Autowave distribution of nitric oxide and its endogenous derivatives in biosystems strongly enhances their biological effects: A working hypothesis

It is hypothesized that in cells producing nitric oxide (NO), NO and its endogenous derivatives (low-molecular S-nitrosothiols and dinitrosyl iron complexes (DNIC) with thiol-containing ligands) can move in the intracellular space not only by diffusion but also in an autowave mode. This hypothesis is based on the previously obtained data on autowave distribution of DNIC with glutathione following application of a drop of a solution of Fe²⁺ + glutathione onto the surface of a thin layer of a S-nitrosoglutathione solution. The appearance of autowaves is conditioned by a self-regulating self-sustained system arising in the process. This system consists of self-convertible DNIC and S-nitrosothiols as well as free ferrous iron ions, thiols and NO and can function in the autowave regime for several seconds with subsequent passage to a steady state maintained by chemical equilibrium between DNIC and their constituent components (free Fe²⁺ ions, thiols, S-nitrosothiols and NO). Possible advantages of autowave distribution of NO and its endogenous derivatives in the intracellular space over free diffusion, which might entail higher efficiency of their biological action, are discussed.     

GC–MS analysis of S-nitrosothiols after conversion to S-nitroso-N-acetyl cysteine ethyl ester and in-injector nitrosation of ethyl acetate

S-Nitrosothiols from low-molecular-mass and high-molecular-mass thiols, including glutathione, albumin and hemoglobin, are endogenous potent vasodilators and inhibitors of platelet aggregation. By utilizing the S-transnitrosation reaction and by using the lipophilic (pK_L 0.78) and strong nucleophilic synthetic thiol N-acetyl cysteine ethyl ester (NACET) we have developed a GC–MS method for the analysis of S-nitrosothiols and their ¹⁵N- or ²H–¹⁵N-labelled analogs as S-nitroso-N-acetyl cysteine ethyl ester (SNACET) and S¹⁵NACET or d₃-S¹⁵NACET derivatives, respectively, after their extraction with ethyl acetate. Injection of ethyl acetate solutions of S-nitrosothiols produced two main reaction products, compound X and compound Y, within the injector in dependence on its temperature. Quantification was performed by selected-ion monitoring of m/z 46 (i.e., [NO₂]^?) for SNACET and m/z 47 (i.e., [¹⁵NO₂]^?) for S¹⁵NACET/d₃-S¹⁵NACET for compound X, and m/z 157 for SNACET and m/z 160 for d₃-S¹⁵NACET for compound Y. In this article we describe the development, validation and in vitro and in vivo applications of the method to aqueous buffered solutions, human and rabbit plasma. Given the ester functionality of SNACET/S¹⁵NACET/d₃-S¹⁵NACET, stability studies were performed using metal chelators and esterase inhibitors. The method was found to be suitable for the quantitative determination of various S-nitrosothiols including SNACET externally added to human plasma (0–10 μM). Nitrite contamination in ethyl acetate was found to interfere. Our results suggest that the concentration of endogenous S-nitrosothiols in human plasma does not exceed about 200 nM in total. Oral administration of S¹⁵NACET to rabbits (40–63 μmol/kg body weight) resulted in formation of ALB-S¹⁵NO, [¹⁵N]nitrite and [¹⁵N]nitrate in plasma.     

Polynuclear water-soluble dinitrosyl iron complexes with cysteine or glutathione ligands: Electron paramagnetic resonance and optical studies

