Influence of association state and DNA binding on the O2-reactivity of [4Fe-4S] fumarate and nitrate reduction (FNR) regulator

The fumarate and nitrate reduction (FNR) regulator is the master switch for the transition between anaerobic and aerobic respiration in Escherichia coli. Reaction of dimeric [4Fe-4S] FNR with O2 results in conversion of the cluster into a [2Fe-2S] form, via a [3Fe-4S] intermediate, leading to the loss of DNA binding through dissociation of the dimer into monomers. In the present paper, we report studies of two previously identified variants of FNR, D154A and I151A, in which the form of the cluster is decoupled from the association state. In vivo studies of permanently dimeric D154A FNR show that DNA binding does not affect the rate of cluster incorporation into the apoprotein or the rate of O2-mediated cluster loss. In vitro studies show that O2-mediated cluster conversion for D154A and the permanent monomer I151A FNR is the same as in wild-type FNR, but with altered kinetics. Decoupling leads to an increase in the rate of the [3Fe-4S]1+ into [2Fe-2S]2+ conversion step, consistent with the suggestion that this step drives association state changes in the wild-type protein. We have also shown that DNA-bound FNR reacts more rapidly with O2 than FNR free in solution, implying that transcriptionally active FNR is the preferred target for reaction with O2

Alternative Oxidase Mediates Pathogen Resistance in Paracoccidioides brasiliensis Infection

Thermally dimorphic pathogenic fungi are responsible for potentially life-threatening diseases of immunocompetent and immunocompromised individuals. These microorganisms grow as conidia-producing mycelia in the environment, which when inhaled by the host convert to the pathogenic yeast form at 37°C. During adaptation and growth, fungi interact with host immune cells and must cope with defense mechanisms such as imposed-oxidative stress (e.g., reactive oxygen species; ROS). Alternative oxidase (AOX) is an enzyme recently implicated in the reduction of ROS production by the mitochondria when triggered by external stimuli, such as temperature and ROS. During this work we have evaluated the relevance of AOX during infection with Paracoccidioides brasiliensis, the etiological agent of one of the most prevalent mycoses in Latin America, paracoccidioidomycosis. We show that PbAOX gene expression is stimulated after interaction with alveolar macrophages or in the presence of H2O2 and is essential for survival against fungicidal activity of both the immune cells and the ROS compound. Moreover, decreasing PbAOX gene expression in P. brasiliensis led to increased survival of infected mice. Altogether, our data supports a relevant role for AOX in the virulence of P. brasiliensis

Carotenoids Play a Positive Role in the Degradation of Heterocycles by Sphingobium yanoikuyae

BACKGROUND: Microbial oxidative degradation is a potential way of removing pollutants such as heterocycles from the environment. During this process, reactive oxygen species or other oxidants are inevitably produced, and may cause damage to DNA, proteins, and membranes, thereby decreasing the degradation rate. Carotenoids can serve as membrane-integrated antioxidants, protecting cells from oxidative stress. FINDINGS: Several genes involved in the carotenoid biosynthetic pathway were cloned and characterized from a carbazole-degrading bacterium Sphingobium yanoikuyae XLDN2-5. In addition, a yellow-pigmented carotenoid synthesized by strain XLDN2-5 was identified as zeaxanthin that was synthesized from β-carotene through β-cryptoxanthin. The amounts of zeaxanthin and hydrogen peroxide produced were significantly and simultaneously enhanced during the biodegradation of heterocycles (carbazole < carbazole + benzothiophene < carbazole + dibenzothiophene). These higher production levels were consistent with the transcriptional increase of the gene encoding phytoene desaturase, one of the key enzymes for carotenoid biosynthesis. CONCLUSIONS/SIGNIFICANCE: Sphingobium yanoikuyae XLDN2-5 can enhance the synthesis of zeaxanthin, one of the carotenoids, which may modulate membrane fluidity and defense against intracellular oxidative stress. To our knowledge, this is the first report on the positive role of carotenoids in the biodegradation of heterocycles, while elucidating the carotenoid biosynthetic pathway in the Sphingobium genus

Cross-talk between S-nitrosylation and S-glutathionylation in control of the Na,K-ATPase regulation in hypoxic heart

Oxygen-induced regulation of Na,K-ATPase was studied in rat myocardium. In rat heart Na,K-ATPase responded to hypoxia with a dose-dependent inhibition in hydrolytic activity. Inhibition of Na,K-ATPase in hypoxic rat heart was associated with decrease in NO production and progressive oxidative stress. Accumulation of oxidized glutathione (GSSG) and decrease in NO availability in hypoxic rat heart were followed by a decrease in S-nitrosylation and up-regulation of S-glutathionylation of the catalytic α subunit of the Na,K-ATPase. Induction of S-glutathionylation of the α subunit by treatment of tissue homogenate with GSSG resulted in complete inhibition of the enzyme in rat a myocardial tissue homogenate. Inhibitory effect of GSSG in rat sarcolemma could be significantly decreased upon activation of NO synthases. we have further tested if oxidative stress and suppression of the Na,K-ATPase activity are observed in hypoxic heart of two subterranean hypoxia-tolerant blind mole species (Spalax galili and Spalax judaei). In both hypoxia tolerant Spalax species activity of the enzyme and tissue redox state were maintained under hypoxic conditions. However, localisation of cysteines within the catalytic subunit of the Na,K-ATPase was preserved and induction of S-glutathionylation by GSSG in tissue homogenate inhibited the Spalax ATPase as efficiently as in rat heart. The obtained data indicate that oxygen-induced regulation of the Na,K-ATPase in the heart is mediated by a switch between S-glutathionylation and S-nitrosylation of the regulatory thiol groups localized at the catalytic subunit of the enzyme