Superoxide reductase from Giardia intestinalis: structural characterization of the first sor from a eukaryotic organism shows an iron centre that is highly sensitive to photoreduction

Superoxide reductase (SOR), which is commonly found in prokaryotic organisms, affords protection from oxidative stress by reducing the superoxide anion to hydrogen peroxide. The reaction is catalyzed at the iron centre, which is highly conserved among the prokaryotic SORs structurally characterized to date. Reported here is the first structure of an SOR from a eukaryotic organism, the protozoan parasite Giardia intestinalis (GiSOR), which was solved at 2.0 Å resolution. By collecting several diffraction data sets at 100 K from the same flash-cooled protein crystal using synchrotron X-ray radiation, photoreduction of the iron centre was observed. Reduction was monitored using an online UV-visible microspectrophotometer, following the decay of the 647 nm absorption band characteristic of the iron site in the glutamate-bound, oxidized state. Similarly to other 1Fe-SORs structurally characterized to date, the enzyme displays a tetrameric quaternary-structure arrangement. As a distinctive feature, the N-terminal loop of the protein, containing the characteristic EKHxP motif, revealed an unusually high flexibility regardless of the iron redox state. At variance with previous evidence collected by X-ray crystallography and Fourier transform infrared spectroscopy of prokaryotic SORs, iron reduction did not lead to dissociation of glutamate from the catalytic metal or other structural changes; however, the glutamate ligand underwent X-ray-induced chemical changes, revealing high sensitivity of the GiSOR active site to X-ray radiation damage

Evidence for detrimental cross interactions between reactive oxygen and nitrogen species in Leber's hereditary optic neuropathy cells

Here we have collected evidence suggesting that chronic changes in the NO homeostasis and the rise of reactive oxygen species bioavailability can contribute to cell dysfunction in Leber’s hereditary optic neuropathy (LHON) patients.We report that peripheral blood mononuclear cells (PBMCs), derived froma female LHON patient with bilateral reduced vision and carrying the pathogenic mutation 11778/ND4, display increased levels of reactive oxygen species (ROS) and reactive nitrogen species (RNS), as revealed by flow cytometry, fluorometric measurements of nitrite/nitrate, and 3-nitrotyrosine immunodetection. Moreover, viability assays with the tetrazolium dye MTT showed that lymphoblasts from the same patient are more sensitive to prolonged NO exposure, leading to cell death. Taken together these findings suggest that oxidative and nitrosative stress cooperatively play an important role in driving LHON pathology when excess NO remains available over time in the cell environment

Antigiardial activity of novel triazolyl-quinolone-based chalcone derivatives:when oxygen makes the difference

Giardiasis is a common diarrheal disease worldwide caused by the protozoan parasite Giardia intestinalis. It is urgent to develop novel drugs to treat giardiasis, due to increasing clinical resistance to the gold standard drug metronidazole (MTZ). New potential antiparasitic compounds are usually tested for their killing efficacy against G. intestinalis under anaerobic conditions, in which MTZ is maximally effective. On the other hand, though commonly regarded as an ‘anaerobic pathogen,’ G. intestinalis is exposed to relatively high O2 levels in vivo, living attached to the mucosa of the proximal small intestine. It is thus important to test the effect of O2 when searching for novel potential antigiardial agents, as outlined in a previous study [Bahadur et al. (2014) Antimicrob. Agents Chemother. 58, 543]. Here, 45 novel chalcone derivatives with triazolyl-quinolone scaffold were synthesized, purified, and characterized by high resolution mass spectrometry, 1H and 13C nuclear magnetic resonance and infrared spectroscopy. Efficacy of the compounds against G. intestinalis trophozoites was tested under both anaerobic and microaerobic conditions, and selectivity was assessed in a counter-screen on human epithelial colorectal adenocarcinoma cells. MTZ was used as a positive control in the assays. All the tested compounds proved to be more effective against the parasite in the presence of O2, with the exception of MTZ that was less effective. Under anaerobiosis eighteen compounds were found to be as effective as MTZ or more (up to three to fourfold); the same compounds proved to be up to >100- fold more effective than MTZ under microaerobic conditions. Four of them represent potential candidates for the design of novel antigiardial drugs, being highly selective against Giardia trophozoites. This study further underlines the importance of taking O2 into account when testing novel potential antigiardial compounds