Electron paramagnetic resonance and optical spectrophotometric studies have demonstrated that low-molecular dinitrosyl iron complexes (DNICs) with cysteine or glutathione exist in aqueous solutions in the form of paramagnetic mononuclear (М-DNICs) and diamagnetic binuclear complexes (B-DNICs). The latter represent Roussin’s red salt esters and can be prepared by treatment of aqueous solutions of Fe²⁺ and thiols (рН 7.4) with gaseous nitric oxide (NO) at the thiol:Fe²⁺ ratio 1:1. М-DNICs are synthesized under identical conditions at the thiol:Fe²⁺ ratios above 20 and produce an EPR signal with an electronic configuration {Fe(NO)₂}⁷ at g_aver. = 2.03. At neutral pH, aqueous solutions contain both M-DNICs and B-DNICs (the content of the latter makes up to 50% of the total DNIC pool). The concentration of B-DNICs decreases with a rise in pH; at рН 9–10, the solutions contain predominantly M-DNICs. The addition of thiol excess to aqueous solutions of B-DNICs synthesized at the thiol:Fe²⁺ ratio 1:2 results in their conversion into М-DNICs, the total amount of iron incorporated into M-DNICs not exceeding 50% of the total iron pool in B-DNICs. Air bubbling of cys-М-DNIC solutions results in cysteine oxidation-controlled conversion of М-DNICs first into cys-B-DNICs and then into the EPR-silent compound Х able to generate a strong absorption band at 278 nm. In the presence of glutathione or cysteine excess, compound Х is converted into B-DNIC/M-DNIC and is completely decomposed under effect of the Fe²⁺ chelator о-phenanthroline or N-methyl-d-glucamine dithiocarbamate (MGD). Moreover, MGD initiates the synthesis of paramagnetic mononitrosyl iron complexes with MGD. It is hypothesized that compound Х represents a polynuclear DNIC with cysteine, most probably, an appropriate Roussin’s black salt thioesters and cannot be prepared by simple substitution of М-DNIC cysteine for glutathione. Treatment of М-DNIC with sodium dithionite attenuates the EPR signal at g_aver. = 2.03 and stimulates the appearance of an EPR signal at g_aver. = 2.0 with a hypothetical electronic configuration {Fe(NO)₂}⁹. These changes can be reversed by storage of DNIC solutions in atmospheric air. The EPR signal at g_aver. = 2.0 generated upon treatment of B-DNICs with dithionite also disappears after incubation of B-DNIC solutions in air. In all probability, the center responsible for this EPR signal represents М-DNIC formed in a small amount during dithionite-induced decomposition of B-DNIC.     

Nitrate removal processes in a constructed wetland treating drainage from dairy pasture

¹⁵N-labelled NO₃^? was used in a surface-flow constructed wetland in spring to examine the relative importance of competing NO₃^? removal processes. In situ mesocosms (0.25 m²) were dosed with 2 l of ¹⁵NO₃^? (NaNO₃, 300 mg N l^?1, 99 atom% ¹⁵N) and bromide (Br^?) solution (LiBr, 4.3 g l^?1, as a conservative tracer). Concentrations of NO₃^?, Br^?, dissolved oxygen and ¹⁵N₂ were monitored periodically and replicate mesocosms were destructively sampled prior to and 6 days after ¹⁵N addition. Denitrification, immobilisation, plant uptake and dissimilatory NO₃^? reduction to NH₄⁺ (DNRA) accounted for 77, 11, 9 and 2% of ¹⁵NO₃^? transformed during the experiment. Only 6% of denitrification gases were directly measured as atmospheric or dissolved ¹⁵N₂; the remainder (71%) was determined via ¹⁵N mass balance. This indicated that a large proportion of the denitrification gases were entrapped within the soil matrix and/or plant aerenchyma. The floating plant Lemna minor exhibited a significantly higher NO₃^? uptake rate (221 mg kg^?1 d^?1) than Typha orientalis (10 mg kg^?1 d^?1), but periodic harvest of plants would remove <3% of annual NO₃^? inputs. Our results suggest that this 6-year-old constructed wetland functions effectively as a sink for NO₃^? during the growing season with less than one-quarter of the NO₃^? processed sequestered into wetland plant, algal and microbial N pools and the balance permanently removed by denitrification.     

