Anaerobic Solution Research Articles

Pertechnetate immobilization in aqueous media with hydrogen sulfide under anaerobic and aerobic environments

Summary The basic chemistry for the immobilization of pertechnetate (TcO4 −) by hydrogen sulfide was investigated in aqueous solution under both aerobic and anaerobic environments. Pertechnetate immobilization was acid dependent, with accelerated rates and increased immobilization yields as the acid concentration increased. Oxygen had no effect under acidic conditions. Under anaerobic alkaline conditions, the pH, and therefore the speciation of sulfide, was the determining factor on the immobilization of pertechnetate. Only 53% of the TcO4 − was immobilized at pH 8, while the yield increased to 83% at pH 9 as HS− became the dominant sulfide species. The immobilization yield then decreased to 73% at pH 13. No reaction was observed between TcO4 − and sulfide under aerobic alkaline conditions, indicating that oxygen suppressed this reaction. Pertechnetate immobilization was found to be first order with respect to both sulfide and pertechnetate in acidic solutions, and in alkaline solution under anaerobic conditions. The results of stoichiometry studies and product analysis under alkaline anaerobic environments indicated that Tc2S7 was obtained at pH 9. EXAFS (extended X-ray absorption fine structure) and XANES (X-ray absorption near edge structure) studies suggested that the samples obtained from acidic, aerobic solution and alkaline anaerobic solution were both Tc2S7. The stability of Tc2S7 is affected by O2 with accelerated dissolution at high pH.

The Release of Phosphorus to Porewater and Surface Water from River Riparian Sediments

Sediments can be both a source and a sink of dissolved phosphorus (P) in surface water and shallow groundwater. Using laboratory mesocosms, we studied the influence of flooding with deionized water and simulated river water on P release to solution using sediment columns taken from a riparian wetland. The mesocosm incubation results showed that rather than retaining nutrients, sediments in the riparian zone may be a significant source of P. Concentrations of dissolved P in porewater reached more than 3 mg L(-1) and in surface water over 0.8 mg L(-1) within a month of sediment inundation. The reductive dissolution of P-bearing iron (Fe) oxides was the likely mechanism responsible for P release. Dissolved P to Fe molar ratios in anaerobic samples were approximately 0.45 when columns were flooded with water that simulated the chemistry of the adjacent river. This suggests there was insufficient Fe in the anaerobic samples to precipitate all P if the solutions were oxygenated or transported to an aerobic environment. If the anaerobic wetland solutions were delivered to oxygenated rivers and streams adjacent to the riparian zone, the equilibrium concentration of P in these systems could rise. The timing of P release was inversely related to the nitrate (NO3-) concentration in floodwater. This indicates that in riparian zones receiving low nitrate loads, or where NO3- loads are being progressively reduced, the risk of dissolved P release may increase. These findings present particular challenges for restoration and management in riparian areas.

Immobilization of hemoglobin on electrodeposited cobalt-oxide nanoparticles: Direct voltammetry and electrocatalytic activity

Cyclic voltammetry at potential range − 1.1 to 0.5 V from aqueous buffer solution (pH 7) containing CoCl 2 produced a well defined cobalt oxide (CoOx) nanoparticles deposited on the surface of glassy carbon electrode. The morphology of the modified surface and cobalt oxide formation was examined with SEM and cyclic voltammetry techniques. Hemoglobin (Hb) was successfully immobilized in cobalt-oxide nanoparticles modified glassy carbon electrode. Immobilization of hemoglobin onto cobalt oxide nanoparticles have been investigated by cyclic voltammetry and UV–visible spectroscopy. The entrapped protein can take direct electron transfer in cobalt-oxide film. A pair of well defined, quasi-reversible cyclic voltammetric peaks at about − 0.08 V vs. SCE (pH 7), characteristic of heme redox couple (Fe(III)/Fe(II)) of hemoglobin, and the response showed surface controlled electrode process. The dependence of formal potential ( E 0′) on the solution pH (56 mV pH − 1 ) indicated that the direct electron transfer reaction of hemoglobin was a one-electron transfer coupled with a one proton transfer reaction process. The average surface coverage of Hb immobilized on the cobalt oxide nanoparticles was about 5.2536 × 10 − 11 mol cm − 2 , indicating high loading ability of nanoparticles for hemoglobin entrapment. The heterogeneous electron transfer rate constant ( k s) was 1.43 s − 1 , indicating great of facilitation of the electron transfer between Hb and electrodeposited cobalt oxide nanoparticles. Modified electrode exhibits a remarkable electrocatalytic activity for the reduction of hydrogen peroxide and oxygen. The Michaels–Menten constant K m of 0.38 mM, indicating that the Hb immobilized onto cobalt oxide film retained its peroxidases activity. The biosensor exhibited a fast amperometric response < 5 s, a linear response over a wide concentration range 5 μM to 700 μM and a low detection limit 0.5 μM. According to the direct electron transfer property and enhanced activity of Hb in cobalt oxide film, a third generation reagentless biosensor without using any electron transfer mediator or specific reagent can be constructed for determination of hydrogen peroxide in anaerobic solutions.

