The genomes of several cyanobacteria show the existence of gene clusters encoding subunits I, II, and III of aa 3 -type cytochrome c oxidase. The enzyme occurs on both plasma and thylakoid membranes of these oxygenic phototrophic prokaryotes. Here we report the expression and purification of a truncated subunit II copper A (Cu A ) domain (i.e. the electron entry and donor binding site) of cytochrome c oxidase from the cyanobacterium Synechocystis PCC 6803 in high yield. The water-soluble purple redox-active bimetallic center displays a relatively low standard reduction potential of 216 mV. Its absorption spectrum at pH 7 is similar to that of other soluble fragments from aa 3 -type oxidases, but the insensitivity of both absorbance and circular dichroism spectra to pH suggests that it is less exposed to the aqueous milieu compared with other Cu A domains. Oxidation of horse heart cytochrome c by the bimetallic center follows monophasic kinetics. At pH 7 and low ionic strength the bimolecular rate constant is (2.1 ؎ 0.3) ؋ 10 4 M ؊1 s ؊1 , and the rates decrease upon the increase of ionic strength. Sequence alignment and modeling of cyanobacterial Cu A domains show several peculiarities such as: (i) a large insertion located between the second transmembrane region and the putative hydrophobic cytochrome c docking site, (ii) the lack of acidic residues shown to be important in the interaction between cytochrome c and Paracoccus Cu A domain, and (iii) an extended C terminus similar to Escherichia coli ubiquinol oxidase.
It has been shown that efficient functioning of photosynthesis and respiration in the cyanobacterium Synechocystis PCC 6803 requires the presence of either cytochrome c6 or plastocyanin. In order to check whether the blue copper protein plastocyanin can act as electron donor to cytochrome c oxidase, we investigated the intermolecular electron transfer kinetics between plastocyanin and the soluble CuA domain (i.e. the donor binding and electron entry site) of subunit II of the aa3-type cytochrome c oxidase from Synechocystis. Both copper proteins were expressed heterologously in Escherichia coli. The forward and the reverse electron transfer reactions were studied yielding apparent bimolecular rate constants of (5.1+/-0.2) x 10(4) M(-1) s(-1) and (8.5+/-0.4) x 10(5) M(-1) s(-1), respectively (20 mM phosphate buffer, pH 7). This corresponds to an apparent equilibrium constant of 0.06 in the physiological direction (reduction of CuA), which is similar to Keq values calculated for the reaction between c-type cytochromes and the soluble fragments of other CuA domains. The potential physiological role of plastocyanin in cyanobacterial respiration is discussed.
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The goal of the development of the current formulation based on 1R-trans Phenothrin and Prallethrin was to show how the microencapsulation can be applied to control the release rate in form of Capsule Suspension (CS) versus "free" non-encapsulated form, therefore to obtain a safer, more sustainable and less toxic biocidal product for its use in household or certain agricultural areas. In this paper, we primarily present the analytical data for the determination of the release rate by applying our microencapsulation technology versus the release of the free material including the biological data for the residual efficacy. In general, this approach represents an effective tool to describe the characteristics of the microcapsule wall in our CS formulation. The release rate was quantified by adopting the Franz cell diffusion method (OECD Test Guideline 428) in order to provide information on release of the active in CS form by using a membrane with a pore size of 0.20 µm.
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