The products of these reactions are often considered to be various NO-dependent modifications (NODMs). The nitrated, nitrosylated, nitrosated, and other NODMs play key roles in the regulation of the most important biochemical processes. In this review, we briefly discuss the metabolic reactions of nitrogen oxides that supply active intermediates for NODMs, the NODM reaction products, and some mechanisms of NODM reparation that allow the recovery of chemically intact biopolymer molecule from a modified (chemically damaged) NODM. For example, residues of 3-nitrotyrosine arising due to the NODM reactions of proteins can be reduced to unsubstituted Tyr residues as a result of alternative NODM reactions through intermediate diazotyrosine derivatives. The heterogeneity of a medium in vivo is an important factor controlling the proceeding of NODM reactions. We showed that many processes determining NODM efficiency proceed differently in the heterogeneous media of organisms and in homogeneous aqueous solutions.     

Radical-induced deamination of 2-amino-2-deoxy-d-glucose in aqueous solution

2-Amino-2-deoxy-d-glucose (10mM) as the free base (pH 8.5) and the hydrochloride (pH 5) were γ-irradiated in aqueous solution under deoxygenated (N₂O-saturated) and oxygenated [N₂O/O₂(4:1)-saturated] conditions. Ammonia and 18 nitrogen-free products were identified and their G-values determined. Mechanisms for the radical-induced deamination are proposed. The radicals centered at C-1, C-2, C-3, and the nitrogen are suggested as initiators in the deamination processes.     

Sterilization of medical productsin low-pressure glow discharges

Results are presented from experimental and theoretical studies of the sterilization of medical products by the plasmas of dc glow discharges in different gas media. The sterilization efficiency is obtained as a function of discharge parameters. The plasma composition in discharges in N₂ and O₂ is investigated under the operating conditions of a plasma sterilizer. It is shown that free surfaces of medical products are sterilized primarily by UV radiation from the discharge plasma, while an important role in sterilization of products with complicated shapes is played by such chemically active particles as oxygen atoms and electronically excited O₂ molecules.     

A comparative study of complex formation in the reactions of gold(III) with Gly-Gly, Gly-l-Ala and Gly-l-His dipeptides

Proton NMR spectroscopy was applied to study the reactions of the dipeptides glycyl-glycine (Gly-Gly) and glycyl-l-alanine (Gly-l-Ala) with hydrogen tetrachloridoaurate(III) (H[AuCl₄]). All reactions were performed at pH 2.0 and 3.0 and at 40 °C. The final products in these reactions were [Au(Gly-Gly-κ³N_G1,N_G2,O_G2)Cl] and [Au(Gly-l-Ala-κ³N_G,N_A,O_A)Cl] complexes. Tridentate coordination of the corresponding dipeptides and square-planar geometry of these Au(III) complexes was confirmed by NMR (¹H and ¹³C) spectroscopy. This study showed that at pH < 3.0 the Au(III) ion was able to deprotonate the amide nitrogen atom. However this displacement reaction was very slow and the total concentration of the corresponding Au(III)-peptide complex formed after 5 days was less than 60% for the Gly-l-Ala or 70% for the Gly-Gly dipeptide. The kinetic data of the reactions between the Gly-Gly and Gly-l-Ala dipeptides and [AuCl₄]⁻ were compared with those for the histidine-containing Gly-l-His dipeptide. The differences in the reactivity of these three dipeptides with the Au(III) ion are discussed.     

Biochemical identification of residues that discriminate between 3,4-dihydroxyphenylalanine decarboxylase and 3,4-dihydroxyphenylacetaldehyde synthase-mediated reactions

