Mediator Methyl Viologen Research Articles

Photocatalytic Removal of the Greenhouse Gas Nitrous Oxide by Liposomal Microreactors.

Nitrous oxide (N2 O) is a potent greenhouse and ozone-reactive gas for which emissions are growing rapidly due to increasingly intensive agriculture. Synthetic catalysts for N2 O decomposition typically contain precious metals and/or operate at elevated temperatures driving a desire for more sustainable alternatives. Here we demonstrate self-assembly of liposomal microreactors enabling catalytic reduction of N2 O to the climate neutral product N2 . Photoexcitation of graphitic N-doped carbon dots delivers electrons to encapsulated N2 O Reductase enzymes via a lipid-soluble biomolecular wire provided by the MtrCAB protein complex. Within the microreactor, electron transfer from MtrCAB to N2 O Reductase is facilitated by the general redox mediator methyl viologen. The liposomal microreactors use only earth-abundant elements to catalyze N2 O removal in ambient, aqueous conditions.

Photocatalytic Removal of the Greenhouse Gas Nitrous Oxide by Liposomal Microreactors.

Nitrous oxide (N2O) is a potent greenhouse and ozone-reactive gas for which emissions are growing rapidly due to increasingly intensive agriculture. Synthetic catalysts for N2O decomposition typically contain precious metals and/or operate at elevated temperatures driving a desire for more sustainable alternatives. Here we demonstrate self-assembly of liposomal microreactors enabling catalytic reduction of N2O to the climate neutral product N2. Photoexcitation of graphitic N-doped carbon dots delivers electrons to encapsulated N2O Reductase enzymes via a lipid-soluble biomolecular wire provided by the MtrCAB protein complex. Within the microreactor, electron transfer from MtrCAB to N2O Reductase is facilitated by the general redox mediator methyl viologen. The liposomal microreactors use only earth-abundant elements to catalyze N2O removal in ambient, aqueous conditions.

Enzyme Electrode Biosensors for N-Hydroxylated Prodrugs Incorporating the Mitochondrial Amidoxime Reducing Component.

Human mitochondrial amidoxime reducing component 1 and 2 (mARC1 and mARC2) were immobilised on glassy carbon electrodes using the crosslinker glutaraldehyde. Voltammetry was performed in the presence of the artificial electron transfer mediator methyl viologen, whose redox potential lies negative of the enzymes' MoVI/V and MoV/IV redox potentials which were determined from optical spectroelectrochemical and EPR measurements. Apparent Michaelis constants obtained from catalytic limiting currents at various substrate concentrations were comparable to those previously reported in the literature from enzymatic assays. Kinetic parameters for benzamidoxime reduction were determined from cyclic voltammograms simulated using Digisim. pH dependence and stability of the enzyme electrode with time were also determined from limiting catalytic currents in saturating concentrations of benzamidoxime. The same electrode remained active after at least 9 days. Fabrication of this versatile and cost-effective biosensor is effective in screening new pharmaceutically important substrates and mARC inhibitors.

Catalytic electrochemistry of the bacterial Molybdoenzyme YcbX

Molybdenum-dependent enzymes that can reduce N-hydroxylated substrates (e.g. N-hydroxyl-purines, amidoximes) are found in bacteria, plants and vertebrates. They are involved in the conversion of a wide range of N-hydroxylated organic compounds into their corresponding amines, and utilize various redox proteins (cytochrome b5, cyt b5 reductase, flavin reductase) to deliver reducing equivalents to the catalytic centre. Here we present catalytic electrochemistry of the bacterial enzyme YcbX from Escherichia coli utilizing the synthetic electron transfer mediator methyl viologen (MV2+). The electrochemically reduced form (MV+.) acts as an effective electron donor for YcbX. To immobilize YcbX on a glassy carbon electrode, a facile protein crosslinking approach was used with the crosslinker glutaraldehyde (GTA). The YcbX-modified electrode showed a catalytic response for the reduction of a broad range of N-hydroxylated substrates. The catalytic activity of YcbX was examined at different pH values exhibiting an optimum at pH 7.5 and a bell-shaped pH profile with deactivation through deprotonation (pKa1 9.1) or protonation (pKa2 6.1). Electrochemical simulation was employed to obtain new biochemical data for YcbX, in its reaction with methyl viologen and the organic substrates 6-N-hydroxylaminopurine (6-HAP) and benzamidoxime (BA).

