Analysis of sequenced bacterial genomes revealed that the genomes encode more than 30% hypothetical and conserved hypothetical proteins of unknown function. Among proteins of unknown function that are conserved in anaerobes, some might be determinants of the anaerobic way of life. This study focuses on two divergent clusters specifically found in anaerobic microorganisms and mainly composed of genes encoding conserved hypothetical proteins. We show that the two gene clusters DVU2103-DVU2104-DVU2105 (orp2) and DVU2107-DVU2108-DVU2109 (orp1) form two divergent operons transcribed by the sigma(54)-RNA polymerase. We further demonstrate that the sigma(54)-dependent transcriptional regulator DVU2106, located between orp1 and orp2, collaborates with sigma(54)-RNA polymerase to orchestrate the simultaneous expression of the divergent orp operons. DVU2106, whose structural gene is transcribed by the sigma(70)-RNA polymerase, negatively retrocontrols its own expression. By using an endogenous pulldown strategy, we identify a physiological complex composed of DVU2103, DVU2104, DVU2105, DVU2108, and DVU2109. Interestingly, inactivation of DVU2106, which is required for orp operon transcription, induces morphological defects that are likely linked to the absence of the ORP complex. A putative role of the ORP proteins in positioning the septum during cell division is discussed.

In this work it is demonstrated that the characterization of c-type haem containing proteins by electrochemical techniques needs to be cautiously performed when using pyrolytic graphite electrodes. An altered form of the cytochromes, which has a redox potential 300 mV lower than that of the native state and displays peroxidatic activity, can be induced by interaction with the pyrolytic graphite electrode. Proper control experiments need to be performed, as altered conformations of the enzymes containing c-type haems can show activity towards the enzyme substrate. The work was focused on the study of the activation mechanism and catalytic activity of cytochrome c peroxidase from Paracoccus pantotrophus. The results could only be interpreted with the assignment of the observed non-turnover and catalytic signals to a non-native conformation state of the electron-transferring haem. The same phenomenon was detected for Met-His monohaem cytochromes (mitochondrial cytochrome c and Desulfovibrio vulgaris cytochrome c-553), as well as for the bis-His multihaem cytochrome c(3) from Desulfovibrio gigas, showing that this effect is independent of the axial coordination of the c-type haem protein. Thus, the interpretation of electrochemical signals of c-type (multi)haem proteins at pyrolytic graphite electrodes must be carefully performed, to avoid misassignment of the signals and incorrect interpretation of catalytic intermediates.

This review focuses on the novel CuZ center of nitrous oxide reductase, an important enzyme owing to the environmental significance of the reaction it catalyzes, reduction of nitrous oxide, and the unusual nature of its catalytic center, named CuZ. The structure of the CuZ center, the unique tetranuclear copper center found in this enzyme, opened a novel area of research in metallobiochemistry. In the last decade, there has been progress in defining the structure of the CuZ center, characterizing the mechanism of nitrous oxide reduction, and identifying intermediates of this reaction. In addition, the determination of the structure of the CuZ center allowed a structural interpretation of the spectroscopic data, which was supported by theoretical calculations. The current knowledge of the structure, function, and spectroscopic characterization of the CuZ center is described here. We would like to stress that although many questions have been answered, the CuZ center remains a scientific challenge, with many hypotheses still being formed.