In vitro anaerobic incubation of Salmonella enterica serotype Typhimurium and laying hen cecal bacteria in poultry feed substrates and a fructooligosaccharide prebiotic

The objective of this study was to investigate the effect of combining a prebiotic with poultry feeds on the growth of Salmonella enterica serotype Typhimurium (ST) in an in vitro cecal fermentation system. Cecal contents from three laying hens were pooled and diluted to a 1:3000 concentration in an anaerobic dilution solution. The cecal dilution was added to sterile test tubes filled with alfalfa and layer ration with and without fructooligosaccharide (FOS). Two controls containing cecal dilutions and anaerobic dilution solution were used. The samples were processed in the anaerobic hood and incubated at 37 °C. Samples were inoculated with Salmonella at 0 and 24 h after in vitro cecal fermentation and plated at 0 and 24 h after inoculation with ST. Plates were incubated for 24 h and colony forming units (CFU) enumerated. The samples immediately inoculated with ST without prior cecal fermentation did not significantly lower ST counts 24 h later. However, samples pre-incubated for 24 h with cecal microflora prior to ST inoculation exhibited reduced ST CFU by approximately 2 logarithms, with the most dramatic decreases seen in alfalfa and layer ration combined with FOS. The addition of FOS to feed substrate diets in combination with cecal contents acted in a synergistic manner to decrease ST growth only after ST was introduced to 24 h cecal incubations.

Photophysical and Photochemical Studies of 2-Phenylbenzimidazole and UVB Sunscreen 2-Phenylbenzimidazole-5-sulfonic Acid¶

The sunscreen agent 2-phenylbenzimidazole-5-sulfonic acid (PBSA) and its parent 2-phenylbenzimidazole (PBI) cause DNA photodamage via both Type-I and Type-II mechanisms when UVB irradiated. We have studied the photophysical and photochemical properties of these compounds and their ability to photogenerate reactive oxygen species including free radicals. PBI and PBSA exhibit both oxidizing and reducing properties in their excited state. The absorption and fluorescence properties of PBSA depend strongly upon pH, and hence the photochemistry of PBSA was studied in both neutral and alkaline solutions. PBSA showed strong oxidizing properties when UV irradiated in neutral aqueous solution (pH 7.4) in the presence of cysteine, glutathione and azide, as evidenced by the detection of the corresponding S-cysteinyl, glutathiyl and azidyl radicals with the aid of the spin trap, 5,5-dimethyl-1-pyrroline N-oxide (DMPO). However, when an aqueous anaerobic solution (pH 10) of PBSA and either nitromethane (NM) or 4-nitrobenzoic acid (4-NBA) were irradiated, the corresponding nitro anion radicals were observed. This finding suggests that both NM and 4-NBA are reduced by direct electron transfer from the excited state PBSA. During UV irradiation of an aerobic solution of PBSA, O2•− and •OH radical were generated and trapped by DMPO. Further, PBI (in ethanol) and PBSA (in ethylene glycol : water 2:1 mixture) showed low temperature (77 K) phosphorescence (λmax= 443, 476 and 509 nm) and also an electron paramagnetic resonance half-field transition (ΔMs=±2), which is evidence for a triplet state. This triplet produced singlet oxygen (1O2) with quantum yields 0.07 and 0.04 in MeCN for PBI and PBSA, respectively. These studies demonstrate that UV irradiation of PBSA and PBI generates a variety of free radicals and active oxygen species that may be involved in the photodamage of DNA.