Encapsulated titanium dioxide nanoparticle-Escherichia coli hybrid system improves light driven hydrogen production under aerobic condition

Utilizing light energy for hydrogen production by combining nano-bio inspired photosynthetic system has received remarkable attention in renewable energy production. In this study, we applied a sodium alginate encapsulation strategy to our previously developed nano-bio hybrid system for photocatalytic hydrogen production under aerobic condition by combining the inorganic semiconductor titanium dioxide (TiO2), electron mediator methylviologen (MV2+), along with E. coli (hydrogenases) in an encapsulated system under the given light intensity of 2000 W m−2 and its hydrogen production efficiency was studied. Under aerobic condition the encapsulated hybrid system produced hydrogen (2.4 mmol H2/ mmol glucose) 3-fold higher than the unencapsulated hybrid system (0.8 mmol H2/ mmol glucose), suggesting that encapsulation is essential to protect oxygen sensitive hydrogenase under aerobic condition. The encapsulated hybrid system was also feasible under direct sunlight for hydrogen production. Overall, this study could serve as a new strategy for biological hydrogen production under aerobic condition.

Dinitrogen Reduction: Interfacing the Enzyme Nitrogenase with Electrodes.

Potential for nitrogenase: Milton, Minteer, and co-workers report the first evidence for the bioelectrochemical reduction of N2 to ammonia by nitrogenase. This complex enzyme could be wired to an electrode by using the soluble mediator methyl viologen; this very simple approach makes it possible to develop a variety of biotechnological devices.

Temperature dependence of an abiotic glucose/air alkaline fuel cell

The temperature dependence of a previously developed glucose fuel cell is explored. This cell uses a small molecule dye mediator to transport oxidizable electrons from glucose to a carbon felt anode. This reaction is driven by an air breathing MnO2 cathode. This research investigates how the temperature of the system affects the power production of the fuel cell. Cell performance is observed using either methyl viologen, indigo carmine, trypan blue, or hydroquinone as a mediator at temperatures of 15, 19, 27, 32, 37, 42, and 49 °C. Cyclic voltammetry of the cell anode at the given temperatures with the individual dyes is also presented. The highest power production amongst all of the cells occurs at 32 °C. This occurs with the mediator indigo carmine or with the mediator methyl viologen. These sustained powers are 2.31 mW cm−2 and 2.39 mW cm−2, respectively. This is approximately a 350% increase for these cells compared to their power produced at room temperature. This dramatic increase is likely due to increased solubility of the mediator dye at higher temperatures.

