An amperometric nitrate reductase-phenosafranin electrode: kinetic aspects and analytical applications

The enzyme-catalysed reduction of nitrate was studied utilising Aspergillus niger nitrate reductase (NR) and phenosafranin in solution as the enzyme regenerator, working at lower potentials than that of the more common methyl viologen mediator. Cyclic voltammograms when enzyme, phenosafranin and substrate were together put in evidence the enzyme-catalysed reduction of nitrate, although with a relatively slow kinetics. From slope values not dependent on mediator concentration, the apparent Michaelis-Menten constant was evaluated. Analytical parameters for the enzyme-modified electrode in the presence of phenosafranin for the determination of nitrate content in water were assessed, including a recovery assay for nitrate added to a river water sample. The stability of the electrode was checked.     

Flavin-mediated photoinactivation of spinach leaf nitrate reductase involving superoxide radical and activating effect of hydrogen peroxide

Irradiation with white light of spinach leaf nitrate reductase (NR) in the presence of flavin mononucleotide (FMN) and ethylenediaminetetraacetic acid (EDTA) resulted in a gradual loss of the enzyme activity, measured with reduced methyl viologen as electron donor. Inactivation of NR was dependent on oxygen and was prevented by superoxide dismutase. On the contrary, the presence of catalase markedly enhanced the rate of inactivation. Nitrate showed a protective effect. NR previously inactivated by irradiation in the presence of FMN and EDTA was greatly reactivated by a short preincubation of the inactive enzyme with either ferricyanide or H₂O₂. These results suggest that spinach leaf NR is reversibly inactivated by photogenerated O₂ , while H₂O₂ has an activating effect.     

LIMITING REACTIONS IN HYDROGEN PHOTOPRODUCTION BY CHLOROPLASTS AND HYDROGENASE

Abstract— Hydrogen was photoproduced from water in a system containing isolated chloroplasts, hy-drogenase, a coupling electron carrier (ferredoxin or methyl viologen), and an oxygen scavenger. The rate and extent of hydrogen production anaerobically was much less than the rate of aerobic electron-carrier reduction by chloroplasts and was not limited by hydrogenase. The limiting reaction in the coupled system was the extent of reduction of methyl viologen anaerobically rather than its oxidation by oxygen produced during the course of the reaction. Inhibition of photosystem II by 3-(3,4dichlorophenyl)-1,1-dimethylurea and addition of a photosystem 1 electron donor did not lead to photoproduction of hydrogen or photoreduction of methyl viologen. Extensive photosystem I hydrogen evolution was obtained when thiols were also present. Platinum asbestos or palladium asbestos replaced hydrogenase in a system coupled to chloroplasts.     

Application of immobilized hydrogenase to h2 storage in concentrated solutions of methyl viologen

It has been found that immobilized cells ofC. pasteurianum possessing hydrogenase activity efficiently catalyze reversible reduction of concentrated (up to 0.5M) solutions of methyl viologen with H₂. A 0.5M aqueous solution of methyl viologen dissolves 240 times as much H₂ as pure water under the same pressure of hydrogen. The experimentally obtained levels of methyl viologen reduction and H₂ evolution are in satisfactory agreement with theoretical calculations. The potential of concentrated solutions of methyl viologen containing immobilized hydrogenase as a H₂ storage medium is discussed.     

Conductometric nitrate biosensor based on methyl viologen/Nafion/nitrate reductase interdigitated electrodes

A highly sensitive, fast and stable conductometric enzyme biosensor for determination of nitrate in water is reported for the first time. The biosensor electrodes were modified by methyl viologen mediator mixed with nitrate reductase (NR) from Aspergillus niger by cross-linking with glutaraldehyde in the presence of bovine serum albumin and Nafion^® cation-exchange polymer. The process parameters for the fabrication of the enzyme electrode and various experimental variables such as pH, the enzyme loading and time of immobilization in glutaralaldehyde vapor were investigated with regard to their influence on sensitivity, limit of detection, dynamic range and operational and storage stability. The biosensor can reach 95% of steady-state conductance value in about 15 s. Linear calibration in the range of 0.02 and 0.25 mM with detection limits of 0.005 mM nitrate was obtained with a signal-to-noise ratio of 3. When stored in 5 mM phosphate buffer (pH 7.5) at 4 °C, the sensor showed good stability over 2 weeks.     