Concerted Nitric Oxide Formation and Release from the Simultaneous Reactions of Nitrite with Deoxy- and Oxyhemoglobin

Recent studies reveal a novel role for hemoglobin as an allosterically regulated nitrite reductase that may mediate nitric oxide (NO)-dependent signaling along the physiological oxygen gradient. Nitrite reacts with deoxyhemoglobin in an allosteric reaction that generates NO and oxidizes deoxyhemoglobin to methemoglobin. NO then reacts at a nearly diffusion-limited rate with deoxyhemoglobin to form iron-nitrosyl-hemoglobin, which to date has been considered a highly stable adduct and, thus, not a source of bioavailable NO. However, under physiological conditions of partial oxygen saturation, nitrite will also react with oxyhemoglobin, and although this complex autocatalytic reaction has been studied for a century, the interaction of the oxy- and deoxy-reactions and the effects on NO disposition have never been explored. We have now characterized the kinetics of hemoglobin oxidation and NO generation at a range of oxygen partial pressures and found that the deoxy-reaction runs in parallel with and partially inhibits the oxy-reaction. In fact, intermediates in the oxy-reaction oxidize the heme iron of iron-nitrosyl-hemoglobin, a product of the deoxy-reaction, which releases NO from the iron-nitrosyl. This oxidative denitrosylation is particularly striking during cycles of hemoglobin deoxygenation and oxygenation in the presence of nitrite. These chemistries may contribute to the oxygen-dependent disposition of nitrite in red cells by limiting oxidative inactivation of nitrite by oxyhemoglobin, promoting nitrite reduction to NO by deoxyhemoglobin, and releasing free NO from iron-nitrosyl-hemoglobin.

A Pollutant Transformation Laboratory Exercise for Environmental Chemistry: The Reduction of Nitrobenzenes by Anaerobic Solutions of Humic Acid

This article presents a laboratory exercise for advanced undergraduate- or graduate-level environmental chemistry in which the student studies the chemical transformation (reduction) of substituted nitrobenzenes by natural organic matter under anaerobic conditions. The exercise uses a reference humic acid (Fluka) prepared in a standardized way yielding reproducible experimental conditions (pH, EH, etc.) and results that allow easy adoption in the teaching laboratory. Instead of focusing on a simple chemical concept, as might be common in lower-level laboratories, this exercise combines multiple concepts such as solution preparation, pH buffers, EH buffers, organic reaction mechanisms, reaction kinetics, and instrumental analysis. Data showing the dependence of the reaction on experimental parameters such as solution pH, solution EH, concentration of bulk electron donors (sulfide), concentration of natural organic matter (the electron mediator), and nitrobenzene substituent are presented. In addition, three journal articles are recommended to tie this laboratory exercise to the lecture portion of an environmental chemistry class.

Aryl radical involvement in amiodarone-induced pulmonary toxicity: Investigation of protection by spin-trapping nitrones

Amiodarone (AM), an antidysrrhythmic drug, can produce serious adverse effects, including potentially fatal AM-induced pulmonary toxicity (AIPT). AM-induced cytotoxicity and pulmonary fibrosis are well recognized, but poorly understood mechanistically. The hypothesis of aryl radical involvement in AM toxicity was tested in non-biological and biological systems. Photolysis of anaerobic aqueous solutions of AM, or N-desethylamiodarone (DEA) resulted in the formation of an aryl radical, as determined by spin-trapping and electron paramagnetic resonance (EPR) spectroscopy experiments. The non-iodinated AM analogue, didesiodoamiodarone (DDIA), did not form aryl radicals under identical conditions. The toxic susceptibility of human lung epithelioid HPL1A cells to AM, DEA, and DDIA showed time- and concentration-dependence. DEA had a more rapid and potent toxic effect (LC(50)=8 microM) than AM (LC(50)=146 microM), whereas DDIA cytotoxicity was intermediate (LC(50)=26 microM) suggesting a minor contribution of the iodine atoms. Incubation of human lung epithelial cells with the spin-trapping nitrones alpha-phenyl-N-t-butylnitrone (PBN, 10 mM) or alpha-(4-pyridyl N-oxide)-N-t-butylnitrone (POBN, 5.0 mM) did not significantly protect against AM, DEA, or DDIA cytotoxicity. Intratracheal administration of AM to hamsters produced pulmonary fibrosis at day 21, which was not prevented by 4 days of treatment with 150 mg/kg/day PBN or 164 mg/kg/day POBN. However, the body weight loss in AM-treated animals was counteracted by PBN. These results suggest that, although AM can generate an aryl radical photochemically, its in vivo formation may not be a major contributor to AM toxicity, and that spin-trapping reagents do not halt the onset of AM toxicity.