S-Nitrosylation of STIM1 by Neuronal Nitric Oxide Synthase Inhibits Store-Operated Ca2 + Entry

Store-operated Ca^2 + entry (SOCE) mediated by stromal interacting molecule-1 (STIM1) and Orai1 represents a major route of Ca^2 + entry in mammalian cells and is initiated by STIM1 oligomerization in the endoplasmic or sarcoplasmic reticulum. However, the effects of nitric oxide (NO) on STIM1 function are unknown. Neuronal NO synthase is located in the sarcoplasmic reticulum of cardiomyocytes. Here, we show that STIM1 is susceptible to S-nitrosylation. Neuronal NO synthase deficiency or inhibition enhanced Ca^2 + release-activated Ca^2 + channel current (I_CRAC) and SOCE in cardiomyocytes. Consistently, NO donor S-nitrosoglutathione inhibited STIM1 puncta formation and I_CRAC in HEK293 cells, but this effect was absent in cells expressing the Cys49Ser/Cys56Ser STIM1 double mutant. Furthermore, NO donors caused Cys49- and Cys56-specific structural changes associated with reduced protein backbone mobility, increased thermal stability and suppressed Ca²⁺ depletion-dependent oligomerization of the luminal Ca^2 +-sensing region of STIM1. Collectively, our data show that S-nitrosylation of STIM1 suppresses oligomerization via enhanced luminal domain stability and rigidity and inhibits SOCE in cardiomyocytes.     

Photochemical NO release from nitrosyl RuII complexes with C-bound imidazoles

The series of nitrosyl complexes trans-[Ru(NH₃)₄L(NO)]Cl₃, L = caffeine, theophylline, imidazole and benzoimidazole in position trans to NO were prepared and their photochemical properties studied. All complexes showed nitric oxide (NO) release under light irradiation at 330–440 nm. Quantum yields for [Ru(NH₃)₄L(H₂O)]³⁺ formation (?_Ru(III)) were sensitive to the natures of L, λ_irr and pH. The major product of the irradiation of trans-[Ru(NH₃)₄L(NO⁺)]³⁺ is the trans-[Ru^III(NH₃)₄L(Cl)]²⁺ and NO as suggested by UV–Vis, electrochemical, and FTIR techniques.     

Nitrogen dioxide oxidizes mitochondrial cytochrome c

We previously reported that high micromolar concentrations of nitric oxide were able to oxidize mitochondrial cytochrome c at physiological pH, producing nitroxyl anion (Sharpe and Cooper, 1998 Biochem. J. 332, 9–19). However, the subsequent re-evaluation of the redox potential of the NO/NO^- couple suggests that this reaction is thermodynamically unfavored. We now show that the oxidation is oxygen-concentration dependent and non stoichiometric. We conclude that the effect is due to an oxidant species produced during the aerobic decay of nitric oxide to nitrite and nitrate. The species is most probably nitrogen dioxide, NO₂^? a well-known biologically active oxidant. A simple kinetic model of NO autoxidation is able to explain the extent of cytochrome c oxidation assuming a rate constant of 3 × 10⁶ M^-1 s^-1 for the reaction of NO₂^? with ferrocytochrome c. The importance of NO₂^? was confirmed by the addition of scavengers such as urate and ferrocyanide. These convert NO₂^? into products (urate radical and ferricyanide) that rapidly oxidize cytochrome c and hence greatly enhance the extent of oxidation observed. The present study does not support the previous hypothesis that NO and cytochrome c can generate appreciable amounts of nitroxyl ions (NO^- or HNO) or of peroxynitrite.     

Synthesis and evaluation of the inhibitory activity of the four stereoisomers of the potent and selective human γ-glutamyl transpeptidase inhibitor GGsTop