Ligand structure, conformational dynamics, and excited-state electron delocalization for control of photoinduced electron transfer rates in synthetic donor-bridge-acceptor systems

Synthesis, ground-, and excited-state properties are reported for two new electron donor-bridge-acceptor (D-B-A) molecules and two new photophysical model complexes. The D-B-A molecules are [Ru(bpy)2(bpy-phi-MV)](PF6)4 (3) and [Ru(tmb)2(bpy-phi-MV)](PF6)4 (4), where bpy is 2,2'-bipyridine, tmb is 4,4',5,5'-tetramethyl-2,2'-bipyridine, MV is methyl viologen, and phi is a phenylene spacer. Their model complexes are [Ru(bpy)2(p-tol-bpy)](PF6)2 (1) and [Ru(tmb)2(p-tol-bpy)](PF6)2 (2), where p-tolyl-bpy is 4-(p-tolyl)-2,2'-bipyridine. Photophysical characterization of 1 and 2 indicates that 2.17 eV and 2.12 eV are stored in their respective (3)MLCT (metal-to-ligand charge transfer) excited state. These values along with electrochemical measurements show that photoinduced electron transfer (D*-B-A-->D (+)-B-A(-)) is favorable in 3 and 4 with DeltaG degrees(ET)=-0.52 eV and -0.62 eV, respectively. The driving force for the reverse process (D(+)-B-A(-) --> D-B-A) is also reported: DeltaG degrees(BET)=-1.7 eV for 3 and -1.5 eV for 4. Transient absorption (TA) spectra for 3 and 4 in 298 K acetonitrile provide evidence that reduced methyl viologen is observable at 50 ps following excitation. Detailed TA kinetics confirm this, and the data are fit to a model to determine both forward (k(ET)) and back (k(BET)) electron transfer rate constants: k(ET)=2.6 x 10(10) s(-1) for 3 and 2.8 x 10(10) s(-1) for 4; k(BET)=0.62 x 10(10) s(-1) for 3 and 1.37 x 10(10) s(-1) for 4. The similar rate constants k ET for 3 and 4 despite a 100 meV driving force (DeltaG degrees(ET)) increase suggests that forward electron transfer in these molecules in room temperature acetonitrile is nearly barrierless as predicted by the Marcus theory. The reduction in electron transfer reorganization energy necessary for this barrierless reactivity is attributed to excited-state electron delocalization in the (3)MLCT excited states of 3 and 4, an effect that is made possible by excited-state conformational changes in the aryl-substituted ligands of these complexes.     

KINETICS AND MECHANISM OF PHOTO-INDUCED METHYL VIOLOGEN REDUCTION WITH ZINC-MESO-TETRAPHENYLPORPHYRINTRISULFONIC ACID AND HYDROGEN EVOLUTION FROM WATER HYDROGENASE

Abstract— The formation of the cation radical of methyl viologen was established when an aqueous solution containing zinc-meso-tetraphenylporphyrintrisulfonic acid, methyl viologen and mercaptoethanol was irradiated by visible light. The reduction of methyl viologen has been studied kinetically and the reduction rate expressed as follows

where [Zn]₀ is the total amount of zinc compounds in the system.
On the basis of the rate expression, the reaction mechanism is discussed.
On the addition of hydrogenase to the above photoirradiation system, hydrogen evolution was observed by both the irradiation of sunlight and a tungsten lamp.     

光敏氧化还原反应——四苯基卟啉类化合物光敏还原甲基紫精

在DMSO-H₂O混合溶剂中,用TPP和它的四种金属络合物作为敏化剂,EDTA作为电子给体,经可见光照射,成功地还原了甲基紫精。反应活性顺序为ZnTPP>TPP>MgTPP》CoTPP>CuTPP。对以ZnTPP为敏化剂的反应,我们测得其量子产率为φ_418nm=0.08。我们考察了ZnTPP、MV²⁺和EDTA的浓度以及氧气对反应的影响。在一定浓度范围内,我们发现,反应速度与MV²⁺和EDTA浓度之间的关系符合于从推测的反应机理导出的动力学方程。     

Reduction of aldehydes and ketones by sodium dithionite using viologens as electron transfer catalyst

It was found that aromatic aldehydes and ketones could be reduced smoothly at room temperature by sodium dithionite using viologen derivatives as an electron transfer catalyst to obtain the corresponding alcohols in good yields. Viologens (methyl viologen and polymer) acted catalytically and active species in the reduction were quinoid forms which were obtained by two-electron reduction of viologens.     

