Design, synthesis, and characterization of a photoactivatable flavocytochrome molecular maquette

We report the construction of a synthetic flavo-heme protein that incorporates two major physiological activities of flavoproteins: light activation of flavin analogous to DNA photolyase and rapid intramolecular electron transfer between the flavin and heme cofactors as in several oxidoreductases. The functional tetra-α-helix protein comprises two 62-aa helix-loop-helix subunits. Each subunit contains a single cysteine to which flavin (7-acetyl-10-methylisoalloxazine) is covalently attached and two histidines appropriately positioned for bis-his coordination of heme cofactors. Both flavins and hemes are situated within the hydrophobic core of the protein. Intramolecular electron transfer from flavosemiquinone generated by photoreduction from a sacrificial electron donor in solution was examined between protoporphyrin IX and 1-methyl-2-oxomesoheme XIII. Laser pulse-activated electron transfer from flavin to meso heme occurs on a 100-ns time scale, with a favorable free energy of approximately −100 meV. Electron transfer from flavin to the lower potential protoporphyrin IX, with an unfavorable free energy, can be induced after a lag phase under continuous light illumination. Thus, the supporting peptide matrix provides an excellent framework for the positioning of closely juxtaposed redox groups capable of facilitating intramolecular electron transfer and begins to clarify in a simplified and malleable system the natural engineering of flavoproteins.

Photochemical energy conversion in a helical oligoproline assembly.

A general method is described for constructing a helical oligoproline assembly having a spatially ordered array of functional sites protruding from a proline-II helix. Three different redox-active carboxylic acids were coupled to the side chain of cis-4-amino-L-proline. These redox modules were incorporated through solid-phase peptide synthesis into a 13-residue helical oligoproline assembly bearing in linear array a phenothiazine electron donor, a tris(bipyridine)ruthenium(II) chromophore, and an anthraquinone electron acceptor. Upon transient 460-nm irradiation in acetonitrile, this peptide triad formed with 53% efficiency an excited state containing a phenothiazine radical cation and an anthraquinone radical anion. This light-induced redox-separated state had a lifetime of 175 ns and stored 1.65 eV of energy.

Potentiation of proton transfer function by electrostatic interactions in photosynthetic reaction centers from Rhodobacter sphaeroides: First results from site-directed mutation of the H subunit.

The x-ray crystallographic structure of the photosynthetic reaction center (RC) has proven critical in understanding biological electron transfer processes. By contrast, understanding of intraprotein proton transfer is easily lost in the immense richness of the details. In the RC of Rhodobacter (Rb.) sphaeroides, the secondary quinone (QB) is surrounded by amino acid residues of the L subunit and some buried water molecules, with M- and H-subunit residues also close by. The effects of site-directed mutagenesis upon RC turnover and quinone function have implicated several L-subunit residues in proton delivery to QB, although some species differences exist. In wild-type Rb. sphaeroides, Glu L212 and Asp L213 represent an inner shell of residues of particular importance in proton transfer to QB. Asp L213 is crucial for delivery of the first proton, coupled to transfer of the second electron, while Glu L212, possibly together with Asp L213, is necessary for delivery of the second proton, after the second electron transfer. We report here the first study, by site-directed mutagenesis, of the role of the H subunit in QB function. Glu H173, one of a cluster of strongly interacting residues near QB, including Asp L213, was altered to Gln. In isolated mutant RCs, the kinetics of the first electron transfer, leading to formation of the semiquinone, QB-, and the proton-linked second electron transfer, leading to the formation of fully reduced quinol, were both greatly retarded, as observed previously in the Asp L213 --> Asn mutant. However, the first electron transfer equilibrium, QA-QB <==> QAQB-, was decreased, which is opposite to the effect of the Asp L213 --> Asn mutation. These major disruptions of events coupled to proton delivery to QB were largely reversed by the addition of azide (N3-). The results support a major role for electrostatic interactions between charged groups in determining the protonation state of certain entities, thereby controlling the rate of the second electron transfer. It is suggested that the essential electrostatic effect may be to "potentiate" proton transfer activity by raising the pK of functional entities that actually transfer protons in a coupled fashion with the second electron transfer. Candidates include buried water (H3O+) and Ser L223 (serine-OH2+), which is very close to the O5 carbonyl of the quinone.

Reaction of the Escherichia coli quinol oxidase cytochrome bo3 with dioxygen: the role of a bound ubiquinone molecule.