2-Amino-4-{[3-(carboxymethyl)phenoxy](methoxy)phosphoryl}butanoic acid (GGsTop) is a potent, highly selective, nontoxic, and irreversible inhibitor of γ-glutamyl transpeptidase (GGT). GGsTop has been widely used in academic and medicinal research, and also as an active ingredient (Nahlsgen) in commercial anti-aging cosmetics. GGsTop consists of four stereoisomers due to the presence of two stereogenic centers, i.e., the α-carbon atom of the glutamate mimic (l/d) and the phosphorus atom (R_P/S_P). In this study, each stereoisomer of GGsTop was synthesized stereoselectively and their inhibitory activity against human GGT was evaluated. The l- and d-configurations of each stereoisomer were determined by a combination of a chiral pool synthesis and chiral HPLC analysis. The synthesis of the four stereoisomers of GGsTop used chiral synthetic precursors that were separated by chiral HPLC on a preparative scale. With respect to the configuration of the α-carbon atom of the glutamate mimic, the l-isomer (k_on = 174 M^?1 s^?1) was ca. 8-fold more potent than the d-isomer (k_on = 21.5 M^?1 s^?1). In contrast, the configuration of the phosphorus atom is critical for GGT inhibitory activity. Based on a molecular modeling approach, the absolute configuration of the phosphorus atom of the active GGsTop isomers was postulated to be S_P. The S_P-isomers inhibited human GGT (k_on = 21.5–174 M^?1 s^?1), while the R_P-isomers were inactive even at concentrations of 0.1 mM.     

Hemodynamic effects of inducible nitric oxide synthase inhibition combined with sildenafil during acute pulmonary embolism

While endogenous nitric oxide (NO) may be relevant to the beneficial hemodynamic effects produced by sildenafil during acute pulmonary embolism (APE), huge amounts of inducible NO synthase (iNOS)-derived NO may contribute to lung injury. We hypothesized that iNOS inhibition with S-methylisothiourea could attenuate APE-induced increases in oxidative stress and pulmonary hypertension and, therefore, could improve the beneficial hemodynamic and antioxidant effects produced by sildenafil during APE. Hemodynamic evaluations were performed in non-embolized dogs treated with saline (n = 4), S-methylisothiourea (0.01 mg/kg followed by 0.5 mg/kg/h, n = 4), sildenafil (0.3 mg/kg, n = 4), or S-methylisothiourea followed by sildenafil (n = 4), and in dogs that received the same drugs and were embolized with silicon microspheres (n = 8 for each group). Plasma nitrite/nitrate (NOx) and thiobarbituric acid reactive substances (TBARS) concentrations were determined by Griess and a fluorometric assay, respectively. APE increased mean pulmonary arterial pressure (MPAP) and pulmonary vascular resistance index (PVRI) by 25 ± 1.7 mm Hg and by 941 ± 34 dyn s cm^?5 m^?2, respectively. S-methylisothiourea neither attenuated APE-induced pulmonary hypertension, nor enhanced the beneficial hemodynamic effects produced by sildenafil after APE (>50% reduction in pulmonary vascular resistance). While sildenafil produced no change in plasma NOx concentrations, S-methylisothiourea alone or combined with sildenafil blunted APE-induced increases in NOx concentrations. Both drugs, either alone or combined, produced antioxidant effects. In conclusion, although iNOS-derived NO may play a key role in APE-induced oxidative stress, our results suggest that the iNOS inhibitor S-methylisothiourea neither attenuates APE-induced pulmonary hypertension, nor enhances the beneficial hemodynamic effects produced by sildenafil.     

Physicochemistry of dinitrosyl iron complexes with thiolate ligands underlying their beneficial effect in endometriosis

Exogenous dinitrosyl iron complexes (DNIC) with thiolate ligands as NO and NO⁺ donors are capable of exerting both regulatory and cytotoxic effects on diverse biological processes similarly to those characteristic of endogenous nitric oxide. Regulatory activity of DNIC (vasodilatory, hypotensive, suppressing thrombosis, increasing erythrocyte elasticity, accelerating skin wound healing, inducing penile erection, etc.) is determined by their capacity of NO and NO⁺ transfer to biological targets of the latter (heme- and thiol-containing proteins, respectively) due to higher affinity of the proteins for NO and NO⁺ than that of DNIC. Cytotoxic activity of DNIC is provided by rapid DNIC decomposition under action of iron-chelating compounds, resulting in appearance of NO and NO⁺ in cells and tissues in high amounts. The latter mechanism is suggested to cause the blocking effect of DNIC as cytotoxic effectors on the development of benign endometrial tumors in rats with experimental endometriosis. It is also proposed that a similar mechanism can operate to cause at least a delay of malignant tumor proliferation under action of DNIC.     