Photochemistry of 2,6-diisopropylphenol (propofol)

The photochemistry of the anaesthetic agent propofol (PPF) was investigated in three different solvents of quite different polarity (cyclohexane, methanol and phosphate buffer pH 7) by means of nanosecond laser flash photolysis and absorption spectroscopy. GC-MS spectrometry measurements of PPF in cyclohexane have revealed the formation of two major products upon low intensity UV continuous irradiation of PPF in aerated solution: the diphenol derivative of PPF and 2,6-diisopropyl-p-benzoquinone (PPFQ). Only the diphenol compound was obtained in anaerobic solution. PPF phenoxyl radical (PPF ) generation has been assigned as the original step leading to the formation of both the diphenol compound and PPFQ in cyclohexane as revealed by laser flash photolysis at 266 nm and by electron paramagnetic resonance spectroscopy as well. Investigation of PPF by nanosecond flash photolysis at 266 nm in the other solvents revealed the occurrence of different photochemical processes depending on the nature and the polarity of the solvent. A reaction scheme is proposed in order to discuss the mechanism of reaction of PPF in all media.

Paired Bacillus anthracis Dps (Mini-ferritin) Have Different Reactivities with Peroxide

Dps (DNA protection during starvation) proteins, mini-ferritins in the ferritin superfamily, catalyze Fe(2+)/H(2)O(2)/O(2) reactions and make minerals inside protein nanocages to minimize radical oxygen-chemistry (metal/osmotic/temperature/nutrient/oxidant) and sometimes to confer virulence. Paired Dps proteins in Bacillus, rare in other bacteria, have 60% sequence identity. To explore functional differences in paired Bacilli Dps protein, we measured ferroxidase activity and DNA protection (hydroxyl radical) for Dps protein dodecamers from Bacillus anthracis (Ba) since crystal structures and iron mineralization (iron-stain) were known. The self-assembled (200 kDa) Ba Dps1 (Dlp-1) and Ba Dps2 (Dlp-2) proteins had similar Fe(2+)/O(2) kinetics, with space for minerals of 500 iron atoms/protein, and protected DNA. The reactions with Fe(2+) were novel in several ways: 1) Ba Dps2 reactions (Fe(2+)/H(2)O(2)) proceeded via an A(650 nm) intermediate, with similar rates to maxi-ferritins (Fe(2+)/O(2)), indicating a new Dps protein reaction pathway, 2) Ba Dps2 reactions (Fe(2+)/O(2) versus Fe(2+)/O(2) + H(2)O(2)) differed 3-fold contrasting with Escherichia coli Dps reactions, with 100-fold differences, and 3) Ba Dps1, inert in Fe(2+)/H(2)O(2) catalysis, inhibited protein-independent Fe(2+)/H(2)O(2) reactions. Sequence similarities between Ba Dps1 and Bacillus subtilis DpsA (Dps1), which is regulated by general stress factor (SigmaB) and Fur, and between Ba Dps2 and B. subtilis MrgA, which is regulated by H(2)O(2) (PerR), suggest the function of Ba Dps1 is iron sequestration and the function of Ba Dps2 is H(2)O(2) destruction, important in host/pathogen interactions. Destruction of H(2)O(2) by Ba Dps2 proceeds via an unknown mechanism with an intermediate similar spectrally (A(650 nm)) and kinetically to the maxi-ferritin diferric peroxo complex.