Alternative Hydride Sources for Ene‐Reductases: Current Trends

Asymmetric hydrogenations are important reactions in industrial synthesis, as up to two stereogenic centres can be generated. Given the current trend towards more “green” or sustainable chemistry, biocatalytic approaches are becoming more important in the production of fine chemicals, pharmaceuticals, and agrochemical products. There are at least four known classes of enzymes that have been explored for their biocatalytic applicability, collectively known as ene-reductases (ERs). Each enzyme requires NAD(P)H as the hydride donor, whereas the mechanism, substrate scope and product stereoand/or enantiospecificity differs between biocatalyst classes. The most widely investigated class is the old yellow enzyme (OYE; EC 1.6.99.1) family and many recent reviews have summarised the (potential) applications of these enzymes for industrial syntheses, for example, Refs. [1–3] . These flavin mononucleotide (FMN)-containing enzymes catalyse the C=C reduction of a wide variety of a,b-unsaturated aldehydes, ketones, nitroalkenes, maleimides, dicarboxylic acids and their esters, and nitriles. A second less well-characterised enzyme class catalysing biocatalytic reductions are enoate reductases (EC 1.3.1.31). These oxygen-sensitive enzymes contain both FAD and [4Fe-4S], and are members of the NADH:flavin oxidoreductase/NADH oxidase family. They have been shown to catalyse the reduction of a variety of a,b-unsaturated monoacids and esters. 5] A more recent class of enzymes investigated are the medium-chain dehydrogenase/reductase (MDR) family of oxidoreductases (1.3.1.-). For example, the flavin-independent double-bond reductase from Nicotiana tabacum (NtDBR) catalyses the reduction of a wide variety of a,b-unsaturated aldehydes, ketones and nitroalkenes. There are a few examples in the literature of potential applications of a fourth class of enzymes, namely the flavin-independent short-chain dehydrogenase/reductases (SDR) from plants. Examples include two menthol dehydrogenases, ( )-menthol dehydrogenase (EC 1.1.1.207) and (+)-neomenthol dehydrogenase (EC 1.1.1.208), which catalyse the reduction of specific menthone isomers to menthol. Unfortunately, the high cost of NAD(P)H nicotinamide coenzymes makes them uneconomical for industrial-scale syntheses. Therefore, alternative methodologies have been adopted to supply the necessary hydride equivalents for alkene reduction. One such method is to include a coenzyme recycling system, in which catalytic levels of NAD(P) are constantly regenerated to NAD(P)H. Several systems have been developed and routinely employed for ER biocatalytic reactions, such as glucose dehydrogenase, glucose-6-phosphate dehydrogenase (G6PDH/glucose-6-phosphate), formate dehydrogenase and phosphite dehydrogenase, and reviewed elsewhere. In each case, only catalytic levels of NAD(P) are needed but stoichiometric levels of a co-substrate are required to drive the recycling enzyme. Faber and coworkers have performed comparative biotransformations by using multiple cofactor recycling systems, substrates and ERs, to determine the ideal hydride source. Product yield and/or enantioselectivity can vary considerably, depending on which coenzyme recycling system has been employed. Additionally, the inclusion of a second enzyme system into large-scale (bio)synthesis may present problems, for example in maintenance of the activity and stability of two enzymes and the high cost of some co-substrates. Electrochemical regeneration of nicotinamide coenzymes has also been investigated, for example in the direct cathodic reduction of NAD(P) . Unfortunately, this simple regeneration method is hampered by low selectivity and the formation of undesired NAD(P) dimeric side products. Several techniques have been developed to bypass the need for nicotinamide coenzymes by using alternative hydride donors to reduce the flavin cofactor of some ERs. Reetz and coworkers described a method whereby the FMN cofactor of the OYE homologue YqjM from Bacillus subtilis was photoreduced, employing free flavin and a sacrificial electron donor. The YqjM bound oxidised FMN was reduced by the free photoreduced FMN, thereby generating the active enzyme. This NAD(P)H-free system was successful in converting ketoisophorone to (R)-levodione, albeit with lower product enantiopurity. Similarly, Hollmann and co-workers described the photoenzymatic flavin reduction of YqjM and NEMR (N-ethylmalemide reductase from E. coli) by using alternative sacrificial electron donors formate and phosphite. These methods required strict anaerobic conditions to prevent the rapid reoxidation of reduced FMN by molecular oxygen. Early attempts at NAD(P)H-independent alkene reduction focussed on the use of artificial mediators, such as N,N-dimethyl4-4-bipyridinium [methyl viologen (MV)] , as a means of reducing the flavin in clostridial enoate reductases. A variety of reduced mediator recycling systems were employed, including direct electron transfer from a cathode and enzymatic-based recycling systems. Successful enzyme-coupled mediator recycling was achieved used the following systems: 1) hydroge[a] Dr. H. S. Toogood, Dr. T. Knaus, Prof. N. S. Scrutton Manchester Institute of Biotechnology, Faculty of Life Sciences University of Manchester 131 Princess Street, Manchester M1 7DN (UK) Fax: (+44)01613068918 E-mail : nigel.scrutton@manchester.ac.uk

Synthesis and Structural Characterization of Some New Porphyrin-Fullerene Dyads and Their Application in Photoinduced H2 Evolution