Prevulcanized natural rubber latex/clay aerogel nanocomposites

Natural rubber latex (NR)/clay aerogel nanocomposites were produced via freeze-drying technique. The pristine clay (sodium montmorillonite) was introduced in 1-3 parts per hundred rubber (phr) in order to study the effect of clay in the NR matrix. The dispersion of the layered clay and the morphology of the nanocomposites were determined by X-ray diffraction (XRD) and scanning electron microscopy (SEM), respectively. Cure characteristics, thermal stability, and the crosslink density of thermal and microwave-cured NR and its composites were investigated. XRD patterns indicated that both intercalated and exfoliated structures were observed at loadings of 1-3 phr clay. SEM studies revealed that the clay aerogel structure was formed at 3 phr clay loading. The increment in Shore A hardness of nanocomposites compared with pure NR signified excellent polymer/filler interaction and the reinforcing effect of the clay to rubber matrix. This was supported by an increase in maximum rheometric torque and crosslink density. The crosslink density of clay-filled NR vulcanizate was found to increase with the pristine clay content in both thermal and microwave curing methods. However, microwave-cured 2 and 3 phr-filled NR vulcanizates exhibited higher crosslink density than those which were thermal-cured under the same curing temperature. In addition, thermal stability studies showed that pristine clay accelerated the decomposition of NR by showing a slight decrease in onset and peak decomposition temperatures along with clay content.     

Fabrication of Redox‐Mediator Supported Potentiometric Nitrate Biosensor with Nitrate Reductase

Fabrication of a more superior nitrate potentiometric biosensor than previously achieved with NaR and NADH has been accomplished by co‐entrapment of redox mediators and NaR into polypyrrole (PPy) film during galvanostatic polymerization of pyrrole. The replacement of NADH with redox mediators such as thionin acetate (ThAc), safranin (Saf), and azure A (AzA) gave more sensitive potentiometric responses, better minimum detectable concentration, linear concentration range and response time for nitrate than possible with NADH. The co‐entrapment of ThAc, Saf, AzA and methyl viologen (MV) with NaR into PPy films also improved the Nernstian behavior of the electrode process beyond the capability of the PPy‐NaR‐NADH biosensor. Substantial reduction in volume and quantity of cofactor/mediator and, hence cost, was achieved by the replacement of NADH with a redox mediator. Only 50 μM of AzA was required to form a PPy‐NaR‐AzA biosensor which gave the most sensitive potentiometric response for nitrate, achieving a minimum detectable concentration of 10 μM, a linear concentration range of 50–5000 μM and a response time of 2–4 s.     

Redox reactivity of photogenerated osmium(II) complexes

Powerful reductants [Os(II)(NH(3))(5)L](2+) (L = OH(2), CH(3)CN) can be generated upon ultraviolet excitation of relatively inert [Os(II)(NH(3))(5)(N(2))](2+) in aqueous and acetonitrile solutions. Reactions of photogenerated Os(II) complexes with methyl viologen to form methyl viologen radical cation and [Os(III)(NH(3))(5)L](3+) were monitored by transient absorption spectroscopy. Rate constants range from 4.9 × 10(4) M(-1) s(-1) in acetonitrile solution to 3.2 × 10(7) (pH 3) and 2.5 × 10(8) M(-1) s(-1) (pH 12) in aqueous media. Photogeneration of five-coordinate Os(II) complexes opens the way for mechanistic investigations of activation/reduction of CO(2) and other relatively inert molecules.     