Identifying redox partners and the interaction surfaces is crucial for fully understanding electron flow in a respiratory chain. In this study, we focused on the interaction of nitrous oxide reductase (N(2)OR), which catalyzes the final step in bacterial denitrification, with its physiological electron donor, either a c-type cytochrome or a type 1 copper protein. The comparison between the interaction of N(2)OR from three different microorganisms, Pseudomonas nautica, Paracoccus denitrificans, and Achromobacter cycloclastes, with their physiological electron donors was performed through the analysis of the primary sequence alignment, electrostatic surface, and molecular docking simulations, using the bimolecular complex generation with global evaluation and ranking algorithm. The docking results were analyzed taking into account the experimental data, since the interaction is suggested to have either a hydrophobic nature, in the case of P. nautica N(2)OR, or an electrostatic nature, in the case of P. denitrificans N(2)OR and A. cycloclastes N(2)OR. A set of well-conserved residues on the N(2)OR surface were identified as being part of the electron transfer pathway from the redox partner to N(2)OR (Ala495, Asp519, Val524, His566 and Leu568 numbered according to the P. nautica N(2)OR sequence). Moreover, we built a model for Wolinella succinogenes N(2)OR, an enzyme that has an additional c-type-heme-containing domain. The structures of the N(2)OR domain and the c-type-heme-containing domain were modeled and the full-length structure was obtained by molecular docking simulation of these two domains. The orientation of the c-type-heme-containing domain relative to the N(2)OR domain is similar to that found in the other electron transfer complexes.

The activation mechanism of Pseudomonas stutzeri cytochrome c peroxidase (CCP) was probed through the mediated electrochemical catalysis by its physiological electron donor, P. stutzeri cytochrome c-551. A comparative study was carried out, by performing assays with the enzyme in the resting oxidized state as well as in the mixed-valence activated form, using cyclic voltammetry and a pyrolytic graphite membrane electrode. In the presence of both the enzyme and hydrogen peroxide, the peak-like signal of cytochrome c-551 is converted into a sigmoidal wave form characteristic of an E(r)C'(i) catalytic mechanism. An intermolecular electron transfer rate constant of (4 +/- 1) x 10(5) M(-1) s(-1) was estimated for both forms of the enzyme, as well as a similar Michaelis-Menten constant. These results show that neither the intermolecular electron transfer nor the catalytic activity is kinetically controlled by the activation mechanism of CCP in the case of the P. stutzeri enzyme. Direct enzyme catalysis using protein film voltammetry was unsuccessful for the analysis of the activation mechanism, since P. stutzeri CCP undergoes an undesirable interaction with the pyrolytic graphite surface. This interaction, previously reported for the Paracoccus pantotrophus CCP, induces the formation of a non-native conformation state of the electron-transferring haem, which has a redox potential 200 mV lower than that of the native state and maintains peroxidatic activity.

This paper describes a new methodology for the conception of urban plans in order for them to fit the principles of Sustainable Development. The methodology herein presented makes use of different software for designing, calculations and dynamic modelling of buildings and urban spaces. The results from these assessments are then interpreted and incorporated in the urban design solutions.
This methodology was applied in a case study in Oeiras, Portugal, in the conception phases of the Oeiras Master Plan. The overall objective of the implementation of this process in this case study was to be able to foresee the area’s final makeover and predict the impacts of the plan on the context through the integrated use of environmental analysis (ArcGIS), simulations of future scenarios (3dsMax) and 3d modelling studies (Revit, Ecotect).
Factors such as geomorphology, solar exposure, prevailing winds and rail and road traffic noise provided information on how to capitalize all favourable conditions of the site, which is a fundamental base for an appropriate land use transformation.
This methodology contributes to the control of urban design solutions and outcomes, and promotes the creation of an interface between urban planning, landscape and architecture projects. The development of a “City Information Modelling”, similar to BIM, which has been successfully applied to Architecture and Engineering projects, is therefore seen as an answer to the new challenges presented by the present and future cities.
This strategy delivers the maximum benefits at all spatial scales by embracing the logic of sustainability so that urban design can be the first tool to optimize future building and infrastructure projects.

