Tryptophan 121 of Subunit II Is the Electron Entry Site to Cytochrome-c Oxidase in Paracoccus denitrificans INVOLVEMENT OF A HYDROPHOBIC PATCH IN THE DOCKING REACTION

To investigate the contribution of hydrophobic residues to the molecular recognition of cytochrome c with cytochrome oxidase, we mutated several hydrophobic amino acids exposed on subunit II of the Paracoccus denitrificans oxidase.KM and kcat values and the bimolecular rate constant were determined under steady- or presteady-state conditions, respectively. We present evidence that Trp-121 which is surrounded by a hydrophobic patch is the electron entry site to oxidase. Mutations in this cluster do not affect the binding of cytochrome c as the KMremains largely unchanged. Rather, the kcat is reduced, proposing that these hydrophobic residues are required for a fine tuning of the redox partners in the initial collisional complex to obtain a configuration optimal for electron transfer

Control of electron transfer by the electrochemical potential gradient in cytochrome-c oxidase reconstituted into phospholipid vesicles.

The kinetics of electron transfer between cytochrome-c oxidase and ruthenium hexamine has been characterized using the native enzyme or its cyanide complex either solubilized by detergent (soluble cytochrome oxidase) or reconstituted into artificial phospholipid vesicles (cytochrome oxidase-containing vesicles). Ru(NH3)2+6 (Ru(II] reduces oxidized cytochrome a, following (by-and-large) bimolecular kinetics; the second order rate constant using the cyanide complex of the enzyme is 1.5 x 10(6) M-1 s-1, for the enzyme in detergent, and slightly higher for COV. In the case of COV the kinetics are not affected by the addition of ionophores. Upon mixing fully reduced cytochrome oxidase with oxygen (in the presence of excess reductants), the oxidation leading to the pulsed enzyme is followed by a steady state phase and (eventually) by complete re-reduction. When the concentrations of dioxygen and oxidase are sufficiently low (micromolar range), the time course of oxidation can be resolved by stopped flow at room temperature, yielding an apparent bimolecular rate constant of 5 x 10(7) M-1 s-1. After exhaustion of oxygen and end of steady state, re-reduction of the pulsed enzyme by the excess Ru(II) is observed; the concentration dependence shows that the rate of re-reduction is limited at 3 s-1 in detergent; this limiting value is assigned to the intramolecular electron transfer process from cytochrome a-Cua to the binuclear center. Using the reconstituted enzyme, the internal electron transfer step is sensitive to ionophores, increasing from 2-3 to 7-8 s-1 upon addition of valinomycin and carbonyl cyanide m-chlorophenylhydrazone. This finding indicates for the first time an effect of the electrochemical potential across the membrane on the internal electron transfer rate; the results are compared with expectations based on the hypothesis formulated by Brunori et al. (Brunori, M., Sarti, P., Colosimo, A., Antonini, G., Malatesta, F., Jones, M.G., and Wilson, M.T. (1985) EMBO J. 4, 2365-2368), and their bioenergetic relevance is discussed with reference to the proton pumping activity of the enzyme

Insights into the Catalytic Mechanism of Glutathione S-Transferase: The Lesson from Schistosoma haematobium

SummaryGlutathione S-transferases (GSTs) are involved in detoxification of xenobiotic compounds and in the biosynthesis of important metabolites. All GSTs activate glutathione (GSH) to GS−; in many GSTs, this is accomplished by a Tyr at H-bonding distance from the sulfur of GSH. The high-resolution structure of GST from Schistosoma haematobium revealed that the catalytic Tyr occupies two alternative positions, one external, involving a π-cation interaction with the conserved Arg21, and the other inside the GSH binding site. The interaction with Arg21 lowers the pKa of the catalytic Tyr10, as required for catalysis. Examination of several other GST structures revealed the presence of an external pocket that may accommodate the catalytic Tyr, and suggested that the change in conformation and acidic properties of the catalytic Tyr may be shared by other GSTs. Arginine and two other residues of the external pocket constitute a conserved structural motif, clearly identified by sequence comparison

Role of Small farming in food security and sustainability: a case study for Tuscany (IT)