We have studied the kinetics of the oxygen reaction of the fully reduced quinol oxidase, cytochrome bo3, using flow-flash and stopped flow techniques. This enzyme belongs to the heme-copper oxidase family but lacks the CuA center of the cytochrome c oxidases. Depending on the isolation procedure, the kinetics are found to be either nearly monophasic and very different from those of cytochrome c oxidase or multiphasic and quite similar to cytochrome c oxidase. The multiphasic kinetics in cytochrome c oxidase can largely be attributed to the presence Of CuA as the donor of a fourth electron, which rereduces the originally oxidized low-spin heme and completes the reduction of O2 to water. Monophasic kinetics would thus be expected, a priori, for cytochrome bo3 since it lacks the CuA center, and in this case we show that the oxygen reaction is incomplete and ends with the ferryl intermediate. Multiphasic kinetics thus suggest the presence of an extra electron donor (analogous to CuA). We observe such kinetics exclusively with cytochrome bo3 that contains a single equivalent of bound ubiquinone-8, whereas we find no bound ubiquinone in an enzyme exhibiting monophasic kinetics. Reconstitution with ubiquinone-8 converts the reaction kinetics from monophasic to multiphasic. We conclude that a single bound ubiquinone molecule in cytochrome bo3 is capable of fast rereduction of heme b and that the reaction with O2 is quite similar in quinol and cytochrome c oxidases.

Electrocatalytically driven omega-hydroxylation of fatty acids using cytochrome P450 4A1.

The cyclic enzymatic function of a cytochrome P450, as it catalyzes the oxygen-dependent metabolism of many organic chemicals, requires the delivery of two electrons to the hemeprotein. In general these electrons are transferred from NADPH to the P450 via an FMN- and FAD-containing flavoprotein (NADPH-P450 reductase). The present paper shows that NADPH can be replaced by an electrochemically generated reductant [cobalt(II) sepulchrate trichloride] for the electrocatalytically driven omega-hydroxylation of lauric acid. Results are presented illustrating the use of purified recombinant proteins containing P450 4A1, such as the fusion protein (rFP450 [mRat4A1/mRatOR]L1) or a system reconstituted with purified P450 4A1 plus purified NADPH-P450 reductase. Rates of formation of 12-hydroxydodecanoic acid by the electrochemical method are comparable to those obtained using NADPH as electron donor. These results suggest the practicality of developing electrocatalytically dependent bioreactors containing different P450s as catalysts for the large-scale synthesis of stereo- and regio-selective hydroxylation products of many chemicals.

Utilização de metanol, etanol e metano como doadores de elétrons para a desnitrificação

Esta tese apresenta e discute os dados obtidos a partir de trabalho experimental projetado para avaliar comparativamente o desempenho de reatores desnitrificantes em batelada, tendo etanol, metanol e gás metano como doadores de elétrons. Os experimentos foram realizados em reatores em escala de bancada. Os ensaios com gás metano objetivaram verificar a efetividade deste sub-produto de reatores anaeróbios em substituir os doadores exógenos de elétrons comumente utilizados, tais como metanol e etanol. Para alcançar o objetivo principal deste trabalho, os parâmetros cinéticos de desnitrificação, para os doadores de elétrons ensaiados, foram determinados nas diferentes condições operacionais. Além disso, as alterações ocorridas na população microbiana, ao longo do período experimental, foram avaliadas em relação à diversidade microbiana, por meio de análises microscópicas (óptica, de fluorescência e eletrônica de varredura) e da técnica de Biologia Molecular de PCR/DGGE. A completa desnitrificação foi alcançada para todos os compostos testados, e o etanol foi o doador de elétrons mais eficiente para a desnitrificação. A melhor razão carbono-nitrogênio para a desnitrificação foi igual a 1,0. Contudo, este parâmetro foi encontrado ser inadequado para utilização no processo de desnitrificação, uma vez que não expressa a capacidade real do composto usado em doar elétrons. A desnitrificação com metano ocorreu tanto na presença como na ausência de oxigênio, embora a baixas velocidades quando comparado com os outros compostos. No entanto, a configuração do reator utilizado neste estudo não foi adequada para promover a efetiva dissolução do gás metano na fase líquida. Por essa razão, sugere-se o desenvolvimento de configurações de reatores apropriadas para minimizar as resistências à transferência de massa da fase gasosa para a líquida e também desta para a biomassa.