In the present work, the proton-transfer reactions of the methylpyranomalvidin-3-O-glucoside pigment in water with different pH values was studied by NMR and UV-visible spectroscopies. The results showed four equilibrium forms: the methylpyranomalvidin-3-O-glucoside cation, the neutral quinoidal base, the respective anionic quinoidal base, and a dianionic base unprotonated at the methyl group. According to the NMR data, it seems that for methylpyranomalvidin-3-O-glucoside besides the acid base equilibrium between the pyranoflavylium cation and the neutral quinoidal base, a new species is formed at pD 4.88-6.10. This is corroborated by the appearance of a new set of signals in the NMR spectrum that may be assigned to the formation of hemiketal/cis-chalcone species to a small extent. The two ionization constants (pK(a1) and pK(a2)) obtained by both methods (NMR and UV-visible) for methylpyranomalvidin-3-O-glucoside are in agreement (pK(a1) = 5.17 +/- 0.03; pK(a2) = 8.85 +/- 0.08; and pK(a1) = 4.57 +/- 0.07; pK(a2) = 8.23 +/- 0.04 obtained by NMR and UV-visible spectroscopies, respectively). Moreover, the fully dianionic unprotonated form (at the methyl group) of the methylpyranomalvidin-3-O-glucoside is converted slowly into a new structure that displays a yellow color at basic pH. On the basis of the results obtained through LC-MS and NMR, the proposed structure was found to correspond to the flavonol syringetin-3-glucoside.

In this work, coloured glasses were produced based on the synthesis of gold and silver nanoparticles by the sol-gel process having in mind their application in art works. Gold and silver were used separately or as a mixture by varying the mole fractions in order to get a range of colours from yellow to red. The gold and silver nanoparticles were prepared by the reduction of tetrachloroauric acid and silver nitrate with sodium citrate in aqueous solutions which were further introduced in the sol-gel system. Attention was focused on the thermal treatment of the sol-gel samples. Different temperatures were used in order to determine their influence on the obtained colour. The glasses were characterized by UV-Vis absorption spectroscopy and the size of the nanoparticles was examined by transmission electron microscopy (TEM). The range of colours mentioned above is obtained either by preparing nanoparticles of each metal and mixing them or by preparing nanoparticles from solutions containing initially ions of both metals. In the former case, two surface plasmon resonance (SPR) bands were observed for temperatures below 200 degrees C while higher temperatures promote the formation of alloys between the Ag and Au nanoparticles. In the latter case, only one SPR band is observed and the nanoparticle size distribution is narrower. The results were explained by nanoparticle aggregation promoted by temperature. Glasses containing only Ag did not present the typical yellow colour above 300 degrees C but it was shown that the colour could be stabilized if Au was added in small amounts (Au/Ag molar ratio 0.1). (C) 2010 Elsevier B.V. All rights reserved.

In moderately acidic water/ethanol solutions, 7-(naphthalen-2-ylmethyl)-5,6-dihydrobenzo[c]xanthen12-ium perchlorate shows C-H proton acidity giving rise to an ethylenic base species, instead of the hydration products of the typical flavylium network of chemical reactions, hemiketal, cis-chalcone and trans-chalcone. The kinetics of this acid-base process is much slower, k(obs)(s(-1)) = 3.5 x 10(-3) + 0.16[H(+)], than those commonly observed for O-H deprotonation. In the presence of the triblock copolymer Pluronic F-127, the ethylenic base is dramatically stabilized shifting the pK(a) from 1.35 (in ethanol : water (1 : 1) to -0.35. Taking profit from the well-described temperature dependence of this polymer, a phase transition thermochromic system was achieved. The system was cycled between 5 and 35 degrees C with negligible changes in absorbance after 20 cycles.