Electrochemical characterization of biosensor based on nitrite reductase and methyl viologen co-immobilized glassy carbon electrode

Nitrite reductase (NiR, nitric-oxide: ferricytochrome c oxidoreductase, EC 1.7.2.1) and methyl viologen (MV) were co-immobilized on glassy carbon electrode (GCE, d=3 mm) by polymer entrapment, and the electrode was tested as an electrochemical biosensor for amperometric determination of nitrite. The immobilization was performed by sequential loading and drying of a homogeneous mixture of poly(vinyl alcohol) (PVA), NiR and MV, followed by poly(allylamine hydrochloride) (PAH) solution, and finally hydrophilic polyurethane (HPU) dissolved in chloroform. The positively charged PAH layer could effectively keep immobilized cationic MV from diffusing through the membrane, holding mediator tightly near or on the electrode surface. The working principle of the biosensor was based on MV mediated electron transfer between electrode and immobilized NiR. The response time (t(90%)) of the biosensor was about 20 s and sensitivity was 11.8 nA/ microM (2.5 mU NiR) with linear response range of 1.5-260 microM (r(2)=0.996) and detection limit of 1.5 microM (S/N=3). Lineweaver-Burk plot showed that Michaelis-Menten constant (K(m,app)) was about 770 microM. The biosensor showed durable storage stability for 24 days (stored in ambient air at room temperature) retaining 80% of its initial activity, and showed satisfactory reproducibility (relative standard deviation (R.S.D.)=3.8%, n=9). Interference study showed that chlorate, chloride, sulfite, sulfate did not interfere with the nitrite determination, however, nitrate interfered with the determination with relative sensitivity of 38% (ratio of sensitivity for nitrate to that for nitrite). In addition to the full characterization of the biosensor, kinetic study was also conducted in solution and the homogeneous rate constant (k(2)) between NiR and MV were determined by chronoamperometry to be 5.8 x 10(5) M(-1) s(-1).     

Hindered fluorescence quenching in an insulated molecular wire

A [3]rotaxane 2 within 1(2) consisting of an anionic phenylene ethynylene dumbbell 2(4-) threaded through two cationic cyclophanes 1(2+) has been prepared using aqueous Glaser coupling. Stern-Volmer analysis of the fluorescence quenching using three different electron-acceptors (methyl viologen 13(2+), dipropyl-4,4'-bipyridinium disulfonate 14 and anthraquinone-2,6-disulfonate 15(2-)) shows that the threaded cyclophanes inhibit electron-transfer from the excited state of the dumbbell by steric shielding, and by electrostatic shielding in the case of methyl viologen.     

Direct observation of the one-electron reduction of methyl viologen mediated by the CO2 radical anion during TiO2 photocatalytic reactions

The one-electron reduction of methyl viologen (MV(2+)) mediated by the carbon dioxide radical anion (CO(2)(*-)) during photocatalytic reactions in a colloidal TiO(2) aqueous solution (pH 2) has been investigated by time-resolved absorption spectroscopy. The formation of MV(*+) generated from the one-electron reduction reaction with CO(2)(*-), which is generated from the one-electron oxidation reactions with the photogenerated holes (h(+)), was directly observed. The spectral features of the photogenerated charge carriers and the kinetic analysis of the formation process of MV(*+) revealed that the CO(2)(*-), desorbed from the surface, reacts with MV(2+) via a homogeneous electron-transfer process in the bulk solution.     

Reduction of mercury(II) by the carbon dioxide radical anion: a theoretical and experimental investigation

The laser flash photolysis technique (λ(exc) = 266 nm) was used to investigate the mechanism of the HgCl(2) reduction mediated by CO(2)(·-) radicals in the temperature range 291.7-308.0 K. For this purpose, the CO(2)(·-) radicals were generated by scavenging of sulfate radicals by formic acid. The absorbance of the reduced radical of methyl viologen, a competitive scavenger of CO(2)(·-), was monitored at 390 nm. Moreover, theoretical calculations, including solvent effects, were also performed within the framework of the density functional theory for various chemical species of Hg(I) and Hg(II) to aid in the modeling of the reaction of reduction of HgCl(2) by CO(2)(·-).     

Electronic interplay on illuminated aqueous carbon nanohorn-porphyrin ensembles

Tetracationic water-soluble porphyrin (H(2)P(4+)) has been immobilized by pi-pi stacking interactions onto the skeleton of carbon nanohorns (CNH), without disrupting their pi-electronic network. The stable aqueous solution of the CNH-H(2)P(4+) nanoensemble was examined by both electron microscopy and spectroscopic techniques. The efficient fluorescence quenching of the H(2)P(4+) moiety in the CNH-H(2)P(4+) nanoensemble was probed by steady-state as well as time-resolved fluorescence emission spectroscopy, suggesting charge separation from the photoexcited H(2)P(4+) to CNH. In the presence of methyl viologen dication (MV(2+)) and a hole trap, accumulation of the reduced species of methyl viologen was observed by the photoillumination of CNH-H(2)P(4+), suggesting that the electron migration from the initially formed charge-separated state takes place. Transient absorption spectroscopy gave further insights on the transient species such as the charge-separated state (CNH(*-))-(H(2)P(4+))(*+), which was consumed in the presence of MV(2+) and hole shifter, leaving the reduced methyl viologen.     