The [3 + 2] cycloaddition reaction of C(60) with ethyl isonicotinoylacetate in the presence of piperidine in PhCl at room temperature or in the presence of Mn(OAc)(3) in refluxing PhCl gave the pyridyl-containing dihydrofuran-fused C(60) derivative (4-C(5)H(4)N)C(O)═C(C(60))CO(2)Et (1), whereas the phenyl-containing C(60) derivative PhC(O)═C(C(60))CO(2)Et (2) was similarly prepared by [3 + 2] cycloaddition reaction of C(60) with ethyl benzoylacetate in the presence of piperidine or Mn(OAc)(3). More interestingly, one of the new porphyrin-fullerene dyads, i.e., [4-C(5)H(4)NC(O)═C(C(60))CO(2)Et]·ZnTPPH (3, ZnTPPH = tetraphenylporphyrinozinc), could be prepared by coordination reaction of the pyridyl-containing C(60) derivative 1 with equimolar ZnTPPH in CS(2)/hexane at room temperature. In addition, the β-keto ester-substituted porphyrin derivative H(2)TPPC(O)CH(2)CO(2)Et (4) was prepared by a sequential reaction of HO(2)CCH(2)CO(2)Et with n-BuLi in 1:2 molar ratio followed by treatment with H(2)TPPC(O)Cl in the presence of Et(3)N and then hydrolysis with diluted HCl, whereas the porphyrinozinc derivative ZnTPPC(O)CH(2)CO(2)Et (5) could be prepared by coordination reaction of 4 with Zn(OAc)(2) in refluxing CHCl(3)/MeOH. Particularly interesting is that the second new porphyrin-fullerene dyad H(2)TPPC(O)═C(C(60))CO(2)Et (6) could be prepared by [3 + 2] cycloaddition reaction of 4 with C(60) in the presence of piperidine in PhCl at room temperature. In addition, treatment of 6 with Zn(OAc)(2) in refluxing CHCl(3)/MeOH afforded the third new dyad ZnTPPC(O)═C(C(60))CO(2)Et (7). All the new compounds 1-7 were characterized by elemental analysis and various spectroscopic methods and particularly for 2, 3, and 5 by X-ray crystallography. The five-component system consisting of an electron donor EDTA, dyad 3, an electron mediator methylviologen (MV(2+)), the catalyst colloidal Pt, and a proton source HOAc was proved to be effective for photoinduced H(2) evolution. A possible pathway for such a type of H(2) evolution was proposed.

Immobilization of the [FeFe]-hydrogenase CrHydA1 on a gold electrode: Design of a catalytic surface for the production of molecular hydrogen

Hydrogenase-modified electrodes are a promising catalytic surface for the electrolysis of water with an overpotential close to zero. The [FeFe]-hydrogenase CrHydA1 from the photosynthetic green alga Chlamydomonas reinhardtii is the smallest [FeFe]-hydrogenase known and exhibits an extraordinary high hydrogen evolution activity. For the first time, we immobilized CrHydA1 on a gold surface which was modified by different carboxy-terminated self-assembled monolayers. The immobilization was in situ monitored by surface-enhanced infrared spectroscopy. In the presence of the electron mediator methyl viologen the electron transfer from the electrode to the hydrogenase was detected by cyclic voltammetry. The hydrogen evolution potential (−290 mV vs NHE, pH 6.8) of this protein modified electrode is close to the value for bare platinum (−270 mV vs NHE). The surface coverage by CrHydA1 was determined to 2.25 ng mm −2 by surface plasmon resonance, which is consistent with the formation of a protein monolayer. Hydrogen evolution was quantified by gas chromatography and the specific hydrogen evolution activity of surface-bound CrHydA1 was calculated to 1.3 μmol H 2 min −1 mg −1 (or 85 mol H 2 min −1 mol −1). In conclusion, a viable hydrogen-evolving surface was developed that may be employed in combination with immobilized photosystems to provide a platform for hydrogen production from water and solar energy with enzymes as catalysts.

In vitro hydrogen photoproduction using Pyrococcus furiosus sulfhydrogenase and TiO 2

The [NiFe] sulfhydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus has been tested as a biological catalyst in a light-driven in vitro hydrogen evolution system, using a solar simulator apparatus as a source of light. The enzyme is active when coupled to photo-induced titanium dioxide (TiO 2) dispersed as submicronic powder in a buffer solution and in the presence of the artificial redox mediator methylviologen (MV), carrying electrons from the TiO 2 particle to the enzyme. To optimize the system towards practical applications, a simplified version which works in the absence of MV was set up and its performance was assayed with different reductants, as a function of time and over a wide range of temperatures. The comparison of MV-mediated and not-mediated conditions in terms of rate and amount of H 2 photoproduced, highlights a lower efficiency, but an improved overall stability of the system when a direct electron transfer from the TiO 2 to the enzyme takes place. As a consequence, the reaction lifetime is productively extended counteracting the effect of the low H 2 evolution rate with regard to the total amount of H 2 photoproduced. In particular, at 60°C the reaction lifetime is prolonged to 8 h, thus resulting in significantly higher amount of H 2 evolved. The flexibility of the P. furiosus sulfhydrogenase/TiO 2 in vitro system with respect to the redox mediator MV and the evidence that the enzyme performance can be maintained over a wide range of temperatures (60–80°C) are promising advances in the identification of the optimal conditions for the development of an economically feasible hydrogen production process exploiting solar light as a source of energy and waste compounds as electron donors.