In available insect genomes, there are several L-3,4-dihydroxyphenylalanine (L-dopa) decarboxylase (DDC)-like or aromatic amino acid decarboxylase (AAAD) sequences. This contrasts to those of mammals whose genomes contain only one DDC. Our previous experiments established that two DDC-like proteins from Drosophila actually mediate a complicated decarboxylation-oxidative deamination process of dopa in the presence of oxygen, leading to the formation of 3,4-dihydroxyphenylacetaldehyde (DHPA), CO₂, NH_3, and H₂O₂. This contrasts to the typical DDC-catalyzed reaction, which produces CO₂ and dopamine. These DDC-like proteins were arbitrarily named DHPA synthases based on their critical role in insect soft cuticle formation. Establishment of reactions catalyzed by these AAAD-like proteins solved a puzzle that perplexed researchers for years, but to tell a true DHPA synthase from a DDC in the insect AAAD family remains problematic due to high sequence similarity. In this study, we performed extensive structural and biochemical comparisons between DHPA synthase and DDC. These comparisons identified several target residues potentially dictating DDC-catalyzed and DHPA synthase-catalyzed reactions, respectively. Comparison of DHPA synthase homology models with crystal structures of typical DDC proteins, particularly residues in the active sites, provided further insights for the roles these identified target residues play. Subsequent site-directed mutagenesis of the tentative target residues and activity evaluations of their corresponding mutants determined that active site His192 and Asn192 are essential signature residues for DDC- and DHPA synthase-catalyzed reactions, respectively. Oxygen is required in DHPA synthase-mediated process and this oxidizing agent is reduced to H₂O₂ in the process. Biochemical assessment established that H₂O₂, formed in DHPA synthase-mediated process, can be reused as oxidizing agent and this active oxygen species is reduced to H₂O; thereby avoiding oxidative stress by H₂O₂. Results of our structural and functional analyses provide a reasonable explanation of mechanisms involved in DHPA synthase-mediated reactions. Based on the key active site residue Asn192, identified in Drosophila DHPA synthase, we were able to distinguish all available insect DHPA synthases from DDC sequences primarily.     

Heparan Sulfate Containing Unsubstituted Glucosamine Residues: BIOSYNTHESIS AND HEPARANASE-INHIBITORY ACTIVITY*

Degradation of heparan sulfate (HS) in the extracellular matrix by heparanase is linked to the processes of tumor invasion and metastasis. Thus, a heparanase inhibitor can be a potential anticancer drug. Because HS with unsubstituted glucosamine residues accumulates in heparanase-expressing breast cancer cells, we assumed that these HS structures are resistant to heparanase and can therefore be utilized as a heparanase inhibitor. As expected, chemically synthetic HS-tetrasaccharides containing unsubstituted glucosamine residues, GlcAβ1–4GlcNH₃⁺(6-O-sulfate)α1–4GlcAβ1–4GlcNH₃⁺(6-O-sulfate), inhibited heparanase activity and suppressed invasion of breast cancer cells in vitro. Bifunctional NDST-1 (N-deacetylase/N-sulfotransferase-1) catalyzes the modification of N-acetylglucosamine residues within HS chains, and the balance of N-deacetylase and N-sulfotransferase activities of NDST-1 is thought to be a determinant of the generation of unsubstituted glucosamine. We also report here that EXTL3 (exostosin-like 3) controls N-sulfotransferase activity of NDST-1 by forming a complex with NDST-1 and contributes to generation of unsubstituted glucosamine residues.     

Simulation of toluene decomposition in a pulse-periodic discharge operating in a mixture of molecular nitrogen and oxygen

The kinetic model of toluene decomposition in nonequilibrium low-temperature plasma generated by a pulse-periodic discharge operating in a mixture of nitrogen and oxygen is developed. The results of numerical simulation of plasma-chemical conversion of toluene are presented; the main processes responsible for C₆H₅CH₃ decomposition are identified; the contribution of each process to total removal of toluene is determined; and the intermediate and final products of C₆H₅CH₃ decomposition are identified. It was shown that toluene in pure nitrogen is mostly decomposed in its reactions with metastable N₂(A₃?? _u ⁺ ) and N₂(a??¹?? _u ^? ) molecules. In the presence of oxygen, in the N₂ : O₂ gas mixture, the largest contribution to C₆H₅CH₃ removal is made by the hydroxyl radical OH which is generated in this mixture exclusively due to plasma-chemical reactions between toluene and oxygen decomposition products. Numerical simulation showed the existence of an optimum oxygen concentration in the mixture, at which toluene removal is maximum at a fixed energy deposition.     

