Reduced Methyl Viologen Research Articles

Use of a Reverse Micelle System for Study of Oligomeric Structure of NAD+-Reducing Hydrogenase from Ralstonia eutropha H16

Inclusion of an oligomeric enzyme, NAD+-dependent hydrogenase from the hydrogen-oxidizing bacterium Ralstonia eutropha, into a system of reverse micelles of different sizes resulted in its dissociation into catalytically active heterodimers and subunits, which were characterized in reactions with various substrates. It was found that: 1) the native tetrameric form of this enzyme catalyzes all types of studied reactions; 2) hydrogenase dimer, HoxHY, is a minimal structural unit catalyzing hydrogenase reaction with an artificial electron donor, reduced methyl viologen; 3) all structural fragments containing FMN and NAD+/NADH-binding sites exhibit catalytic activity in diaphorase reactions with one- and two-electron acceptors; 4) small subunits, HoxY and HoxU also exhibit activity in diaphorase reactions with artificial acceptors. These results can be considered as indirect evidence that the second FMN molecule may be associated with one of the small subunits (HoxY or HoxU) of the hydrogenase from R. eutropha.

Kinetic analyses of the OJIP chlorophyll fluorescence rise in thylakoid membranes.

N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) was previously used to study the kinetics of the OJIP chlorophyll fluorescence rise. The present study is an attempt to elucidate the origin of TMPD-induced delay and quenching of the I-P step of fluorescence rise. For this purpose, we analyzed the kinetics of OJIP rise in thylakoid membranes in which electron transport was modified using ascorbate, methyl viologen (MV), and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB). In the absence of TMPD, the OJIP kinetics of fluorescence induction (FI) was not altered by ascorbate. However, ascorbate eliminated the I-P rise delay caused by high concentrations of TMPD. On the other hand, neither ascorbate nor DBMIB, which blocks the electron release from Photosystem II (PS II) at the cytochrome b6/f complex, could prevent the quenching of I-P rise by TMPD. In control thylakoids, MV suppressed the I-P rise of FI by about 60. This latter effect was completely removed if the electron donation to MV was blocked by DBMIB unless TMPD was present. When TMPD intercepted the linear electron flow from PS II, re-oxidation of TMPD by photosystem I (PS I) and reduction of MV fully abolished the I-P rise. The above is in agreement with the fact that TMPD can act as an electron acceptor for PS II. With MV, the active light-driven uptake of O2 during re-oxidation of TMPD by PS I contributes towards an early decline in the I-P step of the OJIP fluorescence rise.

Metal Nanoparticles on Polymer Surfaces: 5. Catalytic Activity of Colloidal Platinum Films Incorporated in Polystyrene Surface Layer

The fundamental possibility to enlarge Pt nanoparticles in monolayer ensembles formed on polystyrene surfaces by the adsorption from hydrosol in solution of isopropanol and K2PtCl4 is demonstrated for the first time. The enlargement of “seeding” nanoparticles is performed after their preliminary incorporation (partial embedding) into the polymer surface layer by the annealing of a system within the range between “surface” and “bulk” glass transition temperatures of polystyrene. It is shown that a colloidal film of metallic platinum with a thickness up to 200 nm is formed in the course of enlargement and it is mechanically fixed in the polymer surface layer. Such a system exhibits, over a long time, high catalytic activity in the model reaction of methyl viologen reduction with hydrogen.

Isolation of two hydrogenase activities in Chromatium.

Kinetic, chromatographic and electrophoretic studies of Chromatium hydrogenase show the existence in vitro of two different activities (I and II). The two hydrogenases exhibit different kinetic parameters and properties. Using reduced methyl viologen, Km and [S]0.5 values of about 20 microM and 360 microM were calculated for the hydrogenases I and II, respectively. Hill plots revealed that hydrogenase I followed classical hyperbolic (Michaelis-Menten) kinetics. However, a Hill coefficient (h = 0.68) indicating non-hyperbolic kinetics could be shown for hydrogenase II. After several purification steps hydrogenase II still showed kinetics typical of the action of either (a) two enzymes each of which shows Michaelis-Menten kinetics but with different substrate affinities or (b) only one enzyme which shows apparent negative cooperative regulation. The molecular weights of the hydrogenases were about 37,000 (I) and 55,000 (II) when determined by gel filtration. Sodium dodecyl sulphate/polyacrylamide gel electrophoresis revealed that both enzymes give a coincidental single protein band with the same relative mobility indicating a molecular weight of 31,000. Both hydrogenases were able to catalyse the reversible activation of H2 in the presence of artificial electron carriers but with different rates, hydrogenase II being much more active in the H2-uptake mode. The kinetic properties and molecular weight of hydrogenase II are partially modified by high ionic strength resulting in an increased substrate affinity and Hill coefficient and thus resembling hydrogenase I. These results are interpreted as due to the existence in vitro of monomeric and dimeric forms of Chromatium hydrogenase.