The debate on the size of farming, and its relevance for policy purposes, has come back to the fore in recent years. The “International Year of Family Farming and Smallholder Farming”, held by FAO in 2014, aimed to raise the profile of family and smallholder farming worldwide”. During EXPO 2015, a debate followed on structural developments in agriculture and the implications for the competitiveness and sustainability of the agri-food sector and rural areas. Despite a declining number of agricultural holdings and a gradual increase in average farm size, the agricultural sector is largely composed by farms with less than 5 ha of agricultural land and a standard output below 4 000 euro per year. Beyond economic size and value of production, other criteria (e.g. labour units and family involvement can be adopted), alone or in combination, to define size of farming. Academic literature provides a mixed picture on weather a declining number of farms, and a gradual increase in size should be welcomed or contrasted. Oppositional arguments contrasting smaller and larger farm structures with respect to sustainability and food and nutrition security, are nourished by the lack of sufficient or unambiguous scientific evidence. A first line of thought stresses the distinctiveness of smaller farms in delivering food security and sustainability (Rabinowicz, 2014) and the capacity of small farms to mobilize resources additional to those procured through market exchange (van der Ploeg, 2013). A second line of thought considers size as a nonrelevant criterion to assess the performance on food security and sustainability (OECD, 2005), supporting the view on steering behaviours oriented towards improved sustainability, regardless of size. Dualistic debates often focus on some aspects of sustainability and neglect others (Kirwan et al. 2017) or overlook the importance of complementarity between complex agro-food systems and territoriality, as local context largely affects what structural change is desirable at territorial level (Darnhofer et al. 2010). This contribution presents the conceptual and analytical framework adopted in a research project named SALSA, “Small farms, small food businesses and sustainable food and nutrition security (FNS)”, and provides an illustration on one of the 35 reference regions selected. The analysis considers Lucca province in Tuscany and aims at generating preliminary insights in relation to the role of small farms in food and nutrition security

Agricoltori come “custodi del territorio”: il caso della Valle del Serchio in Toscana

The article focuses on the project “Custody of the Territory”, an initiative promoted by a territorial authority of a mountainous area of the Tuscany region (Unione dei Comuni Media Valle del Serchio) which set an agreement with local farmers to improve the hydro-geological management of the district. This innovative flooding prevention strategy, based on farmers’ knowledge and capabilities, shows that an effective hydro-geological management of the territory in mountainous areas should involve not only changes in farmers’ practices, but also a more structural shift regarding their identity, motivations and attitudes. The article highlights the need of implementing integrated policy tools based not only on compensation but also on incentives, communication and learning opportunities, since farmers’ knowledge and joint learning (amongst farmers, institutions, technicians, academics) are very important issues for an effective provision of environmental services through agriculture.L’articolo descrive il progetto Custodia del Territorio, un’iniziativa promossa dall’Unione dei Comuni della Media Valle del Serchio (LU) che ha stipulato contratti di sorveglianza e manutenzione del reticolo idraulico con gli agricoltori locali all’interno della propria area di competenza. Questa strategia innovativa per la prevenzione idraulica, basata sul rafforzamento delle conoscenze e delle abilità degli agricoltori, evidenzia come un’effettiva gestione degli aspetti idraulici nelle aree montane dovrebbe includere non solo cambiamenti nelle pratiche degli agricoltori, ma anche un cambiamento più strutturale che riguarda le loro identità, motivazioni e attitudini. L’articolo sottolinea la necessità di favorire delle politiche integrate basate non solo sulla corresponsione di pagamenti, ma anche su incentivi, comunicazione e opportunità di apprendimento per gli attori locali. Si evidenzia, inoltre, la necessità di sviluppare forme di conoscenze condivise (tra agricoltori, istituzioni, tecnici e ricercatori) per rendere più efficace l’erogazione di servizi ambientali da parte dell’agricoltura

Small farms’ strategies between self-provision and socio-economic integration: effects on food system capacity to provide food and nutrition security