NO-dependent modifications of nucleic acids

This review is devoted to chemical transformations of nucleic acids and their components under the action of nitrogen oxide metabolites. The deamination reaction of bases is discussed in the context of possible competing transformations of its intermediates (nitrosamines, diazonium cations, diazotates, triazenes, and diazoanhydrides) and mechanisms of crosslink formation with proteins and nucleic acids. The oxidation and nitration of bases by NO₂ is considered together with the possibility of radical transfer to domains from the base stacks in DNA. Reduction of redox potentials of bases as a result of stacking interactions explains the possibility of their reactions within nucleic acids with the oxidants whose redox potential is insufficient for the effective reactions with mononucleotides. Modifications of nucleic acids with peroxynitrite derivatives are discussed in the context of the effect of the DNA primary structure and the modification products formed on the reactivity of single bases. The possibility of reduction of nitro groups within modified bases to amino derivatives and their subsequent diazotation is considered. The substitution of oxoguanine for nitroguanine residues may result; the reductive diazotation can lead to undamaged guanine. The intermediate modified bases, e.g., 8-aminoguanine and 8-diazoguanine, were shown to participate in noncanonical base pairing, including the formation of more stable bonds with two bases, which is characteristic of the DNA Z-form. A higher sensitivity of RNA in comparison with DNA to NO-dependent modifications (NODMs) is predicted on the basis of the contribution of medium microheterogeneity and the known mechanisms of nitrosylation and nitration. The possible biological consequences of nucleic acids NODMs are briefly considered. It is shown that the NODMs under the action of nitrogen oxide metabolites generated by macrophages and similar cells in inflammations or infections should lead to a sharp increase in the number of mutations in the case of RNA-containing viruses. As a result, the defense mechanisms of the host organism may contribute to the appearance of new, including more dangerous, variants of infecting viruses.     

In situ 15N-N2O site preference and O2 concentration dynamics disclose the complexity of N2O production processes in agricultural soil

Arable soil continues to be the dominant anthropogenic source of nitrous oxide (N₂O) emissions owing to application of nitrogen (N) fertilizers and manures across the world. Using laboratory and in situ studies to elucidate the key factors controlling soil N₂O emissions remains challenging due to the potential importance of multiple complex processes. We examined soil surface N₂O fluxes in an arable soil, combined with in situ high-frequency measurements of soil matrix oxygen (O₂) and N₂O concentrations, in situ ¹⁵N labeling, and N₂O ¹⁵N site preference (SP). The in situ O₂ concentration and further microcosm visualized spatiotemporal distribution of O₂ both suggested that O₂ dynamics were the proximal determining factor to matrix N₂O concentration and fluxes due to quick O₂ depletion after N fertilization. Further SP analysis and in situ ¹⁵N labeling experiment revealed that the main source for N₂O emissions was bacterial denitrification during the hot-wet summer with lower soil O₂ concentration, while nitrification or fungal denitrification contributed about 50.0% to total emissions during the cold-dry winter with higher soil O₂ concentration. The robust positive correlation between O₂ concentration and SP values underpinned that the O₂ dynamics were the key factor to differentiate the composite processes of N₂O production in in situ structured soil. Our findings deciphered the complexity of N₂O production processes in real field conditions, and suggest that O₂ dynamics rather than stimulation of functional gene abundances play a key role in controlling soil N₂O production processes in undisturbed structure soils. Our results help to develop targeted N₂O mitigation measures and to improve process models for constraining global N₂O budget.     