Catalytic reduction of methyl viologen by sulfide ion within MCM-41

Methyl viologen in the presence of mesoporous MCM-41 and sulfide ions is found to undergo a spontaneous catalytic reduction to methyl viologen radical (MV + ). Various control conditions, including passivation of the external surface and modification of the internal surface of MCM-41, suggest that the catalytic reduction occurs principally within the pores of MCM-41. The source of the sulfide ions can be either an added sulfide salt or coordinatively unsaturated surface sulfide ions of an occluded sulfide nanoparticle. The present system offers a means for the spontaneous formation of methyl viologen radicals, which might serve as a complementary measurement for electron microscopy probing of the location of sulfide nanoparticles synthesized within the pores of mesoporous materials.

NapF Is a Cytoplasmic Iron-Sulfur Protein Required for Fe-S Cluster Assembly in the Periplasmic Nitrate Reductase

The periplasmic nitrate reductase (Nap) is wide-spread in proteobacteria. NapA, the nitrate reductase catalytic subunit, contains a Mo-bisMGD cofactor and one [4Fe-4S] cluster. The nap gene clusters in many bacteria, including Rhodobacter sphaeroides DSM158, contain an napF gene, disruption of which drastically decreases both in vitro and in vivo nitrate reductase activities. In spite its importance in the Nap system, NapF has never been characterized biochemically, and its role remains unknown. The NapF protein has four polycysteine clusters that suggest that it is an iron-sulfur-containing protein. In the present study, a His(6)-tagged NapF protein was overproduced in Escherichia coli and purified anaerobically. The purified NapF protein was used to obtain polyclonal antibodies raised in rabbit, and cellular fractionation of R. sphaeroides followed by immunoprobing with anti-NapF antibodies revealed that the native NapF protein is located in the cytoplasm. This contrasts with the periplasmic location of the mature NapA. However, NapA could not be detected in an isogenic napF(-) strain of R. sphaeroides. The His(6)-tagged NapF protein displayed spectral properties indicative of Fe-S clusters, but these features were rapidly lost, suggesting cluster lability. However, reconstitution of the Fe-S centers into the apo-NapF protein was achieved in the presence of Azotobacter vinelandii cysteine desulfurase (NifS), and this allowed the recovery of nitrate reductase activity in NapA protein that had previously been treated with 2,2'-dipyridyl to remove the [4Fe-4S] cluster. This activity was not recovered in the absence of NapF. Taking into account the cytoplasmic localization of NapF, the presence of labile Fe-S clusters in the protein, the napF(-) strain phenotype, and the NapF-dependent reactivation of the 2,2'-dipyridyl-treated NapA, we propose a role for NapF in assembling the [4Fe-4S] center of the catalytic subunit NapA.

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.

Optical and catalytic properties of Ag2S nanoparticles

Abstract Optical properties of polymer-stabilized aqueous colloids of silver sulfide were studied. Nature and energies of optical transitions responsible for the absorption of visible light by silver sulfide nanoparticles were determined. Catalytic properties of Ag 2 S nanoparticles in methylviologen reduction by sodium sulfide were examined. It was shown that this reaction is reversible, the equilibrium caused by a reverse reaction between the product of direct catalytic process – sulfur (polysulfide anions) and cation-radical of methylviologen. It was found that Ag 2 S nanoparticles can catalyze reduction of Ag + ions by various reductants, in particular, hydroquinone or sodium sulfite, with the formation of silver particles having characteristic plasmon absorption bands in the visible spectral domain. Effect of the conditions of Ag + catalytic reduction on the shape and intensity of plasmon resonance bands of metallic silver were studied, schematic mechanism of this reaction was proposed. It was shown that at the irradiation of solutions containing Ag 2 S nanoparticles, Ag + cations and a reducing agent with the light corresponding to the fundamental absorption band of nanoparticulate silver sulfide, the rate of Ag + catalytic reduction increased whereas kinetic features of catalytic reaction and characteristics of plasmon resonance absorption bands of silver nanoparticles changed. A mechanism of photochemical activation of the catalytic reduction of silver ions with the participation of Ag 2 S nanoparticles was proposed.