Functionalization of carbon nanotubes using aminobenzene acids and electrochemical methods. Electroactivity for the oxygen reduction reaction

Functionalized carbon nanotubes (CNTs) using three aminobenzene acids with different functional groups (carboxylic, sulphonic, phosphonic) in para position have been synthesized through potentiodynamic treatment in acid media under oxidative conditions. A noticeable increase in the capacitance for the functionalized carbon nanotubes mainly due to redox processes points out the formation of an electroactive polymer thin film on the CNTs surface along with covalently bonded functionalities. The CNTs functionalized using aminobenzoic acid rendered the highest capacitance values and surface nitrogen content, while the presence of sulfur and/or phosphorus groups in the aminobenzene structure yielded a lower functionalization degree. The oxygen reduction reaction (ORR) activity of the functionalized samples was similar to that of the parent CNTs, independently of the functional group present in the aminobenzene acid. Interestingly, a heat treatment in N2 atmosphere with a very low O2 concentration (3125 ppm) at 800 °C of the CNTs functionalized with aminobenzoic acid produced a material with high amounts of surface oxygen and nitrogen groups (12 and 4% at., respectively), that seem to modulate the electron-donor properties of the resulting material. The onset potential and limiting current for ORR was enhanced for this material. These are promising results that validates the use of electrochemistry for the synthesis of novel N-doped electrocatalysts for ORR in combination with adequate heat treatments.

Photogeneration of Hydrogen from Water by Hybrid Molybdenum Sulfide Clusters Immobilized on Titania

Two new hybrid molybdenum(IV) Mo3S7 cluster complexes derivatized with diimino ligands have been prepared by replacement of the two bromine atoms of [Mo3S7Br6]2− by a substituted bipyridine ligand to afford heteroleptic molybdenum(IV) Mo3S7Br4(diimino) complexes. Adsorption of the Mo3S7 cores from sample solutions on TiO2 was only achieved from the diimino functionalized clusters. The adsorbed Mo3S7 units were reduced on the TiO2 surface to generate an electrocatalyst that reduces the overpotential for the H2 evolution reaction by approximately 0.3 V (for 1 mA cm−2) with a turnover frequency as high as 1.4 s−1. The nature of the actual active molybdenum sulfide species has been investigated by X-ray photoelectron spectroscopy. In agreement with the electrochemical results, the modified TiO2 nanoparticles show a high photocatalytic activity for H2 production in the presence of Na2S/Na2SO3 as a sacrificial electron donor system.

Kinetic and phylogenetic characterization of an anaerobic dechlorinating microbial community

The reductive dechlorination (RD) of tetrachloroethene (PCE) to vinyl chloride (VC) and, to a lesser extent, to ethene (ETH) by an anaerobic microbial community has been investigated by studying the processes and kinetics of the main physiological components of the consortium. Molecular hydrogen, produced by methanol-utilizing acetogens, was the electron donor for the PCE RD to VC and ETH without forming any appreciable amount of other chlorinated intermediates and in the near absence of methanogenic activity. The microbial community structure of the consortium was investigated by preparing a 1 6S rDNA clone library and by fluorescence in situ hybridization (FISH). The PCR primers used in the clone library allowed the harvest of 16SrDNA from both bacterial and archaeal members in the community. A total of 616 clones were screened by RFLP analysis of the clone inserts followed by the sequencing of RFLP group representatives and phylogenetic analysis. The clone library contained sequences mostly from hitherto undescribed bacteria. No sequences similar to those of the known RD bacteria like 'Dehalococcoides ethenogenes' or Dehalobacter restrictus were found in the clone library, and none of these bacteria was present in the RD consortium according to FISH. Almost all clones fell into six previously described phyla of the bacterial domain, with the majority (56(.)6%) being deep-branching members of the Spirochaetes phylum. Other clones were in the Firmicutes phylum (18(.)5%), the Chloroflexi phylum (16(.)4%), the Bacteroidetes phylum (6(.)3%), the Synergistes genus (11(.)1%) and a lineage that could not be affiliated with existing phyla (11(.)1%). No archaeal clones were found in the clone library. Owing to the phylogenetic novelty of the microbial community with regard to previously cultured microorganisms, no specific microbial component(s) could be hypothetically affiliated with the RD phenotype. The predominance of Spirochaetes in the microbial consortium, the main group revealed by clone library analysis, was confirmed by FISH using a purposely developed probe.