New trans-2-hydroxychalcones bearing a carboxylate group at position 2' (Ct(-)) were synthesized (compounds 2 and 3). These compounds lead to a network of chemical reactions depending on pH value, light, and solvent. In water, when the pH value is lowered, the ionized trans-chalcone is protonated and the flavylium cation AH(+) is formed at very acidic pH values through hemiketal B and cis-chalcone Cc, with global acidity constants of pK'(a) <= -1 and approximate to 0.1, respectively, for 2 and 3. The electron-acceptor character of the carboxylic substituent not only increases the observed acidity of the flavylium cation, but also decreases the rate of the ring-opening/closing from a subsecond timescale to hours relative to model compound 1 (without carboxylate). The photochemistry of the network was studied in detail by means of continuous irradiation, monitored by UV/Vis absorption and H-1 and C-13 NMR spectroscopic analysis. Although compound 3 is only slightly photoactive, compound 2 (Ct(-)) reacts in aqueous solutions (lambda(irr) = 313 nm) to form B- and Cc(-), with a global quantum yield of 0.15, and fully reverts back to Ct(-) with a rate constant of k = 6.7 x 10(-5) s(-1). The flavylium cation is no longer formed in methanol, and irradiation of Ct(-) leads to the formation of B- and the new lactone-trapped chromene species La. The formation of La takes place through a sequence of three photochemical steps: photoisomerization of Ct(-), photo-ring-closing reaction of Cc , and photolactonization of B-. Only the cis/trans isomerization and ring-closing reactions are thermally reversible on a timescale of seconds and hours, respectively. A photochromic system was achieved in rigid matrices of methanol (at 77 K) and 1-dodecanol (5 degrees C) by irradiating lactone La to give a red ortho-quinone allide through a photo-ring-opening reaction; the color disappears with a rate constant of k = 1.25 x 10(-2) s(-1) in 1-dodecanol at 5 degrees C.

A symmetric bis(flavylium) constituted by two 7-hydroxyflavylium moieties linked by a methylviologen bridge was synthesized. The thermodynamic and kinetics of the network of chemical reactions involving bis(flavylium) and the model compound 7-hydroxy-4'-methylflavylium was completely characterized by means of direct and reverse pH jumps (stopped flow) and flash photolysis. Both compounds follow the usual pH-dependent network of chemical reactions of flavylium derivatives. The equilibrium species of the model compound are the flavylium cation (acidic species) and the trans-chalcone (basic species) with an apparent pK(a)' = 2.85. In the case of the bis(flavylium) it was possible to characterize by (1)H NMR spectroscopy three species with different degrees of isomerization: all flavylium, flavylium-trans-chalcone, and all trans-chalcone. Representation of the time-dependent mole fraction distribution of these three forms after a pH jump from equilibrated solutions of all-flavylium cation (lower pH values) to higher pH values, shows that formation of trans-chalcone is not completely stochastic (two independent isomerizations), the isomerization of one flavylium showing a small influence on the isomerization of the other. The radical of the methyl viologen bridge is formed upon reduction of the bis(trans-chalcone) with dithionite. The system is reversible after addition of an oxidant in spite of the occurrence of some decomposition.

The use of cellulosic fibers as load bearing constituents in composite materials has increased over the last decade due to their relative cheapness compared to conventional materials such as glass and aramid fibers, their ability to recycle, and because they compete well in terms of strength per weight of material. All-cellulosic based composites prepared from cellulose derivatives based matrices and microcrystalline cellulosic fibers made by direct coupling between fibers and matrix present interesting mechanical and gas permeation properties, thus being potential candidates for packaging materials. Both the cellulosic matrix and the reinforcing fibers are biocompatible and widely used in the pharmaceutical industry, which is very important for the envisaged application. In addition to their biocompatibility, cellulosic systems have the ability to form both thermotropic and lyotropic chiral nematic phases, and the composites produced from the latter show improved mechanical properties due to fiber orientation induced by the anisotropic matrix. The preparation and characterization (morphological, topographical, mechanical, gas barrier properties) of all-cellulosic based composites are described in this chapter.

Gene knock-out studies on Geobacter sulfurreducens cells showed that the periplasmic triheme cytochrome PpcA is involved in respiratory pathways leading to the extracellular reduction of Fe(III) and U(VI) oxides. The crucial role of this protein in bridging the electron transfer between the cytoplasm and cell exterior was further supported by proteomics studies. In comparison with non-heme proteins, the presence of numerous proton-containing groups in the heme groups causes additional challenges to the full protein assignment and structure calculation. Here, we report the complete assignment of the heme proton signals together with the 1H and 15N backbone and side chain assignments of the reduced form of PpcA.

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