Radiolytic formation of Ag clusters in aqueous polyvinyl alcohol solution and hydrogel matrix

Ag+ ions, in aqueous polyvinyl alcohol (PVA) solution and in PVA hydrogel matrix have been gamma radiolytically reduced to produce Ag clusters. UV-visible absorption spectral characteristics of Ag clusters obtained under different gamma dose, Ag+ concentration, PVA concentration and crosslinking density of the gel used have been studied. The effect of Ag+ ions on the radiation crosslinking of the PVA chains, have also been investigated by viscosity measurements. The radiation-induced Ag+ ion reduction was followed by crosslinking of the PVA chains. PVA was found to be a very efficient stabilizer to prevent aggregation of Ag clusters. The clusters produced in the hydrogel matrix were expected to be smaller than the pore size (∼2–20nm) of the gels used in the study. These Ag clusters were unable to reduce methyl viologen (MV2+) chloride and were stable in air.

Purification and biochemical characterization of simplified eukaryotic nitrate reductase expressed in Pichia pastoris

NAD(P)H:nitrate reductase (NaR, EC 1.7.1.1-3) is a useful enzyme in biotechnological applications, but it is very complex in structure and contains three cofactors—flavin adenine dinucleotide, heme–Fe, and molybdenum–molybdopterin (Mo–MPT). A simplified nitrate reductase (S-NaR1) consisting of Mo–MPT-binding site and nitrate-reducing active site was engineered from yeast Pichia angusta NaR cDNA (YNaR1). S-NaR1 was cytosolically expressed in high-density fermenter culture of methylotrophic yeast Pichia pastoris. Total amount of S-NaR1 protein produced was ∼0.5 g per 10 L fermenter run, and methanol phase productivity was 5 μg protein/g wet cell weight/h. Gene copy number in genomic DNA of different clones showed direct correlation with the expression level. S-NaR1 was purified to homogeneity in one step by immobilized metal affinity chromatography (IMAC) and total amount of purified protein per run of fermentation was ∼180 mg. Polypeptide size was ∼55 kDa from electrophoretic analysis, and S-NaR1 was mainly homo-tetrameric in its active form, as shown by gel filtration. S-NaR1 accepted electrons efficiently from reduced bromphenol blue ( k cat=2081 s −1) and less so from reduced methyl viologen ( k cat=159 s −1). The nitrate K M for S-NaR1 was 30 ± 3 μM, which is very similar to YNaR1. S-NaR1 is capable of specific nitrate reduction, and direct electric current, as shown by catalytic nitrate reduction using protein film cyclic voltammetry, can drive this reaction. Thus, S-NaR1 is an ideal form of this enzyme for commercial applications, such as an enzymatic nitrate biosensor formulated with S-NaR1 interfaced to an electrode system.

Spectral, Optical, and Photocatalytic Characteristics of Quantum-Sized Particles of CdTe

Methods were developed for the synthesis of quantum-sized particles of CdTe, stabilized in aqueous solutions of thioglycolic acid, using H2Te or NaHTe produced in situ by electrochemical reduction of tellurium. The spectral and optical characteristics of the synthesized CdTe nanoparticles were studied. It was found that they have photocatalytic activity in the reduction of methylviologen with sodium sulfite.

Silver clusters in 2-propanol: a radiation chemical study

Pulse radiolysis technique has been used to study the transient absorption spectra of Ag o, Ag 2 +• and lower and higher oligomers of silver in N 2-purged 2-propanol solution of AgClO 4. The rate constant for the reaction of e sol − with Ag + was determined from the decay of solvated electron and was found to be 1.7±0.3×10 10 dm 3 mol −1 s −1. As in the case of methanol, silver atom and its lower oligomers reduce methyl viologen (MV 2+) to MV +•. The higher oligomers were able to produce larger clusters of silver after their reduction by methyl viologen radicals (MV +•). The larger clusters produced in the presence of MV 2+ have an absorption maximum at 410 nm which is red-shifted as compared to that observed in the absence of MV 2+. The coalescence rate was observed to be faster in the presence of MV 2+. As compared to water and methanol systems, the yield of Ag 2 +• was higher in 2-propanol system. In this system, Ag 2 +• could also be produced via the reaction of silver ions with the reducing radical produced from 2-propanol, viz. (CH 3) 2C •OH radical besides the reaction of Ag o with Ag +. γ-radiolysis experiments support the above observations.

Lactic acid production with lactate dehydrogenase using the visible light sensitization of zinc porphyrin.

