Superoxide does react with peroxides : direct observation of the haber-weiss reaction in the gas phase

The fact that nature provides specific enzymes to selectively remove superoxide (O2.−) from aerobic organisms, namely, the superoxide dismutase enzymes,1 has led to the suggestion that this radical ion may cause the oxidative damage associated with degradative disease and aging.2 Intriguingly, however, superoxide itself is relatively unreactive toward most cellular components, which suggests that dismutase enzymes may ultimately protect the cell against more pernicious oxidants formed from superoxide. As such, there is increasing interest in the endogenous chemistry of superoxide and the pathways by which it might beget more reactive oxygen species. Protonation of superoxide to form the hydroperoxyl radical (HOO.) and dismutation of the same species to hydrogen peroxide (HOOH), with subsequent metal-catalyzed reduction to the hydroxyl radical (HO.), are well-characterized processes in which both the HOO. and HO. radicals are significantly more reactive than their common progenitor.2 Recent examples, however, have also linked superoxide to the putative production of singlet oxygen3 and ozone,4, 5 although the definitive characterization of these chemistries in the cellular milieu has proved challenging

Cystine-mediated oxidative defence in Lactobacillus reuteri BR11

Lactobacillus reuteri BR11 possesses an abundant cystine uptake (Cyu) ABC-transporter that was previously found to be involved in a novel mechanism of oxidative defence mediated by cystine. The current study aimed to elucidate this mechanism with a focus on the role of the co-transcribed cystathionine ã-lyase (Cgl). Growth studies of wild-type L. reuteri BR11 and mutants inactivated in cgl and the cystine-binding protein encoding gene cyuC showed that in contrast to the Cyu transporter, whose inactivation led to growth arrest in aerated cultures, Cgl is not crucial for oxidative defence. However, the role of Cgl in oxidative defence became apparent in the presence of severe oxidative damage and cysteine deprivation. Cysteine was found to be protective against oxidative stress, and the action of Cgl in both cysteine biosynthesis and degradation poses a seemingly futile pathway that deprives the intracellular cysteine pool. To further characterise the relationship between Cgl activity and cysteine and their roles in oxidative defence, enzymatic assays were performed on purified Cgl, and intracellular concentrations of cysteine, cystathionine and methionine were determined. Cgl was highly active towards cystine and cystathionine and less active towards cysteine in vitro, suggesting the main function of Cgl to be cysteine biosynthesis. Cysteine was found at high concentrations in the cell, but the levels were not significantly affected by inactivation of cgl or growth under aerobic conditions. It was concluded that both anabolic and catabolic activities of Cgl towards cysteine contribute to oxidative defence, the former by maintaining an intracellular reservoir of thiol analogous to glutathione, and the latter by producing H2S which is readily secreted, thus creating a reducing extracellular environment. The significance of the Cyu transporter to the physiology of L. reuteri BR11 prompted a phylogenetic study to determine its presence in bacteria. Orthologs of the Cyu transporter that are closest matches to the Cyu transporter are only limited to several species of Lactobacillus and Leuconostoc. Outside the Lactobacillales order, the closest matching orthologs belong to Proteobacteria, and there are more orthologs in Proteobacteria than non-Lactobacillales Firmicutes, suggesting that the Cyu transporter locus was present in the ancestor of the Proteobacteria and Firmicutes, and over evolutionary time has been lost or diverged in many Firmicutes. The clustering of the Cyu transporter locus with a gene encoding a Cgl family protein is even rarer. It was only found in L. reuteri, Lactobacillus vaginalis, Weissella paramesenteroides, the Lactobacillus casei group, and several Campylobacter sp. An accompanying phylogenetic study of L. reuteri BR11 using multi-locus sequence analysis showed that L. reuteri BR11 had diverged from more than 100 strains of L. reuteri isolated from various hosts and geographical locations. However, comparison with other Lactobacillus species supported the current classification of BR11 as L. reuteri. The most closely related species to L. reuteri is L. vaginalis or Lactobacillus antri, depending on the housekeeping gene used for analysis. The close evolutionary relationship of L. vaginalis to L. reuteri and the high degree of sequence identity between the cgl-cyuABC loci in both species suggest that the Cyu system is highly likely to perform similar functions in L. vaginalis. In search of other genes that function in oxidative defence, a number of mutants which were inactivated in genes that confer increased resistance to oxidative stress in other bacteria were constructed. The genes targeted were ahpC (peroxidase component of the alkyl hydroperoxide reductase system), tpx (thiol peroxidase), osmC (osmotically induced protein C), mntH (Mn2+/Fe2+ transporter), gshA (ã-glutamylcysteine synthetase) and msrA (methionine sulfoxide reductase). The ahpC and mntH mutants had slightly lower minimum inhibitory concentrations of organic peroxides, suggesting these genes might be involved in resistance to organic peroxides in L. reuteri. However, none of the mutants exhibited growth defects in aerated cultures, in stark contrast to the cyuC mutant. This may be due to compensatory functions of other genes, a hypothesis which cannot be tested until a robust protocol for constructing markerless multiple gene deletion mutants in L. reuteri is developed. These results highlight the importance of the Cyu transporter in oxidative defence and provide a foundation for extending the research of this system in other bacteria.

Direct Detection of a Persistent Carbonyloxyl Radical in the Gas Phase

Long lived: Carbonyloxyl radicals (RCO2 .) are reactive intermediates that play key roles in initiating polymerization reactions. This reactivity also makes their direct observation difficult. For the first time a persistent organic RCO2 . radical is detected in the gas phase, its extraordinary longevity is attributed to the high barrier towards fragmentation owing to the endothermicity of the decarboxylation products. Grant Numbers ARC/DP0986738, ARC/DP120102922, ARC/DE120100467

Fragmentations of deprotonated alkyl hydroperoxides (ROO-) upon collisional activation : A combined experimental and computational study

The collision-induced dissociation ( CID) mass spectra of the \[M-H](-) anions of methyl, ethyl, and tert-butyl hydroperoxides have been measured over a range of collision energies in a flowing afterglow - selected ion flow tube (FA-SIFT) mass spectrometer. Activation of the CH3OO- anion is found to give predominantly HO- fragment anions whilst CH3CH2OO- and (CH3)(3)COO- produce HOO- as the major ionic fragment. These results, and other minor fragmentation pathways, can be rationalized in terms of unimolecular rearrangement of the activated anions with subsequent decomposition. The rearrangement reactions occur via initial abstraction of a proton from the alpha-carbon in the case of CH3OO- or the beta-carbon for CH3CH2OO- and (CH3)(3)COO-. Electronic structure calculations suggest that for the CH3CH2OO- anion, which can theoretically undergo both alpha- and beta-proton abstraction, the latter pathway, resulting in HOO- + CH2CH2, is energetically preferred.