nitrophenols and quinone

p-Nitrophenol 4-monooxygenase PnpA, the key enzyme in the hydroquinone pathway of p-nitrophenol (PNP) degradation, catalyzes the monooxygenase reaction of PNP to p-benzoquinone in the presence of FAD and NADH. Here, we determined the first crystal structure of PnpA from Pseudomonas putida DLL-E4 in its apo and FAD-complex forms to a resolution of 2.04 Å and 2.48 Å, respectively. The PnpA structure shares a common fold with hydroxybenzoate hydroxylases, despite a low amino sequence identity of 14-18%, confirming it to be a member of the Class A flavoprotein monooxygenases. However, substrate docking studies of PnpA indicated that the residues stabilizing the substrate in an orientation suitable for catalysis are not observed in other homologous hydroxybenzoate hydroxylases, suggesting PnpA employs a unique catalytic mechanism. This work expands our understanding on the reaction mode for this enzyme class.

Topics: Bacterial Proteins; Benzoquinones; Binding Sites; Biocatalysis; Crystallography, X-Ray; Flavin-Adenine Dinucleotide; Models, Molecular; Molecular Structure; Nitrophenols; Oxygenases; Protein Binding; Protein Conformation; Pseudomonas putida; Substrate Specificity

Pulsed discharge plasma (PDP) combined with charcoal (PDP-charcoal) was employed to treat dye wastewater, with methyl orange (MO) as the model pollutant. The charcoal was prepared using spent tea leaves and was characterized by scanning electron microscopy, Fourier-transform infrared spectroscopy, and Boehm titration to investigate the adsorption and catalytic characteristics before and after adsorption and PDP treatment. The prepared charcoal exhibited a high MO adsorption capacity, and the adsorption process followed the pseudo-second-order kinetic model and the Freundlich model. The MO decoloration efficiency reached 69.8 % within 7.5 min of treatment in the PDP-charcoal system, whereas values of 29.2 and 25.9 % were achieved in individual PDP and charcoal systems, respectively. The addition of n-butanol and H2PO4 (-) presented inhibitive effects on MO decoloration in the PDP system. However, these effects were much weaker in the PDP-charcoal system. In addition, the effects of charcoal on O3 and H2O2 formation were evaluated, and the results showed that both the O3 and H2O2 concentrations decreased in the presence of charcoal. The MO decomposition intermediates were analyzed using UV-Vis spectrometry and GC-MS. 1,4-Benzoquinone, 4-nitrophenol, 4-hydroxyaniline, and N,N'-dimethylaniline were detected. A possible pathway for MO decomposition in this system was proposed.

Topics: Adsorption; Aminophenols; Azo Compounds; Benzoquinones; Camellia sinensis; Charcoal; Color; Coloring Agents; Hydrogen Peroxide; Kinetics; Microscopy, Electron, Scanning; Nitrophenols; Ozone; Plant Leaves; Spectroscopy, Fourier Transform Infrared; Waste Disposal, Fluid; Wastewater; Water Pollutants, Chemical

This research investigated the removal mechanisms of p-nitrophenol, p-methoxyphenol, and p-benzoquinone at a porous Ti4O7 reactive electrochemical membrane (REM) under anodic polarization. Cross-flow filtration experiments and density functional theory (DFT) calculations indicated that p-benzoquinone removal was primarily due to reaction with electrochemically formed OH(•), while the dominant removal mechanism of p-nitrophenol and p-methoxyphenol was a function of the anodic potential. At low anodic potentials (1.7-1.8 V/SHE), p-nitrophenol and p-methoxyphenol were removed primarily by an electrochemical adsorption/polymerization mechanism on the REM. Increasing anodic potentials (1.9-3.2 V/SHE) resulted in the electroassisted adsorption mechanism contributing far less to p-methoxyphenol removal compared to p-nitrophenol. DFT calculations indicated that an increase in anodic potential resulted in a shift in p-methoxyphenol removal from a 1e(-) direct electron transfer (DET) reaction that resulted in radical formation and significant adsorption/polymerization, to a 2e(-) DET reaction that formed nonadsorbing products (i.e., p-benzoquinone). However, the anodic potentials were too low for the 2e(-) DET reaction to be thermodynamically favorable for p-nitrophenol. The decreased COD adsorption for p-nitrophenol at higher anodic potentials was attributed to reaction of soluble/adsorbed organics with OH(•). These results provide the first mechanistic explanation for p-substituted phenolic compound removal during advanced electrochemical oxidation processes.