Lactic acid production with L-lactate dehydrogenase from Pig heart (LDH) and reduced methyl viologen produced by the visible light photosensitization of zinc tetrakis(4-methylpyridyl)porphyrin (ZnTMPyP) was investigated. When the sample solution containing triethanolamine as an electron-donating reagent, ZnTMPyP, methyl viologen as an electron carrier, pyruvic acid and LDH in potassium phosphate buffer (pH 7) was irradiated, lactic acid production was observed. After 240 min irradiation, the amount of lactic acid production and the yield of pyruvic acid to lactic acid were estimated to be 0.17 mmol dm(-3) and 17.0%, respectively.

Yields and Migration Distances of Reducing Equivalents in the Radiolysis of Silica Nanoparticles

The reduction of methyl viologen ions, MV2+, adsorbed on SiO2 nanoparticles in N2O saturated colloidal suspensions has been studied using the pulse radiolysis technique. Reduced methyl viologen, MV+, was produced within the electron pulse, followed by slower reactions of MV2+ with OH and H radicals. The fast formation of MV+ was taken as evidence that electrons produced in the initial ionization events within the SiO2 particles rapidly migrate to the surface and reduce the adsorbed molecules; however, yields of MV+ as high as 6.5 radicals per 100 eV were measured, significantly exceeding common yields for ionization of less than 4.5 per 100 eV. The high yields suggest that an additional source for reduction equivalents is generated in the silica and is scavenged by the adsorbed MV2+ acceptor. Monte Carlo track structure calculations point to radiation-induced excitons as the scavengeable reducing species. The yield of MV+ radicals decreases linearly with increasing particle size at a given surface coverage by MV2+. This decrease in yield was attributed to a competition between electron−hole recombination within the silica bulk and scavenging of electrons at the interface. The limiting size at which no reducing equivalents arrive at the aqueous side of the interface was estimated to be d ∼ 30 nm.

Effect of Zn on acetyl coenzyme a synthase: evidence for a conformational change in the alpha subunit during catalysis.

Acetyl coenzyme A synthase (ACS) is an alpha2beta2 tetramer in which the active-site A-cluster, located in the alpha subunits, consists of an Fe4S4 cubane bridged to a {Nip Nid} binuclear site. The alpha subunits exist in two conformations. In the open conformation, Nip is surface-exposed, while the proximal metal is buried in the closed conformation. Nip is labile and can be replaced by Cu. In this study, the effects of Zn are reported. ACS in which Zn replaced Nip was inactive and did not exhibit the so-called NiFeC EPR signal nor the ability to accept a methyl group from the corrinoid-iron-sulfur protein (CoFeSP). Once Zn-bound, it could not be replaced by subsequently adding Ni. The Zn-bound A-cluster cannot be reduced and bound with CO or become methylated, probably because Zn (like Cu) is insufficiently nucleophilic for these functions. Unexpectedly, Zn replaced Nip only while ACS was engaged in catalysis. Under these conditions, replacement occurred with kapp approximately 0.6 min-1. Replacement was blocked by including EDTA in the assay mix. Zn appears to replace Nip when ACS is in an intermediate state (or states) of catalysis but this(these) state(s) must not be present when ACS is reduced in CO alone, or in the presence of CoA, CoFeSP, or reduced methyl viologen. Nip appears susceptible to Zn-attack when the alpha subunit is in the open conformation and protected from attack when it is in the closed conformation. This is the first evidence that the structurally-characterized conformations of the alpha subunit change during catalysis, indicating a mechanistic role for this conformational change.

Reductively activated nitrous oxide reductase reacts directly with substrate.

In the terminal step of bacterial denitrification, N2O is converted to N2 at the mu4-sulfide bridged tetranuclear CuZ center of nitrous oxide reductase. The enzyme can be activated by reduced methyl viologen, with up to a 15-fold increase in specific activity. The reductively activated nitrous oxide reductase from Achromobacter cycloclastes was isolated and characterized by visible absorption and EPR spectroscopy, and both methods showed that the CuZ center can attain a [4Cu(I)] oxidation state. When N2O was added to the activated, reductant-free enzyme, distinct spectral changes were observed, indicating that this state of the enzyme interacts with substrate. This was further supported by the detection of 15N-labeled product in the absence of steady-state turnover conditions. A new absorption band around 970 nm appeared following reaction of activated nitrous oxide reductase with N2O, which may represent a catalytic intermediate state of the enzyme.

Properties of stable hydrogenase from the purple sulfur bacterium Lamprobacter modestohalophilus.

