No abstract

Proteins containing reactive cysteine residues (protein-Cys) are receiving increased attention as mediators of hydrogen peroxide signaling. These proteins are mainly identified by mining the thiol proteomes of oxidized protein-Cys in cells and tissues. However, it is difficult to determine if oxidation occurs through a direct reaction with hydrogen peroxide or by thiol-disulfide exchange reactions. Kinetic studies with purified proteins provide invaluable information about the reactivity of protein-Cys residues with hydrogen peroxide. Previously, we showed that the characteristic UV-Vis spectrum of horseradish peroxidase compound I, produced from the oxidation of horseradish peroxidase by hydrogen peroxide, is a simple, reliable, and useful tool to determine the second-order rate constant of the reaction of reactive protein-Cys with hydrogen peroxide and peroxynitrite. Here, the method is fully described and extended to quantify reactive protein-Cys residues and micromolar concentrations of hydrogen peroxide. Members of the peroxiredoxin family were selected for the demonstration and validation of this methodology. In particular, we determined the pK(a) of the peroxidatic thiol of rPrx6 (5.2) and the second-order rate constant of its reactions with hydrogen peroxide ((3.4 ± 0.2) × 10⁷M⁻¹ s⁻¹) and peroxynitrite ((3.7 ± 0.4) × 10⁵ M⁻¹ s⁻¹) at pH 7.4 and 25°C.

Deletion of COQ10 in Saccharomyces cerevisiae elicits a respiratory defect characterized by the absence of cytochrome c reduction, which is correctable by the addition of exogenous diffusible coenzyme Q2. Unlike other coq mutants with hampered coenzyme Q6 (Q6) synthesis, coq10 mutants have near wild-type concentrations of Q6. In the present study, we use Q-cycle inhibitors of the coenzyme QH2-cytochrome c reductase (bc1 complex) to assess electron transfer properties of coq10 cells. Our results show that coq10 mutants respond to antimycin A, indicating an active Q cycle in these mutants, even though they are unable to transport electrons through cytochrome c and are not responsive to myxothiazol. EPR spectroscopic analysis also suggests that wild type and coq10 mitochondria accumulate similar amounts of Q6 semiquinone, despite a lower steady state level of bc1 complex in the coq10 cells. Confirming the reduced respiratory chain state in coq10 cells, we found that the expression of the Aspergillus fumigatus alternative oxidase in these cells leads to a decrease in antimycin-dependent H2O2 release and improves their respiratory growth.

Peroxiredoxins are receiving increasing attention as defenders against oxidative damage and sensors of hydrogen peroxide-mediated signaling events. In the yeast Saccharomyces cerevisiae, deletion of one or more isoforms of the peroxiredoxins is not lethal but compromises genome stability by mechanisms that remain under scrutiny. Here, we show that cytosolic peroxiredoxin-null cells (tsa1⌬tsa2⌬) are more resistant to hydrogen peroxide than wildtype (WT) cells and consume it faster under fermentative conditions. Also, tsa1⌬tsa2⌬ cells produced higher yields of the 1-hydroxyethyl radical from oxidation of the glucose metabolite ethanol, as proved by spin-trapping experiments. A major role for Fenton chemistry in radical formation was excluded by comparing WT and tsa1⌬tsa2⌬ cells with respect to their levels of total and chelatable metal ions and of radical produced in the presence of chelators. The main route for 1-hydroxyethyl radical formation was ascribed to the peroxidase activity of Cu,Znsuperoxide dismutase (Sod1), whose expression and activity increased ϳ5-and 2-fold, respectively, in tsa1⌬tsa2⌬ compared with WT cells. Accordingly, overexpression of human Sod1 in WT yeasts led to increased 1-hydroxyethyl radical production. Relevantly, tsa1⌬tsa2⌬ cells challenged with hydrogen peroxide contained higher levels of DNA-derived radicals and adducts as monitored by immuno-spin trapping and incorporation of 14 C from glucose into DNA, respectively. The results indicate that part of hydrogen peroxide consumption by tsa1⌬tsa2⌬ cells is mediated by induced Sod1, which oxidizes ethanol to the 1-hydroxyethyl radical, which, in turn, leads to increased DNA damage. Overall, our studies provide a pathway to account for the hypermutability of peroxiredoxin-null strains.Living organisms are constantly exposed to oxygen-and nitrogen-derived reactive species that are produced by normal metabolic activity and in response to external stimuli. To protect themselves against the toxicity of these species, aerobic organisms have evolved a range of defense mechanisms (1). Among these, a family of cysteine-based peroxidases, currently named peroxiredoxins, has attracted considerable attention because of its ubiquity and versatility. These enzymes have been shown to detoxify hydrogen peroxide, organic peroxides and peroxynitrite through oxidation of their reactive cysteine residues, which are recycled back by reducing equivalents provided by thioredoxin and other thiol-electron donors (reviewed in Refs. 2-5). In addition to detoxifying peroxides, specific peroxiredoxins have been shown to act as molecular chaperones (6, 7) and to play roles in regulating hydrogen peroxide-mediated cell signaling events (3,8,9).Many organisms have multiple peroxiredoxins. Six peroxiredoxins have been identified in human cells, and five in the yeast Saccharomyces cerevisiae. Peroxiredoxin isoforms are distributed to different locations within the cell, and two of the yeast peroxiredoxins, Tsa1 and Tsa2, are cytosolic (10). Tsa1 was the first peroxiredox...

A reciclagem de papel diminui o corte de árvores e os gastos com água e energia naprodução de papel. O “papel semente” é um papel reciclado que contém sementesem sua composição e pode ser “plantado” após o uso. No IFC–Campus Camboriú,parte dos papéis descartados não pode ser reaproveitado para rascunho e por estemotivo, este projeto propôs a produção de papel semente para diminuição dovolume de resíduos sólidos. Foram testadas diferentes estratégias para a confecçãode papel, com diferentes sementes – o que permitiu a produção de papéis para usosbreves como crachás e cartões.