Oxygen production and consumption by chloroplasts in situ and in vitro as studied with microscopic spin label probes

A new spin-label oximetry approach able to measure the oxygen partial pressure in complex photosynthetic systems has been developed using bovine serum albumin (BSA)-coated light paraffin oil particles containing cholestane spin label (CSL). Paraffin oil particles protect the spin label against the action of chemically active metabolites. The amplitude of the electron paramagnetic resonance (EPR) signal from CSL measured at a saturating microwave power is sensitive to the concentration of oxygen. We demonstrate here the ability of this method to monitor the kinetics of light-induced oxygen production in situ, i.e., in the interior of a bean leaf. The oxygen release, observed during leaf illumination with continuous light, exhibits an overshoot that correlates with the well-known nonmonotonous behavior of the Photosystem I reaction center, P700. Short-term illumination of isolated bean chloroplasts, suspended in the presence of the electron mediator methylviologen, induces a reversible uptake of oxygen. However, after prolonged illumination, chloroplasts lose their ability to regenerate oxygen in the dark. The exhaustion of oxygen (and oxygen active forms) is accompanied by the loss of CSL paramagnetism and the capacity to photooxidize P700. Comparison of the kinetics of P700 redox transients with oximetric data demonstrates that oxygen concentration is the essential factor controlling electron transport in leaves and isolated chloroplasts.

Reductive dehalogenation as a respiratory process.

Anaerobic bacteria can reductively dehalogenate aliphatic and aromatic halogenated compounds in a respiratory process. Only a few of these bacteria have been isolated in pure cultures. However, long acclimation periods, substrate specificity, high dehalogenation rates, and the possibility to enrich for the dehalogenation activity by subcultivation in media containing an electron donor indicate that many of the reductive dehalogenations in the environment are catalyzed by specific bacteria. Molecular hydrogen or formate appear to be good electron donors for the enrichment of such organisms. Furthermore, systems have to be employed which supply the cultures with the halogenated compounds beyond their toxicity level. All bacteria that are presently available in pure culture and grow with a halogenated compound as electron acceptor are members of new genera. Based on experimental results with the membrane-impermeable electron mediator methyl viologen, a model of the respiration system of Dehalobacter restrictus, a tetrachloroethene-dechlorinating bacterium, is presented. Further studies of the biochemistry and energetics of respiratory-dehalogenating strains will help to understand the mechanisms involved and perhaps reveal the evolutionary origin of the dehalogenating enzyme systems.

LIGHT‐DRIVEN H2 PRODUCTION WITH PROFLAVIN AND HYDROGENASE: COMPARISON OF CYTOCHROME C3 AND METHYL VIOLOGEN AS e‐ MEDIATORS

AbstractWe have studied the hydrogenase‐catalyzed production of H, when either its natural electron mediator cytochrome c3 (c3) or the artificial mediator methyl viologen (MV) was reduced by illumination of proflavin (PF) in the presence of ethylenediaminetetraacetate (EDTA). The reduction rates of MV and c3 were comparable at equivalent concentrations of PF, taking into account the four redox sites of c3. However, when the concentration of c3 exceeded that of PF, the reduction rate decreased. We explain this by light quenching. In the H2‐producing system, MV was more efficient than c3 as electron mediator when the initial reaction rates were compared. However, under certain conditions with MV, the rate of H2 production decreased rapidly with time of illumination, whereas with c3 it consistently remained stable. Possible reasons for this difference are discussed.Severe inactivation of hydrogenase was observed in the absence of the primary electron donor EDTA. It is concluded that this inactivation is caused by the excited state of PF

Characterization and purification of a hydrogenase from the eukaryotic green alga Scenedesmus obliquus

Several catalytic properties of the hydrogenase from Scenedesmus obliquus have been examined to optimize the purification conditions. The K m -value for H 2-evolution in the presence of the most effective electron mediator methylviologen is 0.66 mM. The pH-optimum is 6.3, the temperature-optimum is 50°C and the energy of activation is 38.4 ± 2 kJ·mol −1. The soluble hydrogenase from the green alga, Scenedesmus obliquus, was purified 1290-fold to homogeneity. The enzyme consists of two subunits with molecular masses of 55 kDa and 36 kDa. The molecular weight of the native enzyme, determined by gel filtration, is 150 ± 5 kDa.