Topics: Adsorption; Anisoles; Benzoquinones; Biological Oxygen Demand Analysis; Electrochemistry; Membranes, Artificial; Models, Chemical; Molecular Conformation; Nitrophenols; Oxidation-Reduction; Phenols; Porosity; Quantum Theory; Thermodynamics; Time Factors; Titanium

Burkholderia sp. strain SJ98 (DSM 23195) utilizes 2-chloro-4-nitrophenol (2C4NP) or para-nitrophenol (PNP) as a sole source of carbon and energy. Here, by genetic and biochemical analyses, a 2C4NP catabolic pathway different from those of all other 2C4NP utilizers was identified with chloro-1,4-benzoquinone (CBQ) as an intermediate. Reverse transcription-PCR analysis showed that all of the pnp genes in the pnpABA1CDEF cluster were located in a single operon, which is significantly different from the genetic organization of all other previously reported PNP degradation gene clusters, in which the structural genes were located in three different operons. All of the Pnp proteins were purified to homogeneity as His-tagged proteins. PnpA, a PNP 4-monooxygenase, was found to be able to catalyze the monooxygenation of 2C4NP to CBQ. PnpB, a 1,4-benzoquinone reductase, has the ability to catalyze the reduction of CBQ to chlorohydroquinone. Moreover, PnpB is also able to enhance PnpA activity in vitro in the conversion of 2C4NP to CBQ. Genetic analyses indicated that pnpA plays an essential role in the degradation of both 2C4NP and PNP by gene knockout and complementation. In addition to being responsible for the lower pathway of PNP catabolism, PnpCD, PnpE, and PnpF were also found to be likely involved in that of 2C4NP catabolism. These results indicated that the catabolism of 2C4NP and that of PNP share the same gene cluster in strain SJ98. These findings fill a gap in our understanding of the microbial degradation of 2C4NP at the molecular and biochemical levels.

Topics: Bacterial Proteins; Benzoquinones; Burkholderia; Mixed Function Oxygenases; Multigene Family; Nitrophenols; Quinone Reductases

The electrochemical oxidation of pesticide, o-nitrophenol (ONP) as one kind of pesticide that is potentially dangerous and biorefractory, was studied by galvanostatic electrolysis using boron-doped diamond (BDD) as anode. The influence of several operating parameters, such as applied current density, supporting electrolyte, and initial pH value, was investigated. The best degradation occurred in the presence of Na2SO4 (0.05 M) as conductive electrolyte. After 8h, nearly complete degradation of o-nitrophenol was achieved (92%) using BDD electrodes at pH 3 and at current density equals 60 mA cm(-2). The decay kinetics of o-nitrophenol follows a pseudo-first-order reaction. Aromatic intermediates such as catechol, resorcinol, 1,2,4-trihydroxybenzene, hydroquinone and benzoquinone and carboxylic acids such as maleic glycolic, malonic, glyoxilic and oxalic, have been identified and followed during the ONP treatment by chromatographic techniques. From these anodic oxidation by-products, a plausible reaction sequence for ONP mineralization on BDD anodes is proposed.

Topics: Benzoquinones; Boron; Carboxylic Acids; Catechols; Diamond; Electrodes; Electrolysis; Hydrogen-Ion Concentration; Hydroquinones; Kinetics; Models, Chemical; Nitrophenols; Oxygen; Pesticides; Resorcinols; Water Pollutants, Chemical; Water Purification

Monooxygenation is an important route of nitroaromatic compound (NAC) biodegradation and it is widely found for cometabolic transformations of NACs and other aromatic pollutants. We investigated the C and N isotope fractionation of nitrophenol monooxygenation to complement the characterization of NAC (bio)degradation pathways by compound-specific isotope analysis (CSIA). Because of the large diversity of enzymes catalyzing monooxygenations, we studied the combined C and N isotope fractionation and the corresponding (13)C- and (15)N-apparent kinetic isotope effects (AKIEs) of four nitrophenol-biodegrading microorganisms (Bacillus spharericus JS905, Pseudomonas sp. 1A, Arthrobacter sp. JS443, Pseudomonas putida B2) in the pH range 6.1-8.6 with resting cells and crude cell extracts. While the extent of C and N isotope fractionation and the AKIE-values varied considerably for the different organisms, the correlated C and N isotope signatures (δ(15)N vs δ(13)C) revealed trends, indicative of two distinct monooxygenation pathways involving hydroxy-1,4-benzoquinone or 1,2- and 1,4-benzoquinone intermediates, respectively. The distinction was possible based on larger secondary (15)N-AKIEs associated with the benzoquinone pathway. Isotope fractionation was neither masked substantially by nitrophenol speciation nor transport across cell membranes. Only when 4-nitrophenol was biodegraded by Pseudomonas sp. 1A did isotope fractionation become negligible, presumably due to rate-limiting substrate binding steps pertinent to the catalytic cycle of flavin-dependent monooxygenases.