Purification,kinetic and thermodynamic characterization of soluble acid invertase from sugarcane (Saccharum officinarum L.)

We report for the first time kinetic and thermodynamic properties of soluble acid invertase (SAI) of sugarcane (Saccharum officinarum L.) salt sensitive local cultivar CP 77-400 (CP-77). The SAI was purified to apparent homogeneity on FPLC system. The crude enzyme was about 13 fold purified and recovery of SAI was 35%. The invertase was monomeric in nature and its native molecular mass on gel filtration and subunit mass on SDS-PAGE was 28 kDa. SAI was highly acidic having an optimum pH lower than 2. The acidic limb was missing. Proton transfer (donation and receiving) during catalysis was controlled by the basic limb having a pKa of 2.4. Carboxyl groups were involved in proton transfer during catalysis. The kinetic constants for sucrose hydrolysis by SAI were determined to be: k_m = 55 mg ml^?1, k_cat = 21 s^?1, k_cat/k_m = 0.38, while the thermodynamic parameters were: ΔH* = 52.6 kJ mol^?1, ΔG* = 71.2 kJ mol^?1, ΔS* = ?57 J mol^?1 K^?1, ΔG*_E–S = 10.8 kJ mol^?1 and ΔG*_E–T = 2.6 kJ mol^?1. The kinetics and thermodynamics of irreversible thermal denaturation at various temperatures 53–63 °C were also determined. The half -life of SAI at 53 and 63 °C was 112 and 10 min, respectively. At 55 °C, surprisingly the half -life increased to twice that at 53 °C. ΔG*, ΔH* and ΔS* of irreversible thermal stability of SAI at 55 °C were 107.7 kJ mol^?1, 276.04 kJ mol^?1 and 513 J mol^?1K^?1, respectively.     

Oxygen regulates the effective diffusion distance of nitric oxide in the aortic wall

Endothelium-derived nitric oxide (NO) is critical in maintaining vascular tone. Accumulating evidence shows that NO bioavailability is regulated by oxygen concentration. However, it is unclear to what extent the oxygen concentration regulates NO bioavailability in the vascular wall. In this study, a recently developed experimental setup was used to measure the NO diffusion flux across the aortic wall at various oxygen concentrations. It was observed that for a constant NO concentration at the endothelial surface, the measured NO diffusion flux out of the adventitial surface at [O₂] = 0 μM is around fivefold greater than at [O₂] = 150 μM, indicating that NO is consumed in the aortic wall in an oxygen-dependent manner. Analysis of experimental data shows that the rate of NO consumption in the aortic wall is first order with respect to [NO] and first order with respect to [O₂], and the rate constant k₁ was determined as (4.0 ± 0.3) × 10³ M^?1 s^?1. Computer simulations demonstrate that NO concentration distribution significantly changes with oxygen concentration and the effective NO diffusion distance at low oxygen level ([O₂] ≤ 25 μM) is significantly longer than that at high oxygen level ([O₂] = 200 μM). These results suggest that oxygen-dependent NO consumption may play an important role in dilating blood vessels during hypoxia by increasing the effective NO diffusion distance.     

The trans-labilization of nitric oxide in RuII complexes by C-bound imidazoles

The synthesis, characterization and reactivity of trans-[NO(L)(NH₃)₄Ru]Cl₃ (L=imidazole, theophylline and caffeine) are presented. ¹H NMR spectroscopy indicates that the imidazole ligands are coordinated to the Ru^II through a carbon atom (imκ²_, 1,3Me₂Xanκ⁸ and 1,3,7Me₃Xanκ⁸). The nitrosyl stretching frequencies (ν_NO≅1913 cm⁻¹) suggest the coordinated nitrosyl has substantial NO⁺ character. The complexes undergo a single-electron reduction (E°≅−0.50 V versus Ag/AgCl), which involves the coordinated nitrosyl. Dissociation of NO^· in the reduced species is facilitated by the trans-imidazolylidene ligand. The lower than expected reduction potentials of these complexes may account for their inactivity in evoking neuronal firing in the hippocampus by releasing NO following reduction.     