Conditions leading to high CO2 (>5 kPa) in waterlogged-flooded soils and possible effects on root growth and metabolism.

Soil waterlogging impedes gas exchange with the atmosphere, resulting in low P(O2) and often high P(CO2). Conditions conducive to development of high P(CO2) (5-70 kPa) during soil waterlogging and flooding are discussed. The scant information on responses of roots to high P(CO2) in terms of growth and metabolism is reviewed. P(CO2) at 15-70 kPa has been reported for flooded paddy-field soils; however, even 15 kPa P(CO2) may not always be reached, e.g. when soil pH is above 7. Increases of P(CO2) in soils following waterlogging will develop much more slowly than decreases in P(O2); in soil from rice paddies in pots without plants, maxima in P(CO2) were reached after 2-3 weeks. There are no reliable data on P(CO2) in roots when in waterlogged or flooded soils. In rhizomes and internodes, P(CO2) sometimes reached 10 kPa, inferring even higher partial pressures in the roots, as a CO2 diffusion gradient will exist from the roots to the rhizomes and shoots. Preliminary modelling predicts that when P(CO2) is higher in a soil than in roots, P(CO2) in the roots would remain well below the P(CO2) in the soil, particularly when there is ventilation via a well-developed gas-space continuum from the roots to the atmosphere. The few available results on the effects of P(CO2) at > 5 kPa on growth have nearly all involved sudden increases to 10-100 kPa P(CO2); consequently, the results cannot be extrapolated with certainty to the much more gradual increases of P(CO2) in waterlogged soils. Nevertheless, rice in an anaerobic nutrient solution was tolerant to 50 kPa CO2 being suddenly imposed. By contrast, P(CO2) at 25 kPa retarded germination of some maize genotypes by 50%. With regard to metabolism, assuming that the usual pH of the cytoplasm of 7.5 was maintained, every increase of 10 kPa CO2 would result in an increase of 75-90 mM HCO3(-) in the cytoplasm. pH maintenance would depend on the biochemical and biophysical pH stats (i.e. regulatory systems). Furthermore, there are indications that metabolism is adversely affected when HCO3(-) in the cytoplasm rises above 50 mM, or even lower; succinic dehydrogenase and cytochrome oxidase are inhibited by HCO3(-) as low as 10 mM. Such effects could be mitigated by a decrease in the set point for the pH of the cytoplasm, thus lowering levels of HCO3(-) at the prevailing P(CO2) in the roots. Measurements are needed on P(CO2) in a range of soil types and in roots of diverse species, during waterlogging and flooding. Species well adapted to high P(CO2) in the root zone, such as rice and other wetland plants, thrive even when P(CO2) is well over 10 kPa; mechanisms of adaptation, or acclimatization, by these species need exploration.

Tuning Supported Catalyst Reactivity with Dendrimer-Templated Pt−Cu Nanoparticles

The effects of particle composition on heterogeneous catalysis were studied using dendrimer-encapsulated nanoparticles (DENs) as precursors to supported Pt-Cu catalysts. Bimetallic Pt-Cu DENs with varying Pt/Cu ratios were prepared in an anaerobic aqueous solution and deposited onto a high-purity commercial alumina support. The dendrimer template was then thermally removed to yield supported nanoparticle catalysts, which were studied with toluene hydrogenation and CO oxidation catalysis as well as infrared spectroscopy of adsorbed CO. Incorporating Cu into Pt nanoparticles had opposite effects on the two test reactions. Cu acted as a mild promoter for CO oxidation catalysis, and the promoting effect was independent of the amount of Cu present. Conversely, Cu acted as a strong poison for toluene hydrogenation catalysis, and the normalized rate tracked inversely with Cu content. Infrared spectroscopy of the supported nanoparticles indicated that electronic effects (electron donation from Cu to Pt) were minimal for these materials. Consequently, the catalysis results are interpreted in terms of potential structural differences as a function of Cu incorporation and reaction conditions.