The Nitric-oxide Reductase from Paracoccus denitrificans Uses a Single Specific Proton Pathway

The NO reductase from Paracoccus denitrificans reduces NO to N₂O (2NO + 2H⁺ + 2e⁻ → N₂O + H₂O) with electrons donated by periplasmic cytochrome c (cytochrome c-dependent NO reductase; cNOR). cNORs are members of the heme-copper oxidase superfamily of integral membrane proteins, comprising the O₂-reducing, proton-pumping respiratory enzymes. In contrast, although NO reduction is as exergonic as O₂ reduction, there are no protons pumped in cNOR, and in addition, protons needed for NO reduction are derived from the periplasmic solution (no contribution to the electrochemical gradient is made). cNOR thus only needs to transport protons from the periplasm into the active site without the requirement to control the timing of opening and closing (gating) of proton pathways as is needed in a proton pump. Based on the crystal structure of a closely related cNOR and molecular dynamics simulations, several proton transfer pathways were suggested, and in principle, these could all be functional. In this work, we show that residues in one of the suggested pathways (denoted pathway 1) are sensitive to site-directed mutation, whereas residues in the other proposed pathways (pathways 2 and 3) could be exchanged without severe effects on turnover activity with either NO or O₂. We further show that electron transfer during single-turnover reduction of O₂ is limited by proton transfer and can thus be used to study alterations in proton transfer rates. The exchange of residues along pathway 1 showed specific slowing of this proton-coupled electron transfer as well as changes in its pH dependence. Our results indicate that only pathway 1 is used to transfer protons in cNOR.     

Current dynamics and mechanisms for the instability of a non-self-sustained glow discharge in nitrogen

The current dynamics in a non-self-sustained glow discharge in atmospheric-pressure nitrogen (with a small admixture of oxygen) at cryogenic and room temperatures is studied experimentally and theoretically. For the first time, the theoretical model incorporates the processes of the decomposition of O ₂ ⁺ ·N₂ and NO⁺·N₂ complex ions in collisions with vibrationally excited nitrogen molecules and the associative ionization reactions with the participation of excited nitrogen and oxygen atoms. The computation results agree quite satisfactorily with the experimental data on the current dynamics and the duration of the stable phase of a non-self-sustained discharge for various applied voltages. Even a small (0.01%) oxygen admixture is found to greatly affect the dynamics of the ion composition and the characteristic duration of the stable phase of a non-self-sustained discharge in atmospheric-pressure nitrogen.     

Isolation and Characterization of an Oxygen Sensitive Mutant of Azorhizobium caulinodans

An oxygen sensitive mutant of Azorhizobium caulinodans strain IRBG 46 was isolated by NTG mutagenesis. It was defective in N₂ fixation under 3% O₂ level, while under 1% O₂ it was almost as active as the parent strain IRBG 46. The mutant was also found to be a slow grower with reduced respiratory activity, low azide tolerance and no catalase activity. However, it did not differ from its parent strain with respect to nitrate respiration. Under symbiotic condition the mutant formed smaller, light green nodules as compared to bigger, dark green nodules formed by the wild type strain. The mutant was also defective in N₂ fixation under symbiotic condition. Complementation analysis showed that the mutation might be in either fixL or fixJ gene which are involved in O₂ regulation of nif/fix gene expression. A possible role of all these factors in conferring a highly O₂ tolerant nitrogen fixing system in the organism, has been discussed.     

Investigation of the mechanism for rapid heating of nitrogen and air in gas discharges

A rapid heating of nitrogen-oxygen mixtures excited by gas discharges is investigated numerically with allowance for the following main processes: the reactions of predissociation of highly excited electronic states of oxygen molecules (which are populated via electron impact or via the quenching of the excited states of N₂ molecules), the reactions of quenching of the excited atoms O(¹ D) by nitrogen molecules, the VT relaxation reactions, etc. The calculated results adequately describe available experimental data on the dynamics of air heating in gas-discharge plasmas. It is shown that, over a broad range of values of the reduced electric field E/N, gas heating is maintained by a fixed fraction of the discharge power that is expended on the excitation of the electronic degrees of freedom of molecules (for discharges in air, η_E?28%). The lower the oxygen content of the mixture, the smaller the quantity η_E. The question of a rapid heating of nitrogen with a small admixture of oxygen is discussed.     