Origin of magnetization tunneling in single-molecule magnets as determined by single-crystal high-frequency EPR

We review an extensive body of single-crystal high-frequency electron paramagnetic resonance (HFEPR) data in order to determine the transverse spin Hamiltonian parameters that control the tunneling of the direction of magnetization in a variety of integer and half-integer-spin single-molecule magnets (SMMs). The SMMs studied are members of the following families: S = 9/2 [Mn₄O₃Cl]⁶⁺; S = 5 [Mn₃NiO₄]⁶⁺; S = 6 [Mn₃ZnO₄]⁶⁺; and S = 4 [Ni₄(OR)₄]⁴⁺. HFEPR spectra for the half-integer S = 9/2 Mn₄ complexes that have C₃ symmetry do not provide measurable evidence for transverse spin Hamiltonian terms. This finding is consistent with the relatively large coercive field seen in the magnetization hysteresis loops for these complexes. On the other hand, a low symmetry S = 9/2 complex exhibits a much faster rate of ground-state magnetization tunneling, in agreement with HFEPR spectra for a powder sample that gives a rhombic zero-field splitting (ZFS) parameter of E = 0.140 cm^?1. The S = 5 Mn₃Ni systems exhibit magnetization tunneling that is much faster than seen for the high-symmetry S = 9/2 Mn₄ complexes. This can be attributed to their integer-spin ground states. Like the C₃ symmetry Mn₄ SMMs, the HFEPR spectra for high-symmetry Mn₃Ni complexes do not provide measurable evidence for transverse ZFS terms. However, the spectra exhibit broad peaks, suggesting distributions in the local molecular environments brought about by disordered solvate molecules. This disorder likely explains the fast tunneling in the high-symmetry S = 5 Mn₃Ni systems, though one cannot rule out fourth- (and higher-) order interactions that cannot be detected by HFEPR due to the broad resonances. The one S = 6 Mn₃Zn complex shows an even faster rate of tunneling compared to the isostructural S = 5 Mn₃Ni complex. Finally, the S = 4 [Ni(hmp)(dmb)Cl]₄ complex provides unique insights into the origin of fourth- (and higher-) order interactions found for many SMMs on the basis of analysis using a giant spin Hamiltonian (GSH) approximation. We conclude that the fourth-order anisotropy found for the S = 4 ground state of [Ni(hmp)(dmb)Cl]₄ originates from the second-order ZFS interactions associated with the individual Ni^II ions, but only as a result of higher-order processes that occur via S-mixing between the ground state and higher-lying (S < 4) spin-multiplets. The S-mixing is relatively strong in this system because of comparable exchange and anisotropy energy scales. The relatively fast tunneling is a direct consequence of this S-mixing, as opposed to any intrinsic fourth-order (spin–orbit) anisotropy associated with Ni^II.     

Availability of N and S affect nutrient acquisition efficiencies differently by Trifolium repens and Lolium perenne when grown in monoculture or in mixture

Sulphur (S) deficiency is recognized as a limiting factor for crop production in many regions in the world. In grasslands, S availability has been shown to alter the biomass production of Trifolium repens and Lolium perenne and their specific interactions. To establish the role of N and S availabilities on the competitive interaction for these minerals by T. repens and L. perenne when grown together, two S rates (0 and 30 kg S ha^?1) combined with three N rates (0, 50 and 180 kg N ha^?1) were investigated in a cut/regrowth experiment over a period of 4 months under glasshouse conditions. N was applied as ¹⁵NH₄ ¹⁵NO₃ to determine their actual N fertilizer recovery in the harvested fraction of the shoot. S yields were used to estimate their apparent S fertilizer recovery. At final harvest, N reserves of T. repens stolons were analyzed to estimate their implication in the regrowth process. In monoculture and in both cuts (1 and 2), N benefited both species by increasing their N and S yields. S benefited only T. repens. In mixture, at cut 1, L. perenne behaved as a better competitor than T. repens thanks to N, while at cut 2, T. repens dominated the community thanks to strong positive S effect. N recovery of L. perenne grown in mixture was greatly improved by S supply. For T. repens, S enhanced its ability to fix N₂ and improved the accumulation of soluble proteins in its stolons. It is clear that the N:S ratio of soil may affect the functionality of grassland plant communities and their structure. Results suggest that (i) the limitations in the availability of soil S could restrict leguminous species growth in high N soil conditions, and (ii) the modulation of S level could be used as a tool to modify the composition of grassland communities.     