Two electron utilization of methyl viologen anolyte in nonaqueous organic redox flow battery

Methyl viologen(MV) as a bench-mark anolyte material has been frequently applied in aqueous organic redox flow batteries(AORFBs) towards large-scale renewable energy storage. However, only the first reduction of MV was utilized in aqueous electrolytes because of the insoluble MV~0 generated from the second reduction of MV. Herein, we report that methyl viologen with bis(trifluoromethane)sulfonamide counter anion, MVTFSI, can achieve two reversible reductions in a nonaqueous supporting electrolyte. Paired with(Ferrocenylmethyl)trimethylammonium bis(trifluoromethanesulfonyl)imide, FcNTFSI as catholyte, the MVTFS/FcNTFSI nonaqueous organic redox flow battery(NOARFB) can take advantage of either one electron or two electron storage of the methyl viologen moiety and provide theoretical energy density of 24.9 Wh/L and a cell voltage of up to 1.5 V. Using a highly conductive LiTFSI/CH_3CN supporting electrolyte and a porous Daramic separator, the NOARFB displayed excellent cycling performance, including up to a 68.3% energy efficiency at 40 mA/cm~2, and more than 88% total capacity retention after 100 cycles.     

Encapsulation of ferrocene and peripheral electrostatic attachment of viologens to dimeric molecular capsules formed by an octaacid, deep-cavity cavitand

In aqueous media the deep-cavity cavitand octaacid 1 forms stable dimeric molecular capsules 1(2), which are stabilized by hydrophobic effects. In this work we investigate the binding interactions in aqueous solution between these capsules and the redox active guests, ferrocene (Fc) and three 4,4'-bipyridinium (viologen) dications: methyl viologen (MV(2+)), ethyl viologen (EV(2+)), and butyl viologen (BV(2+)). Using NMR spectroscopic and electrochemical techniques we clearly show that the hydrophobic Fc guest is encapsulated inside 1(2). An interesting effect of this encapsulation is that the reversible voltammetric response of Fc is completely eliminated when it resides inside the 1(2) capsular assembly, a finding that is attributed to very slow electrochemical kinetics for the oxidation of Fc@1(2). Diffusion coefficient measurements (PGSE NMR spectroscopy) reveal that all three viologen guests are strongly bound to the dimeric capsules. However, the (1)H NMR spectroscopic data are not consistent with encapsulation and the measured diffusion coefficients indicate that two viologen guests can strongly associate with a single dimeric capsule. Furthermore, the (V(2+))(2)*1(2) complex is capable of encapsulating ferrocene, clearly suggesting that the viologen guests are bound externally, via coulombic interactions, to the anionic polar ends of the capsule. The electrochemical kinetic rate constants for the reduction of the viologen residue in the V(2+)*1(2) complexes were measured and found to be substantially lower than those for the free viologen guests.     

The effect of the concentration of platinum nanocolloid stabilizer on the rate of catalytic reactions in aqueous solution

The reduction of methyl viologen with hydrogen in the presence of NaOH and neptunium (VI) ions with formic acid in aqueous solutions catalyzed by platinum nanocolloids is studied. It is established that the rate of reaction with the participation of methyl viologen exponentially decreases with an increase in the concentration of a colloid stabilizer (sodium polyacrylate). The rate of neptunium reduction catalyzed by platinum nanoparticles of various sizes also decreases with an increase in the polyacrylate concentration to 4 × 10⁻³ M. A further increase in the content of stabilizer leads to some acceleration of the reaction. The mechanism of the effect of the stabilizer concentration on the catalytic activity of platinum nanocolloids is discussed. Original Russian Text ? B.G. Ershov, A.V. Anan’ev, N.L. Sukhov, 2006, published in Kolloidnyi Zhurnal, 2006, Vol. 68, No. 2, pp. 172–179.