Multiple Active Site Conformers in the Carbon Monoxide Complexes of Trematode Hemoglobins

Sequence alignment of hemoglobins of the trematodes Paramphistomum epiclitum and Gastrothylax crumenifer with myoglobin suggests the presence of an unusual active site structure in which two tyrosine residues occupy the E7 and B10 helical positions. In the crystal structure of P. epiclitum hemoglobin, such an E7-B10 tyrosine pair at the putative helical positions has been observed, although the E7 Tyr is displaced toward CD region of the polypeptide. Resonance Raman data on both P. epiclitum and G. crumenifer hemoglobins show that interactions of heme-bound ligands with neighboring amino acid residues are unusual. Multiple conformers in the CO complex, termed the C, O, and N conformers, are observed. The conformers are separated by a large difference (approximately 60 cm(-1)) in the frequencies of their Fe-CO stretching modes. In the C conformer the Fe-CO stretching frequency is very high, 539 and 535 cm(-1), for the P. epiclitum and G. crumenifer hemoglobins, respectively. The Fe-CO stretching of the N conformer appears at an unusually low frequency, 479 and 476 cm(-1), respectively, for the two globins. A population of an O conformer is seen in both hemoglobins, at 496 and 492 cm(-1), respectively. The C conformer is stabilized by a strong polar interaction of the CO with the distal B10 tyrosine residue. The O conformer is similar to the ones typically seen in mutant myoglobins in which there are no strong interactions between the CO and residues in the distal pocket. The N conformer possesses an unusual configuration in which a negatively charged group, assigned as the oxygen atom of the B10 Tyr side chain, interacts with the CO. In this conformer, the B10 Tyr assumes an alternative conformation consistent with one of the conformers seen the crystal structure. Implications of the multiple configurations on the ligand kinetics are discussed.

Models for the Molybdenum Hydroxylases: Synthesis, Characterization and Reactivity of cis-Oxosulfido-Mo(VI) Complexes

Atom transfer reactions have been employed to convert Tp(i)(Pr)MoO(2)(OAr) into monomeric cis-oxosulfido-Mo(VI) and dimeric mu-disulfido-Mo(V) species, [Tp(i)(Pr)MoOS(OAr)](n)() (Tp(i)(Pr) = hydrotris(3-isopropylpyrazol-1-yl)borate; OAr = phenolate or naphtholate derivative; n = 1 and 2, respectively). Dark red, monomeric Tp(i)(Pr)MoOS(OAr) complexes contain distorted octahedral cis-oxosulfido-Mo(VI) centers, with d(Mo=O) = 1.692(5) A, d(Mo=S) = 2.132(2) A, and angle(O=Mo=S) = 103.68(16) degrees for the 2-sec-butylphenolate derivative. Dark red-purple, dimeric [Tp(i)(Pr)MoOS(OAr)](2) complexes undergo S-S bond cleavage forming monomeric oxosulfido-Mo(VI) species in solution. In the solid state, the 3,5-di-tert-butylphenolate derivative exhibits a centrosymmetric structure, with distorted octahedral anti oxo-Mo(V) centers bridged by a disulfido-kappaS,kappaS' ligand. Hydrolysis of the oxosulfido-Mo(VI) complexes results in the formation of [Tp(i)(Pr)MoO](2)(mu-S(2))(mu-O). In anaerobic solutions, certain oxosulfido-Mo(VI) complexes convert to molybdenyl complexes bearing bidentate 2-mercaptophenolate or related naphtholate ligands formed via intramolecular attack of the sulfido ligand on a coligand C-H group. The oxosulfido-Mo(VI) complexes serve as precursors to biologically relevant Mo(V) and heterobimetallic MoO(mu-S)Cu species and undergo a range of biomimetic reactions.