Microscale structure and function of anaerobic-aerobic granules containing glycogen accumulating organisms

The spatial arrangement and metabolic activity of 'Candidatus Competibacter phosphatis' was investigated in granular sludge from an anaerobic-aerobic sequencing batch reactor enriched for glycogen-accumulating organisms. In this process, the electron donor (acetate) and the electron acceptor (oxygen) were supplied sequentially in each phase. The organism, identified by fluorescence in situ hybridisation, was present throughout the granules; however, metabolic activity was limited to a 100-mum-thick layer immediately below the surface of the granules. To investigate the cause of this, oxygen microsensors and a novel microscale biosensor for volatile fatty acids were used in conjunction with chemical staining for intracellular storage polymers. It was found that the limited distribution of activity was caused by mass transport limitation of oxygen into the granules during the aerobic phase. (C) 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Efficient and stable nitritation and denitritation of ammonium-rich sludge dewatering liquor using an SBR with continuous loading

Separate treatment of dewatering liquor from anaerobic sludge digestion significantly reduces the nitrogen load of the main stream and improves overall nitrogen elimination. Such ammonium-rich wastewater is particularly suited to be treated by high rate processes which achieve a rapid elimination of nitrogen with a minimal COD requirement. Processes whereby ammonium is oxidised to nitrite only (nitritation) followed by denitritation with carbon addition can achieve this. Nitrogen removal by nitritation/denitritation was optimised using a novel SBR operation with continuous dewatering liquor addition. Efficient and robust nitrogen elimination was obtained at a total hydraulic retention time of 1 day via the nitrite pathway. Around 85-90% nitrogen removal was achieved at an ammonium loading rate of 1.2 g NH4+-N m(-3) d(-1). Ethanol was used as electron donor for denitritation at a ratio of 2.2gCODg(-1) N removed. Conventional nitritation/denitritation with rapid addition of the dewatering liquor at the beginning of the cycle often resulted in considerable nitric oxide (NO) accumulation during the anoxic phase possibly leading to unstable denitritation. Some NO production was still observed in the novel continuous mode, but denitritation was never seriously affected. Thus, process stability can be increased and the high specific reaction rates as well as the continuous feeding result in decreased reactor size for full-scale operation. (c) 2006 Elsevier Ltd. All rights reserved.

The mechanisms of melt stabilisation of polyolefins

The effects of melt stabilisers on the oxidative degradation of polyolefins (polypropylene, low density polyethylene) have been studied under a variety of processing conditions . The changes in the both chemical and physical properties of unstabilised polymers occurring during processing were found to be strongly dependent on the amount of oxygen present in the mixer. 2 ,6 ,3' ,5' -tetra-tert-butyl-4'-phenoxy-4-methylene-2, 5-cyclohexadiene-1- one (galvinoxyl), iodine, nitroxyl radicals and cupric stearate were found to be very efficient melt stabilisers particularly when processed in a restricted amount of air. The mechanisms of their melt stabilising action have been investigated and a common cyclical regenerative mechanism involving both chain-breaking electron acceptor (CB-A) and chain-breaking electron donor (CB-D) antioxidant activity was found to be involved in each case. 2,6,3',5'-tetra-tert-butyl-4'-hydroxy phenyl-4-rrethylene-2,5-cyclohexadiene- 1-one (hydrogalvinoxy1), 4-hydroxy, 2,2,6, 6-tetra methyl-N-hydroxy piperidine and hydrogen iodide were formed together with olefinic unsaturation in the substrates during the melt processing of the polymers containing galvinoxyl, 4-hydroxy, 2,2,6, 6-tetra methyl piperidine oxyl and iodine respectively. No bonding of the melt stabilisers to the polymers was found to occur. Cupric stearate was found to undergo a similar redox reaction during its action as a melt stabiliser with the formation of unsaturation in the polymer. Evidence for the above processes is presented. The behaviours of melt stabilisers in the subsequent thermal and photooxidation of polyolefins have also been studied. Galvinoxyl which is very effective under both mild and severe processing canditions has been found to be an effective antioxidant during thermal oxidation (oven ageing) and it is also moderately good. as a photo-stabiliser. Iodine and cupric stearate acted efficiently during melt stabilisation of polymers, however they were both ineffective as thermo-oxidative antioxidants and UV stabilisers. Although the melt stabilisation effectiveness of stable nitroxyl radicals (e.g. 4-hydroxy, 2,2,6,6-tetra methyl piperidineoxyl and Bis- (2,2,6 ,6-tetra methyl-4- piperidinyl-N-oxyl) sebacate) is not as high as that of galvinoxyl during processing particularly in excess of air, they have been found to be much more efficient as UV stabilisers for polyolefins. The reasons for this are discussed.