Hydrogen and oxygen trapping at the H-cluster of [FeFe]-hydrogenase revealed by site-selective spectroscopy and QM/MM calculations

[FeFe]-hydrogenases are superior hydrogen conversion catalysts. They bind a cofactor (H-cluster) comprising a four-iron and a diiron unit with three carbon monoxide (CO) and two cyanide (CN^?) ligands. Hydrogen (H₂) and oxygen (O₂) binding at the H-cluster was studied in the C169A variant of [FeFe]-hydrogenase HYDA1, in comparison to the active oxidized (Hox) and CO-inhibited (Hox-CO) species in wildtype enzyme. ⁵⁷Fe labeling of the diiron site was achieved by in vitro maturation with a synthetic cofactor analogue. Site-selective X-ray absorption, emission, and nuclear inelastic/forward scattering methods and infrared spectroscopy were combined with quantum chemical calculations to determine the molecular and electronic structure and vibrational dynamics of detected cofactor species. Hox reveals an apical vacancy at Fe_d in a [4Fe4S-2Fe]^3 ? complex with the net spin on Fe_d whereas Hox-CO shows an apical CN^? at Fe_d in a [4Fe4S-2Fe(CO)]^3 ? complex with net spin sharing among Fe_p and Fe_d (proximal or distal iron ions in [2Fe]). At ambient O₂ pressure, a novel H-cluster species (Hox-O₂) accumulated in C169A, assigned to a [4Fe4S-2Fe(O₂)]^3 ? complex with an apical superoxide (O₂^?) carrying the net spin bound at Fe_d. H₂ exposure populated the two-electron reduced Hhyd species in C169A, assigned as a [(H)4Fe4S-2Fe(H)]^3 ? complex with the net spin on the reduced cubane, an apical hydride at Fe_d, and a proton at a cysteine ligand. Hox-O₂ and Hhyd are stabilized by impaired O₂^– protonation or proton release after H₂ cleavage due to interruption of the proton path towards and out of the active site.     

Removal of nutrients from wastewater with Canna indica L. under different vertical-flow constructed wetland conditions

Constructed wetlands are becoming increasingly popular worldwide for removing contaminants from domestic wastewater. This study investigated the removal efficiency of nitrogen (N) and phosphorus (P) from wastewater with the simulated vertical-flow constructed wetlands (VFCWs) under three different substrates (i.e., BFAS or blast furnace artificial slag, CBAS or coal burn artificial slag, and MSAS or midsized sand artificial slag), hydraulic loading rates (i.e., 7, 14, and 21 cm d^?1), and wetland operational periods (0.5, 1, and 2 years) as well as with and without planting Canna indica L. The wastewater was collected from the campus of South China Agricultural University, Guangzhou, China. Results show that the percent removal of total P (TP) and ammonium N (NH₄⁺-N) by the substrates was BFAS > CBAS > MSAS due to the high contents of Ca and Al in substrate BFAS. In contrast, the percent removal of total N (TN) by the substrates was CBAS > MSAS > BFAS due to the complicated nitrification/denitrification processes. The percent removal of nutrients by all of the substrates was TP > NH₄⁺-N > TN. About 10% more TN was removed from the wastewater after planting Canna indica L. A lower hydraulic loading rate or longer hydraulic retention time (HRT) resulted in a higher removal of TP, NH₄⁺-N, and TN because of more contacts and interactions among nutrients, substrates, and roots under the longer HRT. Removal of NO₃^?N from the simulated VFCWs is a complex process. A high concentration of NO₃^?N in the effluent was observed under the high hydraulic loading rate because more NH₄⁺-N and oxygen were available for nitrification and a shorter HRT was unfavorable for denitrification. In general, a longer operational period had a highest removal rate for nutrients in the VFCWs.     