Physical and morphological constraints on transport in nodules

For active nodule nitrogen fixation, O₂, N₂, and carbohydrate must be transported throughout the nodule. No quantitative analysis of these transport processes in the nodules has been presented. By invoking several simplifying assumptions, a second-order differential equation for the various gradients and concentrations in the nodule was solved. Even though the nodule can only be approximated in this analysis, it indicates clearly that intercellular gas spaces must exist in nodules for adequate O₂ distribution. To preserve low O₂ concentrations and protect the nitrogenase, these gas spaces cannot be in direct contact with the ambient atmosphere. It is hypothesized that a gas barrier exists in the cortical region of the nodule to limit O₂ diffusion. This barrier would not substantially inhibit N₂ transport. Carbohydrate transport from the vascular tissue via diffusion in the liquid phase can adequately accommodate the requirements within the nodule.     

Reaction of N-hydroxyguanidine with the ferrous-oxy state of a heme peroxidase cavity mutant: A model for the reactions of nitric oxide synthase

Yeast cytochrome c peroxidase was used to construct a model for the reactions catalyzed by the second cycle of nitric oxide synthase. The R48A/W191F mutant introduced a binding site for N-hydroxyguanidine near the distal heme face and removed the redox active Trp-191 radical site. Both the R48A and R48A/W191F mutants catalyzed the H₂O₂ dependent conversion of N-hydroxyguanidine to N-nitrosoguanidine. It is proposed that these reactions proceed by direct one-electron oxidation of NHG by the Fe⁺⁴O center of either Compound I (Fe⁺⁴O, porph⁺) or Compound ES (Fe⁺⁴O, Trp⁺). R48A/W191F formed a Fe⁺²O₂ complex upon photolysis of Fe⁺²CO in the presence of O₂, and N-hydroxyguanidine was observed to react with this species to produce products, distinct from N-nitrosoguanidine, that gave a positive Griess reaction for nitrate + nitrite, a positive Berthelot reaction for urea, and no evidence for formation of NO. It is proposed that HNO and urea are produced in analogy with reactions of nitric oxide synthase in the pterin-free state.     

Diurnal variation in the functioning of cowpea nodules

Nitrogenase (EC 1.7.99.2) activity of nodules of cowpea (Vigna unguiculata [L.] Walp), maintained under conditions of a 12-hour day at 30°C and 800 to 1,000 microeinsteins per square meter per second (photosynthetically active radiation) and a 12-hour night at 20°C, showed a marked diurnal variation with the total electron flux through the enzyme at night being 60% of that in the photoperiod. This diurnal pattern was, however, due to changes in hydrogen evolution. The rate of nitrogen fixation, measured by short-term ¹⁵N₂ assimilation or estimated from the difference in hydrogen evolution in air or Ar:O₂ (80:20; v/v), showed no diurnal variation. Carbon dioxide released from nodules showed a diurnal variation synchronized with that of nitrogenase functioning and, as a consequence, the apparent `respiratory cost' of nitrogen fixation in the photoperiod was almost double that at night (9.74 ± 0.38 versus 5.70 ± 0.90 moles CO₂ evolved per mole N₂ fixed). Separate carbon and nitrogen balances constructed for nodules during the photoperiod and dark period showed that, at night, nodule functioning required up to 40% less carbohydrate to achieve the same level of nitrogen fixation as during the photoperiod (2.4 versus 1.4 moles hexose per mole N₂ fixed).

Stored reserves of nonstructural carbohydrate of the nodule only partly satisfied the requirement for carbon at night, and fixation was dependent on continued import of translocated assimilates at all times. Measurements of the soluble nitrogen pools of the nodule together with ¹⁵N studies indicated that, both during the day and night, nitrogenous products of fixation were effectively translocated to all organs of the host plant despite low rates of transpiration at night. Reduced fluxes of water through the plant at night were apparently counteracted by increased concentration of nitrogen, especially as ureides, in the xylem stream.

     

Evolution of Nitrogen Oxide(s) during In Vivo Nitrate Reductase Assay of Soybean Leaves

Studies were conducted to quantitate the evolution of nitrogen oxides (NO_(x)) from soybean [Glycine max (L.) Merr.] leaves during in vivo nitrate reductase (NR) assays with aerobic and anaerobic gas purging. Anaerobic gas purging (N₂ and argon) consistently resulted in greater NO_(x) evolution than did aerobic gas purging (air and O₂). The evolution of NO_(x) was dependent on gas flow rate and on NO₂⁻ formation in the assay medium; although a threshold level of NO₂⁻ appeared to exist beyond which the rate of NO_(x) evolution did not increase further.     