Reconstitution and Characterization of Aminopyrrolnitrin Oxygenase, a Rieske N-Oxygenase That Catalyzes Unusual Arylamine Oxidation

Rieske oxygenases catalyze a wide variety of important oxidation reactions. Here we report the characterization of a novel Rieske N-oxygenase, aminopyrrolnitrin oxygenase (PrnD) that catalyzes the unusual oxidation of an arylamine to an arylnitro group. PrnD from Pseudomonas fluorescens Pf5 was functionally expressed in Escherichia coli, and the activity of the purified PrnD was reconstituted, which required in vitro assembly of the Rieske iron-sulfur cluster into the protein and the presence of NADPH, FMN, and an E. coli flavin reductase SsuE. Biochemical and bioinformatics studies indicated that the reconstituted PrnD contains a Rieske iron-sulfur cluster and a mononuclear iron center that are formed by residues Cys(69), Cys(88), His(71), His(91), Asp(323), His(186), and His(191), respectively. The enzyme showed a limited range of substrate specificity and catalyzed the conversion of aminopyrrolnitrin into pyrrolnitrin with K(m) = 191 microM and k(cat) = 6.8 min(-1). Isotope labeling experiments with (18)O(2) and H(2)(18)O suggested that the oxygen atoms in the pyrrolnitrin product are derived exclusively from molecular oxygen. In addition, it was found that the oxygenation of the arylamine substrates catalyzed by PrnD occurs at the enzyme active site and does not involve free radical chain reactions. By analogy to known examples of arylamine oxidation, a catalytic mechanism for the bioconversion of amino pyrrolnitrin into pyrrolnitrin was proposed. Our results should facilitate further mechanistic and crystallographic studies of this arylamine oxygenase and may provide a new enzymatic route for the synthesis of aromatic nitro compounds from their corresponding aromatic amines.

Synthesis and thermochemical study of ligand substitution reactions of aminobis(phosphines), Ph 2P(R)NPPh 2, with [Me 2Pt(COD)

The reactions of aminobis(phosphines), Ph 2PN(R)PPh 2 (R = H, Me, Et, n Pr, n Bu, Ph), with Me 2Pt(COD) led to the formation of four-membered metallacycles in good yield. The enthalpies of the ligand substitution reactions are measured by the anaerobic solution calorimetry in THF at 30 °C.

The Three Nitric-oxide Synthases Differ in Their Kinetics of Tetrahydrobiopterin Radical Formation, Heme-Dioxy Reduction, and Arginine Hydroxylation

The nitric-oxide synthases (NOSs) make nitric oxide and citrulline from l-arginine. How the bound cofactor (6R)-tetrahydrobiopterin (H4B) participates in Arg hydroxylation is a topic of interest. We demonstrated previously that H4B radical formation in the inducible NOS oxygenase domain (iNOSoxy) is kinetically coupled to the disappearance of a heme-dioxy intermediate and to Arg hydroxylation. Here we report single turnover studies that determine and compare the kinetics of these transitions in Arg hydroxylation reactions catalyzed by the oxygenase domains of endothelial and neuronal NOSs (eNOSoxy and nNOSoxy). There was a buildup of a heme-dioxy intermediate in eNOSoxy and nNOSoxy followed by a monophasic transition to ferric enzyme during the reaction. The rate of heme-dioxy decay matched the rates of H4B radical formation and Arg hydroxylation in both enzymes. The rates of H4B radical formation differed such that nNOSoxy (18 s(-1)) > iNOSoxy (11 s(-1)) > eNOSoxy (6 s(-1)), whereas the lifetimes of the resulting H4B radical followed an opposite rank order. 5MeH4B supported a three-fold faster radical formation and greater radical stability relative to H4B in both eNOSoxy and nNOSoxy. Our results indicate the following: (i) the three NOSs share a common mechanism, whereby H4B transfers an electron to the heme-dioxy intermediate. This step enables Arg hydroxylation and is rate-limiting for all subsequent steps in the hydroxylation reaction. (ii) A direct correlation exists between pterin radical stability and the speed of its formation in the three NOSs. (iii) Uncoupled NO synthesis often seen for eNOS at low H4B concentrations may be caused by the slow formation and poor stability of its H4B radical.