Propriedades bioativas dos polissacarídeos sulfatados da alga comestível Gracilaria birdiae

Red marine algae of the genus Gracilaria synthesize sulfated polysaccharides (PS) bioactive. But many of these PS were not properly assessed, as is the case of PS synthesized by edible seaweed Gracilaria birdiae. Previous studies showed that sulfated galactans this alga has anti-inflammatory effect. In this work, a galactan (GB) of G. birdiae was obtained and evaluated by different tests. GB showed anticoagulant activity in APTT assay. GB showed no toxicity to normal cells (3T3), but inhibited the survival of cells of adenocarcinoma of the cervix (HeLa) and human pancreatic cancer (Panc-1) 80% (1.5 mg / ml). GB was not able to hijack the OH radical or the superoxide radical. However, showed activity electron donor in two different tests and presented iron chelator activity (70% and 1.0 mg / ml) and Copper (70% at 0.5 mg / ml). The presence of a higher GB promotes formation of crystals of calcium oxalate dihydrate small size, which is less aggressive, because GB is able to interact with and stabilize the crystal that form. Furthermore, GB (2.0 mg / mL) was not cytotoxic to human renal cells (HEK-293). The data lead us to propose that GB has a great potential for the treatment of urolithiasis

(Table 1) Downcore age and bulk geochemistry of sediment core GS7202-35

The distribution of redox-sensitive metals in sediments is potentially a proxy for past ocean ventilation and productivity, but deconvolving these two major controls has proved difficult to date. Here we present a 740 kyr long record of trace element concentrations from an archived sediment core collected at ~15°S on the western flank of the East Pacific Rise (EPR) on 1.1 Myr old crust and underlying the largest known hydrothermal plume in the world ocean. The downcore trace element distribution is controlled by a variable diagenetic overprint of the inferred primary hydrothermal plume input. Two main diagenetic processes are operating at this site: redox cycling of transition metals and ferrihydrite to goethite transition during aging. The depth of oxidation in these sediments is controlled by fluctuations in the relative balance of bottom water oxygen and electron donor input (organic matter and hydrothermal sulfides). These fluctuations induce apparent variations in the accumulation of redox-sensitive species with time. Subsurface U and P peaks in glacial age sediments, in this and other published data sets along the southern EPR, indicate that basin-wide changes in deep ocean ventilation, in particular at glacial-interglacial terminations II, III, IV, and V, alter the depth of the oxidation front in the sediments. These basin-wide changes in the deep Pacific have significant implications for carbon partitioning in the ocean-atmosphere system, and the distribution of redox-sensitive metals in ridge crest sediment can be used to reconstruct past ocean conditions at abyssal depths in the absence of alternative proxy records.

Design of Transparent Conducting Electrodes and Surface Relief Gratings for Plasmonic Polymer Solar Cells

As the concept of renewable energy becomes increasingly important in the modern society, a considerable amount of research has been conducted in the field of organic photovoltaics in recent years. Although organic solar cells generally have had lower efficiencies compared to silicon solar cells, they have the potential to be mass produced via solution processing. A common polymer solar cell architecture relies on the usage of P3HT (electron donor) and PCBM (electron acceptor) bulk heterojunction. One of the main issues with this configuration is that in order to compensate for the high exciton recombination rate, the photoactive layer is often made very thin (on the order of 100 $%). This results in low solar cell photocurrents due to low absorption. This thesis investigates a novel method of light trapping by coupling surface plasmons at the electrode interface via surface relief gratings, leading to EM field enhancements and increased photo absorption. Experimental work was first conducted on developing and optimizing a transparent electrode of the form &'()/+,/&'() to replace the traditional ITO electrode since the azopolymer gratings cannot withstand the high temperature processing of ITO films. It was determined that given the right thickness profiles and deposition conditions, the MAM stack can achieve transmittance and conductivity similar to ITO films. Experimental work was also conducted on the fabrication and characterization of surface relief gratings, as well as verification of the surface plasmon generation. Surface relief gratings were fabricated easily and accurately via laser interference lithography on photosensitive azopolymer films. Laser diffraction studies confirmed the grating pitch, which is dependent on the incident angle and wavelength of the writing beam. AFM experiments were conducted to determine the surface morphology of the gratings, before and after metallic film deposition. It was concluded that metallic film deposition does not significantly alter the grating morphologies.