Small farms’ contribution to food and nutrition security (FNS) is widely acknowledged; however, the diversity of context-specific characteristics of small farms is still barely documented in terms of farm strategies and household dynamics. The paper analyses this contribution in connection with the strategies related to the destination of the produce, with specific attention to the balance between food self-provisioning and economic integration. The analysis of self-provisioning relies on the assumptions that (i) production and consumption decisions cannot be analysed separately when they are attributed to the same entity and that (ii) family farm strategic choices are influenced by both business outcomes and household’’s welfare. The analysis of economic integration hinges on Polanyi’’s categories of market, reciprocity and redistribution as the three main modes of economic integration of a farm within its environment. We have collected information from a range of farmers in the Lucca province (northern Tuscany, Italy) and key stakeholders, through interviews, focus groups and field visits. The results of our analysis highlight the different ways small farms’ contribution to FNS in relation to each mode of economic integration adopted by the small farms. The different forms of this contribution can be identified at two levels: (i) internal to the farming household and (ii) external (i.e. referred to the community and broader society). A concept of food quality encompassing local sustainability, cultural heritage and social cohesion, is crucial to valorise, through appropriate policies, the specificities of small farms’ contribution to FNS

Sulfolobus acidocaldarius terminal oxidase. A kinetic investigation and its structural interpretation.

Abstract The thermoacidophilic archaebacterium Sulfolobus acidocaldarius possesses a very unusual terminal oxidase. We report original kinetic experiments on membranes of this microorganism carried out by stopped flow, using time-resolved optical spectroscopy combined with singular value decomposition analysis. The reduced-oxidized kinetic difference spectrum of the Sulfolobus membranes is characterized by three significant peaks in the visible region at 605, 586, and 560 nm. The 605-nm peak and part of the 586-nm peak (cytochrome aa3-type quinol oxidase) are reduced synchronously by both ascorbate plus N,N,N',N'-tetramethyl-p-phenylendiamine (TMPD) and dithionite, and they are very rapidly oxidized by molecular oxygen. A second pool of cytochromes seems to contribute to the 586-nm peak which is not reduced by ascorbate plus TMPD and reacts very slowly with dithionite. The b-type cytochromes (560 nm peak) are reduced by both reductants and are essentially "non-autoxidizable" at room temperature. Only one CO binding site with spectral features, kinetic properties, and ligand affinity not very dissimilar from those of mammalian cytochrome oxidase can be detected in the ascorbate-reduced membranes. On the contrary, a second CO binding site having unusual properties for aa3 terminal oxidases can be detected in the dithionite-reduced membranes

Nitrite reductase from Pseudomonas aeruginosa: Sequence of the gene and the protein

AbstractThe gene coding for nitrite reductase of Pseudomonas aeruginosa has been cloned and its sequence determined. The coding region is 1707 bp long and contains information for a polypeptide chain of 568 amino acids. The sequence of the mature protein has been confirmed independently by extensive amino acid sequencing. The amino-terminus of the mature protein is located at Lys-26; the preceding 25 residue long extension shows the features typical of signal peptides. Therefore the enzyme is probably secreted into the periplasmic space. The mature protein is made of 543 amino acid residues and has a molecular mass of 60204 Da. The c-heme-binding domain, which contains the only two Cys of the molecule, is located at the amino-terminal region. Analysis of the protein sequence in terms of hydrophobicity profile gives results consistent with the fact that the enzyme is fully water soluble and not membrane bound; the most hydrophilic region appears to correspond to the c-heme domain. Secondary structure predictions are in general agreement with previous analysis of circular dichroic data

Polarclean as a Sustainable Reaction Medium for the Waste Minimized Synthesis of Heterocyclic Compounds

Herein we report the use of Rhodiasolv© Polarclean as a novel polar aprotic solvent for the synthesis of decorated heterocycles via dipolar cycloaddition (isooxazoles) or intramolecular C–H functionalization processes (benzo-fused chromenes). The use of Polarclean allowed to isolate the final products in good yields by simple solid filtration or liquid-liquid phase separation, avoiding the need for chromatographic purification. Moreover, since in the synthesis of benzo-fused chromenes, the metal catalyst is retained in Polarclean, the catalyst/reaction medium can be easily reused for consecutive reaction runs, without any apparent loss in efficiency. This methodology is associated with a limited waste production. These results extend the applicability of Polarclean as a promising reaction medium for the replacement of toxic petrol-based solvent