Some properties of a hydrogenase from the recently isolated phototrophic sulfur bacterium Lamprobacter modestohalophilus strain Syvash and its resistance to a number of inactivating factors have been investigated. The enzyme consists of two subunits, 64 and 30 kD; pI = 4.5. The optimal pH was 8.5-9.5 for hydrogen uptake and 4.0 for H2 evolution. Hydrogenase preparations were resistant to the effects of O2, CO, and temperature, revealing high stability under storage. A considerable inactivation of the enzyme was observed at temperatures above 80 degrees C; the temperature optimum of methyl viologen reduction by H2 was 85 degrees C. Inhibitory effects of Ni2+, Cd2+, and Mg2+ on the hydrogenase activity were shown to be reversible and competitive with respect to methyl viologen in the hydrogen oxidation reaction.

Complex formation between ferredoxin and Synechococcus ferredoxin:nitrate oxidoreductase

The ferredoxin-dependent nitrate reductase from the cyanobacterium Synechococcus sp. PCC 7942 has been shown to form a high-affinity complex with ferredoxin at low ionic strength. This complex, detected by changes in both the absorbance and circular dichroism (CD) spectra, did not form at high ionic strength. When reduced ferredoxin served as the electron donor for the reduction of nitrate to nitrite, the activity of the enzyme declined markedly as the ionic strength increased. In contrast, the activity of the enzyme with reduced methyl viologen (a non-physiological electron donor) was independent of ionic strength. These results suggest that an electrostatically stabilized complex between Synechococcus nitrate reductase and ferredoxin plays an important role in the mechanism of nitrate reduction catalyzed by this enzyme. Treatment of Synechococcus nitrate reductase with either an arginine-modifying reagent or a lysine-modifying reagent inhibited the ferredoxin-dependent activity of the enzyme but did not affect the methyl viologen-dependent activity. Treatment with these reagents also resulted in a large decrease in the affinity of the enzyme for ferredoxin. Formation of a nitrate reductase complex with ferredoxin prior to treatment with either reagent protected the enzyme against loss of ferredoxin-dependent activity. These results suggest that lysine and arginine residues are present at the ferredoxin-binding site of Synechococcus nitrate reductase. Results of experiments using site-specific, charge reversal variants of the ferredoxin from the cyanobacterium Anabaena sp. PCC 7119 as an electron donor to nitrate reductase were consistent with a role for negatively charged residues on ferredoxin in the interaction with Synechococcus nitrate reductase.

Photoinduced Reduction of Methylviologen with TiO2/Polymer Films

Abstract TiO2/polymer films on quartz substrate were fabricated by layer-by-layer method and the ultrathin films showed photocatalytic activity for reduction of 1,1′-dimethyl-4,4′-bipyridinium dichloride on the irradiation of UV light.

Monomeric NarB is a dual-affinity nitrate reductase, and its activity is regulated differently from that of nitrate uptake in the unicellular diazotrophic cyanobacterium Synechococcus sp. strain RF-1.

Synechococcus sp. strain RF-1 is a unicellular freshwater cyanobacterium that fixes N(2) aerobically and exhibits a circadian rhythm for nitrogenase activity under a light-dark regimen. Synechococcus sp. strain RF-1 also utilizes nitrate, nitrite, or ammonium for growth. Under the diazotrophic growth, the nitrate uptake in Synechococcus sp. strain RF-1 was induced by nitrate or nitrite but repressed by ammonium. In contrast, a prominent nitrate reductase (NR) activity was detected in diazotrophically grown cells using the reduced methyl viologen assay. The NR activity was not inhibited by ammonium and only slightly enhanced by nitrate. The different expression patterns of nitrate uptake and NR in Synechococcus sp. strain RF-1 were reflected in general at the transcript level determined by reverse transcriptase PCR. Under both nitrate-induced and uninduced conditions, the in situ NR activity exhibited similar biphasic kinetics for nitrate. The recombinant NR encoded by the narB gene of Synechococcus sp. strain RF-1, expressed in E. coli, also showed the biphasic kinetics with similar pH and temperature profiles. By in-gel NR activity assay, the recombinant NarB was found to exist as a single form. Both the high- and low-affinity NR activities of the recombinant NarB showed the same thermostability. When modified at the N terminus by a polyhistidine tag, the recombinant NR activity was shifted from biphasic to hyperbolic kinetics and showed only a single K(m) for nitrate, indicating the functional importance of the NarB N-terminal structure in NR kinetics.