Influence of Osmotic and Nutritional Stress on Physiology of Penicillium fellutanum in Removal of Phosphocholine and Modification of Phospho-1-O-[N-Peptidyl-(2-Aminoethanol)] Phosphodiesters of Peptidophosphogalactomannan Species

Addition of 3 M NaCl to 72-h cultures of Penicillium fellutanum in 2 mM phosphate resulted in an increase in percentage of extracellular peptidophosphogalactomannan III (pP_xGMⁱⁱⁱ) and a decrease in that of pP_xGMⁱⁱ. The magnitude of ³¹P nuclear magnetic resonance signals at 1.47 and 1.33 ppm of phospho-1-O-[N-peptidyl-(2-aminoethanol)] phosphodiesters pP_xGMⁱⁱ and pP_xGMⁱⁱⁱ decreased compared with controls. The data suggest that serine, glycine, and threonine residues from the 3-kDa peptide and from galactofuranosyl-6-O-phospho-1′-O-[N-peptidyl-(2-aminoethanol)] residues were the precursors of the needed choline-derived osmolytes.     

Bioenergy by‐products as soil amendments? Implications for carbon sequestration and greenhouse gas emissions

An important but little understood aspect of bioenergy production is its overall impact on soil carbon (C) and nitrogen (N) cycling. Increased energy production from biomass will inevitably lead to higher input of its by‐products to the soil as amendments or fertilizers. However, it is still unclear how these by‐products will influence microbial transformation processes in soil, and thereby its greenhouse gas (GHG) balance and organic C stocks. In this study, we assess C and N dynamics and GHG emissions following application of different bioenergy by‐products to soil. Ten by‐products were selected from different bioenergy chains: anaerobic digestion (manure digestates), first generation biofuel by‐products (rapeseed meal, distilled dried grains with solubles), second‐generation biofuel by‐products (nonfermentables from hydrolysis of different lignocellulosic materials) and pyrolysis (biochars). These by‐products were added at a constant N rate (150 kg N ha^?1) to a sandy soil and incubated at 20 °C. After 60 days, >80% of applied C had been emitted as CO₂ in the first‐generation biofuel residue treatments. For second‐generation biofuel residues this was approximately 60%, and for digestates 40%. Biochars were the most stable residues with the lowest CO₂ loss (between 0.5% and 5.8% of total added C). Regarding N₂O emissions, addition of first‐generation biofuel residues led to the highest total N₂O emissions (between 2.5% and 6.0% of applied N). Second‐generation biofuel residues emitted between 1.0% and 2.0% of applied N, with the original feedstock material resulting in similar N₂O emissions and higher C mineralization rates. Anaerobic digestates resulted in emissions <1% of applied N. The two biochars used in this study decreased N₂O emissions below background values. We conclude that GHG dynamics of by‐products after soil amendment cannot be ignored and should be part of the lifecycle analysis of the various bioenergy production chains.     

Electrochemical and spectroelectrochemical studies of complexes of 1,10-phenanthroline-5,6-dione

Electrochemical and spectroelectrochemical (UV-Vis, IR, EPR) of pd (pd = 1,10-phenanthroline-5,6-dione), Pt(N,N′-pd)Cl2, Pd(N,N′-pd)Cl₂, [Ru(bpy)₂(N,N′-pd)]Cl₂ (bpy = 2,2′-bipyridine) and Pt(O,O′-pd)(PPh₃)₂, where N,N′ and O,O′ refers to coordination of pd to the metal centre via N and O atoms, respectively, reveals that the electron transfer processes between +0.5 and −1.25 V all occur at the pd ligand in agreement with DFT calculations. The two CO groups carry a significant amount of the negative charge in mono-reduced pd¹⁻. The mode of coordination of pd has a greater influence on its redox chemistry than the metal centre or the ancillary ligands.