Topics: Arthrobacter; Bacillus; Benzoquinones; Biocatalysis; Biodegradation, Environmental; Carbon Isotopes; Chemical Fractionation; Environmental Pollutants; Kinetics; Metabolic Networks and Pathways; Mixed Function Oxygenases; Nitrogen Isotopes; Nitrophenols; Pseudomonas putida

Pseudomonas sp. strain WBC-3 utilizes para-nitrophenol (PNP) as a sole source of carbon, nitrogen, and energy. In order to identify the genes involved in this utilization, we cloned and sequenced a 12.7-kb fragment containing a conserved region of NAD(P)H:quinone oxidoreductase genes. Of the products of the 13 open reading frames deduced from this fragment, PnpA shares 24% identity to the large component of a 3-hydroxyphenylacetate hydroxylase from Pseudomonas putida U and PnpB is 58% identical to an NAD(P)H:quinone oxidoreductase from Escherichia coli. Both PnpA and PnpB were purified to homogeneity as His-tagged proteins, and they were considered to be a monomer and a dimer, respectively, as determined by gel filtration. PnpA is a flavin adenine dinucleotide-dependent single-component PNP 4-monooxygenase that converts PNP to para-benzoquinone in the presence of NADPH. PnpB is a flavin mononucleotide-and NADPH-dependent p-benzoquinone reductase that catalyzes the reduction of p-benzoquinone to hydroquinone. PnpB could enhance PnpA activity, and genetic analyses indicated that both pnpA and pnpB play essential roles in PNP mineralization in strain WBC-3. Furthermore, the pnpCDEF gene cluster next to pnpAB shares significant similarities with and has the same organization as a gene cluster responsible for hydroquinone degradation (hapCDEF) in Pseudomonas fluorescens ACB (M. J. Moonen, N. M. Kamerbeek, A. H. Westphal, S. A. Boeren, D. B. Janssen, M. W. Fraaije, and W. J. van Berkel, J. Bacteriol. 190:5190-5198, 2008), suggesting that the genes involved in PNP degradation are physically linked.

Topics: Benzoquinones; Chromatography, Affinity; Chromatography, Gel; Cloning, Molecular; Coenzymes; Dimerization; DNA, Bacterial; Gene Order; Genes, Bacterial; Hydroquinones; Molecular Sequence Data; Multigene Family; NADP; Nitrophenols; Oxygenases; Pseudomonas; Quinone Reductases; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Synteny

This study deals with the degradation of various nitrophenols by cathode reduction and electro-Fenton methods. Phenols (Poh), 2-nitrophenol (2-NP), 3-nitrophenol (3-NP), 4-nitrophenol (4-NP), and 2,4-dinitrophenol (2,4-DNP) are treated and different degradation sequences are obtained. The relationship between the structure and activities of nitrophenols is discussed. Using 4-NP as a model nitrophenol, the electrochemical behaviors on graphite cathode and Pt anode are analyzed by cyclic voltammetry. The contribution of different reactions to the degradation of 4-NP is investigated in divided cells. The degradation of 4-NP is much faster in the cathode cell than in the anodic cell. In the cathode cell, the degradation of 4-NP is significantly enhanced by the introduction of aeration and Fe(2+). Ultraviolet-visible (UV-vis) spectra reveal different reaction pathways for the degradation in the anodic cell and cathode cell. Treatment of high concentration of 4-NP in the undivided cell shows that more than 98% removal of 4-NP and about 13% removal of total organic carbon (TOC) are obtained for both processes, while the subsequent biodegradability test shows that electro-Fenton can eliminate the toxicity and improve the biodegradability of 4-NP. Negligible quantity of nitrate and nitrite ions detected in both processes indicates that there is no direct release of -NO(2) and -NO groups from 4-NP and its degradation intermediates. Intermediates such as hydroquinone and bezoquinone are detected by gas chromatography/mass spectrum (GC/MS). The degradation pathway of 4-NP in electro-Fenton process is proposed as the cathode reduction followed by hydroxyl oxidation.

Topics: Benzoquinones; Biodegradation, Environmental; Carbon; Electrochemistry; Electrodes; Hydroquinones; Nitrates; Nitrites; Nitrophenols; Spectrophotometry; Time Factors; Ultraviolet Rays; Waste Disposal, Fluid; Water Purification