Acute and chronic nitrite toxicity in juvenile pike-perch (Sander lucioperca) and its compensation by chloride

Pike-perch Sander lucioperca is currently considered as one of the most promising candidates for production in freshwater recirculation aquaculture systems (RAS). Here, due to the lack of studies on nitrite (NO₂^?) toxicity in pike-perch, a flow-through exposure at 0, 0.44, 0.88, 1.75, 3.5, 7, 14 and 28 mg/L NO₂^?–N was carried out to determine the acute and chronic toxicity over a period of 32 days. In juvenile pike-perch, 120 h LC₅₀ was 6.1 mg/L NO₂^?–N and at ≥ 14 mg/L NO₂^?–N all fish had died within 24 h. Chronic exposure revealed a significant build up of NO₂^? in the plasma as well as in the muscles at ≥ 0.44 mg/L NO₂^?–N peaking in fish exposed to the highest concentration of 3.5 mg/L NO₂^?–N after 32 days. Still, due to high individual variation methemoglobin (MetHb) was only significantly increased (p < 0.01) at 3.5 mg/L NO₂^?–N. No adverse effects on red blood cells (RBC) and hematocrit were observed in any of the treatments. In a second experiment, compensation of NO₂^? toxicity at increasing chloride concentrations (40 (freshwater), 65, 90, 140, 240, 440 mg/L Cl^?) was observed at a constant exposure of 10 mg/L NO₂^?–N for 42 days. At ≥ 240 mg/L Cl^?, NO₂^? build-up in blood plasma and muscle was completely inhibited. At lower Cl^? concentrations (≤ 140 mg/L), NO₂^? was significantly increased in plasma, but only insignificantly elevated in muscle due to high individual variation. MetHb was increased significantly difference only at 40 mg/L Cl^? (freshwater control) compared to the control. Again, high individual variations were observed. As a conclusion, S. lucioperca is moderately sensitive towards NO₂^? and acceptable levels in RAS should hence not exceed 1.75 mg/L NO₂^?–N to avoid MetHb formation. However, based on the 120 h LC₅₀ and a factor of 0.01 according to Sprague (1971), a NO₂^? concentration of ≤ 0.061 mg/L NO₂^?–N is considered as “safe.” Thereby, no NO₂^? should accumulate in the plasma or muscle tissue during chronic exposure. For 10 mg/L NO₂^?–N, ≥ 240 mg/L chloride compensates for NO₂^? uptake in plasma and muscle.     

Phenol and sulfide oxidation in a denitrifying biofilm reactor and its microbial community analysis

A microbial consortium attached onto a polyethylene support was used to evaluate the simultaneous oxidation of sulfide and phenol by denitrification. The phenol, sulfide and nitrate loading rates applied to an inverse fluidized bed reactor were up to 168 mg phenol–C/(l d), 37 mg S^2?/(l d) and 168 mg NO₃^?–N/(l d), respectively. Under steady state operation the consumption efficiencies of phenol, sulfide and nitrate were 100%. The N₂ yield (g N₂/g NO₃^?–N) was 0.89. The phenol was mineralized resulting in a yield of 0.82 g bicarbonate–C/g phenol–C and sulfide was completely oxidized to sulfate with a yield of 0.99 g SO₄^2?–S/g S^2?. 16S rRNA gene-based microbial community analysis of the denitrifying biofilm showed the presence of Thauera aromatica, Thiobacillus denitrificans, Thiobacillus sajanensis and Thiobacillus sp. This is the first work reporting the simultaneous oxidation of sulfide and phenol in a denitrifying biofilm reactor.