Enrichment experiments and primary production at Sagres (SW Portugal)

Water was collected from the Sagres station (SW Portugal) in September 2002, at a site adjacent to the upwelling centre of Cabo São Vicente, during relaxation of upwelling conditions. Surface and depth samples were enriched with inorganic nutrients in order to evaluate their relative influence on the microalgal assemblage. Small-scale, short-term bioassays involved separate in vitro additions of nitrogen and phosphorus. Enrichments with nitrogen led to a general increase of primary production, suggesting nitrogen as the primary potential nutrient limiting microalgal growth during this period, as well as altering the relative microplanktonic composition in favour of diatoms.     

An Enzymatic Platform for the Synthesis of Isoprenoid Precursors

The isoprenoid family of compounds is estimated to contain ∼65,000 unique structures including medicines, fragrances, and biofuels. Due to their structural complexity, many isoprenoids can only be obtained by extraction from natural sources, an inherently risky and costly process. Consequently, the biotechnology industry is attempting to genetically engineer microorganisms that can produce isoprenoid-based drugs and fuels on a commercial scale. Isoprenoid backbones are constructed from two, five-carbon building blocks, isopentenyl 5-pyrophosphate and dimethylallyl 5-pyrophosphate, which are end-products of either the mevalonate or non-mevalonate pathways. By linking the HMG-CoA reductase pathway (which produces mevalonate) to the mevalonate pathway, these building block can be synthesized enzymatically from acetate, ATP, NAD(P)H and CoA. Here, the enzymes in these pathways are used to produce pathway intermediates and end-products in single-pot reactions and in remarkably high yield, ∼85%. A strategy for the regio-specific incorporation of isotopes into isoprenoid backbones is developed and used to synthesize a series of isotopomers of diphosphomevalonate, the immediate end-product of the mevalonate pathway. The enzymatic system is shown to be robust and capable of producing quantities of product in aqueous solutions that meet or exceed the highest levels achieved using genetically engineered organisms in high-density fermentation.     

EPR characterization of the new Qrc complex from sulfate reducing bacteria and its ability to form a supercomplex with hydrogenase and TpIc3

The Quinone-reductase complex (Qrc) is a respiratory complex with Type I cytochrome c(3):menaquinone reductase activity, recently described in sulfate-reducing bacteria. Qrc is related to the complex iron-sulfur molybdoenzyme family and to the alternative complex III. In this work we report a detailed characterization of the redox properties of the metal cofactors of Qrc using EPR spectroscopy, which allowed the determination of the reduction potentials of five out of six hemes c, one [3Fe-4S](1+/0) center and the three [4Fe-4S](2+/1+) centers. In addition, we show that Qrc forms a supercomplex with [NiFe] hydrogenase and TpIc(3), its physiological electron donors.     

Artefacts induced on <Emphasis Type="Italic">c</Emphasis>-type haem proteins by electrode surfaces

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.