Mechanistic Studies of Rhodobacter sphaeroides Me2SO Reductase

Studies of the molybdenum-containing dimethyl sulfoxide reductase from Rhodobacter sphaeroides have yielded new insight into its catalytic mechanism. A series of reductive titrations, performed over the pH range 6-10, reveal that the absorption spectrum of reduced enzyme is highly sensitive to pH. The reaction of reduced enzyme with dimethyl sulfoxide is found to be clearly biphasic throughout the pH range 6-8 with a fast, initial substrate-binding phase and substrate-concentration independent catalytic phase. The intermediate formed at the completion of the fast phase has the characteristic absorption spectrum of the established dimethyl sulfoxide-bound species. Quantitative reductive and oxidative titrations of the enzyme demonstrate that the molybdenum center takes up only two reducing equivalents, implying that the two pyranopterin equivalents of the molybdenum center are not formally redox active. Finally, the visible spectrum associated with the catalytically relevant "high-g split" Mo(V) species has been determined. Spectral deconvolution and EPR quantitation of enzyme-monitored turnover experiments with trimethylamine N-oxide as substrate reveal that no substrate-bound intermediate accumulates and that Mo(V) content remains near unity for the duration of the reaction. Similar experiments with dimethyl sulfoxide show that significant quantities of both the Mo(V) species and the dimethyl sulfoxide-bound complex accumulate during the course of reaction. Accumulation of the substrate-bound complex in the steady-state with dimethyl sulfoxide arises from partial reversal of the physiological reaction in which the accumulating product, dimethyl sulfide, reacts with oxidized enzyme to yield the substrate-bound intermediate, a process that significantly slows turnover.

Polyvinylferrocenium modified Pt electrode for anaerobic glucose monitoring

An amperometric enzyme electrode for the determination of glucose under anaerobic solution conditions was developed by immobilizing glucose oxidase and then by adsorbing ferrocene in polyvinylferrocenium matrix coated on a Pt electrode surface. The amperometric response due to the electrooxidation of ferrocene that the reduced flavin adenine dinucleotide centers of glucose oxidase was measured at a constant potential. The response characteristics of the enzyme electrode were investigated. The effects of the thickness of the polymeric film, the amount of the enzyme immobilized, the amount of the mediator, the glucose concentration, the applied potential, operating pH and temperature on the response of the enzyme electrode were studied. The response time and the optimum pH were found to be 30–40 s and pH 7.4 at 25 °C, respectively. The linear response was observed up to 5.0 mM glucose concentration that the produced detectable current was 0.0075 mM glucose concentration. The activation energy ( E a) of immobilized enzyme reaction was calculated to be 41.3 kJ mol −1 from the Arrhenius plot. The apparent Michaelis-Menten constant ( K Mapp) was found to be 6.05 mM glucose according to the Lineweaver-Burk graph of the Michaelis-Menten equation under the optimum conditions. The interference signal due to the most common electrochemical interfering species was also evaluated.

Metal-catalyzed anaerobic disproportionation of hydroxylamine. Role of diazene and nitroxyl intermediates in the formation of N2, N2O, NO+, and NH3.

The catalytic disproportionation of NH(2)OH has been studied in anaerobic aqueous solution, pH 6-9.3, at 25.0 degrees C, with Na(3)[Fe(CN)(5)NH(3)].3H(2)O as a precursor of the catalyst, [Fe(II)(CN)(5)H(2)O](3)(-). The oxidation products are N(2), N(2)O, and NO(+) (bound in the nitroprusside ion, NP), and NH(3) is the reduction product. The yields of N(2)/N(2)O increase with pH and with the concentration of NH(2)OH. Fast regime conditions involve a chain process initiated by the NH(2) radical, generated upon coordination of NH(2)OH to [Fe(II)(CN)(5)H(2)O](3)(-). NH(3) and nitroxyl, HNO, are formed in this fast process, and HNO leads to the production of N(2), N(2)O, and NP. An intermediate absorbing at 440 nm is always observed, whose formation and decay depend on the medium conditions. It was identified by UV-vis, RR, and (15)NMR spectroscopies as the diazene-bound [Fe(II)(CN)(5)N(2)H(2)](3)(-) ion and is formed in a competitive process with the radical path, still under the fast regime. At high pH's or NH(2)OH concentrations, an inhibited regime is reached, with slow production of only N(2) and NH(3). The stable red diazene-bridged [(NC)(5)FeHN=NHFe(CN)(5)](6)(-) ion is formed at an advanced degree of NH(2)OH consumption.