How bacteria breathe in hydrogen sulfide-rich environments

Hydrogen sulfide (H2S) is now universally recognized as an endogenous signalling molecule playing a central role in human physiology. This gas, although it controls a number of physiological processes at low (submicromolar) concentrations, is toxic at high concentrations as it blocks cell respiration by potently inhibiting cytochrome c oxidase, the terminal enzyme of the mitochondrial respiratory chain. In a recent study on the model micro-organism Escherichia coli, it was shown that the bacterial respiratory oxidase cytochrome bd is resistant to H2S inhibition, thus enabling bacterial O2 respiration and growth in the presence of sulfide. This may be relevant because many microbes are H2S producers and some of them live in sulfide-rich environments, such as the human gut and other natural habitats. The potential impact of this finding in different areas (environment, life evolution and human health) is discussed

The molecular mechanisms by which nitric oxide controls mitochondrial complex IV.

This mini-review of focussed on the information available on the molecular mechanisms by which NO controls the function of mitochondrial cytochrome c oxidase and thereby cell respiration. The reaction mechanisms are described as dissected in vitro and recently confirmed in cell cultures, whereby two reaction pathways have been identified, leading to accumulation of either the [a3(2+)NO]-nitrosyl or the [a3(3+)NO2-]-nitrite derivative of the enzyme. The experimental data and the theoretical computation analysis, supporting the hypothesis that one pathway prevails on the other depending on the electron flow level through the respiratory chain, are discussed. Finally, the patho-physiological implications of the reaction between NO and CcOX have been also outlined

Mechanism of S-nitrosothiol formation and degradation mediated by copper ions

Experimental evidence is presented supporting a mechanism of S-nitrosothiol formation and degradation mediated by copper ions using bovine serum albumin, human hemoglobin and glutathione as models. We found that Cu(2+), but not Fe(3+), induces in the presence of NO a fast S-nitrosation of bovine serum albumin and human hemoglobin, and the reaction is prevented by thiol blocking reagents. During the reaction, Cu(+) is accumulated and accounts for destabilization of the S-nitrosothiol formed. In contrast, glutathione rapidly dimerizes in the presence of Cu(2+), the reaction competing with S-nitrosation and therefore preventing the formation of S-nitrosoglutathione. We have combined the presented role of Cu(2+) in S-nitrosothiol formation with the known destabilizing effect of Cu(+), providing a unique simple picture where the redox state of copper determines either the NO release from S-nitrosothiols or the NO scavenging by thiol groups. The reactions described are fast, efficient, and may occur at micromolar concentration of all reactants. We propose that the mechanism presented may provide a general method for in vitro S-nitrosation I.F. 7.

Cytochrome c oxidase, ligands and electrons

Abstract We present hereby an overview of the reactions of cytochrome c oxidase, the terminal enzyme of the mitochondrial respiratory chain, with ligands (primarily oxygen) and electrons, pointing out where necessary unresolved facts or questionable interpretations

Investigating the mechanism of electron transfer to the binuclear center in Cu-heme oxidases

Novel experimental evidence is presented further supporting the hypothesis that, starting with resting oxidized cytochrome c oxidase, the internal electron transfer to the oxygen binding site is kinetically controlled. The reduction of the enzyme was followed spectroscopically and in the presence of NO or CO, used as trapping ligands for reduced cytochrome a3; ruthenium hexamine was used as a spectroscopically silent electron donor. Consistent with the high combination rate constant for reduced cytochrome a3, NO proved to be a very efficient trapping ligand, while CO did not. The results are discussed in view of two alternative (thermodynamic and kinetic) hypotheses of control of electron transfer to the binuclear (cyt.a3-CuB) center. Fulfilling the prediction of the kinetic control hypothesis: i) the reduction of cytochrome a3 and ligation are synchronous and proceed at the intrinsic rate of cytochrome a3 reduction, ii) the measured rate of formation of the nitrosyl derivative is independent of the concentration of both the reductant and NO I.F.4.3

Cytochrome c oxidase does not catalyze the anaerobic reduction of NO

A possible role of reduced cytochrome c oxidase in the metabolism of nitric oxide (NO) has been examined with amperometric and stopped-flow photometric techniques. Reduced purified cytochrome c oxidase and mitochondria showed no catalytic reaction with NO under anaerobic conditions within more than 30 minutes. Only fast binding of NO to the reduced enzyme in a 1:1 stoichiometric ratio was observed. The NO binding rate was strongly decreased in the presence of 1 mM cyanide. These data indicate that, contrary to previous proposals, cytochrome c oxidase in the absence of oxygen does not contribute to physiological NO metabolism I.F.2.7