Hydrogen peroxide photoproduction by immobilized cells of the blue-green alga Anabaena variabilis: A way to solar energy conversion

A photosystem for hydrogen peroxide photoproduction formed by immobilized cells of the blue-green alga, Anabaena variabilis and the redox mediator methyl viologen is described. Hydrogen peroxide is produced in a redox catalyst cycle in which methyl viologen is reduced by electrons from water obtained by the photosynthetic apparatus of the algae using solar energy, and reoxidized by the introduction of oxygen into the solution. Hydrogen peroxide is produced during methyl viologen re-oxidation in two steps by means of the formation of superoxide. Experimental conditions for maximum photoproduction (catalyst charge, chlorophyll, and agar final concentration for cell immobilization) have been investigated using a continuous photosystem with immobilized A. variabilis as photocatalyst. Under the determined optimum conditions, the photosystem produces hydrogen peroxide at a rate of 100 μmoles/mg Chl · h, maintaining the production for several hours, and with an energy conversion efficiency of about 2%. Taking into account the use of hydrogen peroxide as fuel, this photosystem can be a useful tool in the storage of solar energy.

Continuous photosynthetic production of hydrogen peroxide by the blue-green algae Anacystis nidulans R2 as a way to solar energy conversion

Continuous production of hydrogen peroxide by a culture of the blue-green algae Anacystis nidulans R2 is described as a model system for the conversion of solar energy. The redox power from the photolysis of water, promoted by the photosynthetic apparatus of algae, is driven by the redox mediator methyl viologen to oxygen which is reduced, in two successive steps, to superoxide and hydrogen peroxide. Hydrogen peroxide is a chemical compound with a high value since it is extensively used in industry, especially as bleaching agent. Moreover, this product is a powerful source of energy which releases more than 100 kJ per mol upon decomposition into water and oxygen; it is considered a fuel able to drive rockets and engines. Optimum pH and temperature of the photosystem, as well as the effect on H 2O 2 production of the concentration of methyl viologen, amount of cell, and intensity of light have been studied. Under saturation of light and methyl viologen, and and at the temperature of 45°C, a production of hydrogen peroxide higher than 200 μmol/mg Chl · h was reached. In these conditions, 2% of the light energy absorbed by the photosystem is converted in chemical energy as H 2O 2.

Efficiency of hydrogen photoproduction by photosystem I-enriched subchloroplast vesicles combined with Proteus mirabilis cells. Effects of some exogenous electron donors

High rates of hydrogen photoproduction are obtained when glutaraldehyde-fixed Photosystem I-enriched vesicles (Photosystem II-depleted) are added to hydrogenase-containing cells of Proteus mirabilis in the presence of the mediator methylviologen and a suitable electron donating system. This donor system includes ascorbate, dithioerythritol (DTE) and the mediator tetramethylphenylene-diamine (TMPD) and reduces the photosynthetic electron transfer chain at the level of plastocyanin. Both DTE and ascorbate are required for hydrogen photoproduction, DTE being the ultimate electron donor and ascorbate only having a catalytic function. Whereas the aerobic photoreduction of methylviologen is similar in the presence of DTE, ascorbate or both, under anaerobic conditions only combination of both compounds results in a high and stable amount of reduced methylviologen that can be utilized by the hydrogenase. It is concluded that oxidation reactions of reduced methylviologen, competing with the hydrogenase, rather than methylviologen photoreduction, limit hydrogen photoproduction in the presence of either DTE or ascorbate. These oxidation reactions are suggested to involve back reactions to the oxidized form(s) of ascorbate and DTE but backflow to the photosynthetic electron transfer chain (i.e. cyclic electron transfer) can not be excluded.