Cytochrome bd oxidase and nitric oxide: from reaction mechanisms to bacterial physiology.

International audience; Experimental evidence suggests that the prokaryotic respiratory cytochrome bd quinol oxidase is responsible for both bioenergetic functions and bacterial adaptation to different stress conditions. The enzyme, phylogenetically unrelated to the extensively studied heme-copper terminal oxidases, is found in many commensal and pathogenic bacteria. Here, we review current knowledge on the catalytic intermediates of cytochrome bd and their reactivity towards nitric oxide (NO). Available information is discussed in the light of the hypothesis that, owing to its high NO dissociation rate, cytochrome bd confers resistance to NO-stress, thereby providing a strategy for bacterial pathogens to evade the NO-mediated host immune attack

Cytochrome bd oxidase from Escherichia coli displays high catalase activity: An additional defense against oxidative stress

AbstractCytochrome bd oxygen reductase from Escherichia coli has three hemes, b558, b595 and d. We found that the enzyme, as-prepared or in turnover with O2, rapidly decomposes H2O2 with formation of approximately half a mole of O2 per mole of H2O2. Such catalase activity vanishes upon cytochrome bd reduction, does not compete with the oxygen-reductase activity, is insensitive to NO, CO, antimycin-A and N-ethylmaleimide (NEM), but is inhibited by cyanide (Ki ∼2.5μM) and azide. The activity, possibly associated with heme-b595, was also observed in catalase-deficient E. coli cells following cytochrome bd over-expression suggesting a protective role against oxidative stress in vivo

Role of membrane potential on the control of cytochrome c oxidase over respiration in intact hepatoma HepG2 cells

Metabolic control analysis (MCA) has been largely applied to the analysis of oxidative phosphorylation, in order to investigate the control exerted by each individual reaction step on the whole pathway. Using this approach, the control exerted by the electron transport chain complexes was shown to be higher in experimental systems closer to in vivo conditions than in mitochondria. To study the effect of the mitochondrial transmembrane proton electrochemical gradient (ΔμH+) on the control of respiration by cytochrome c oxidase (CcOX) in intact cells, we applied MCA to mitochondrial respiration of HepG2 cells. Both the overall O2 consumption and specific CcOX activity of actively phosphorylating cells were progressively inhibited by cyanide titration under conditions in which the electrical (Δψ) and/or the chemical (ΔpH) component of ΔμH+ was selectively modulated by addition of ionophores. Under endogenous conditions, i.e., in the absence of ionophores, CcOX displayed a high control coefficient value, thus representing an important site of regulation of mitochondrial oxidative phosphorylation. A high CcOX control coefficient value was also measured in the presence of nigericin, when Δψ is maximal, and in the presence of nigericin and valinomycin, when ΔμH+ is abolished. On the contrary and interestingly CcOX displayed a markedly lower control coefficient in the presence valinomycin converting Δψ into ΔpH. These results show that CcOX activity and its control over oxidative phosphorylation critically depend on Δψ in actively phosphorylating cells

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

Enzymatic detoxification of O2 and NO in the human parasite, Giardia intestinalis: A mini review

Giardia intestinalis is the etiological agent of giardiasis, a common human intestinal disease with 280 million cases per year. Giardiasis is typically treated with the broad-range antibiotic metronidazole; however, the emergence of drug-resistant strains calls for the development of new anti-parasitic drugs. Very little is known regarding the molecular mechanisms adopted by Giardia to cope with the oxidative/nitrosative environmental stress, encountered by the parasite during colonization of the human intestine. Giardia is particularly sensitive to oxidative stress, as it lacks some of the most common ROS-detoxifying enzymes and it is endowed with O-2-labile key metabolic enzymes. Surprisingly, it colonizes a fairly aerobic (up to 50 mu M O-2) tract of the human gut (the upper part of the small intestine). Accordingly, survival of the parasite relies on antioxidant systems, though, as yet, the only two H2O-forming and O-2-consuming enzymes described in Giardia are NADH oxidase and flavodiiron protein (FDP). Nitric oxide (NO) is an antimicrobial agent produced, together with ROS, by the host immune system to fight pathogens. In vitro NO-stress has been reported to have cytostatic, rather than cytotoxic, effects on Giardia. This effect leads to the suggestion that Giardia is endowed with defense mechanisms against NO and, very recently, the NO-detoxifying flavohemoglobin from it has been characterized

Cytochrome c oxidase and nitric oxide in action: Molecular mechanisms and pathophysiological implications

Background: The reactions between Complex IV (cytochrome coxidase, CcOX) and nitric oxide (NO) were described in the early 60's. The perception, however, that NO could be responsible for physiological or pathological effects, including those on mitochondria, lags behind the 80's, when the identity of the endothelial derived relaxing factor (EDRF) and NO synthesis by the NO synthases were discovered. NO controls mitochondrial respiration, and cytotoxic as well as cytoprotective effects have been described. The depression of OXPHOS ATP synthesis has been observed, attributed to the inhibition of mitochondria! Complex I and IV particularly, found responsible of major effects. Scope of review: The review is focused on CcOX and NO with some hints about pathophysiological implications. The reactions of interest are reviewed, with special attention to the molecular mechanisms underlying the effects of NO observed on cytochrome c oxidase, particularly during turnover with oxygen and reductants. Major conclusions and general significance: The NO inhibition of CcOX is rapid and reversible and may occur in competition with oxygen. Inhibition takes place following two pathways leading to formation of either a relatively stable nitrosyl-derivative (CcOX-NO) of the enzyme reduced, or a more labile nitrite-derivative (CcOX-NO2- of the enzyme oxidized, and during turnover. The pathway that prevails depends on the turnover conditions and concentration of NO and physiological substrates, cytochrome c and O-2. All evidence suggests that these parameters are crucial in determining the CcOX vs NO reaction pathway prevailing in vivo, with interesting physiological and pathological consequences for cells. This article is part of a Special Issue entitled: Respiratory Oxidases. (C) 2011 Elsevier B.V. All rights reserved

Enzymatic detoxification of O₂ and NO in the human parasite, <i>Giardia intestinalis</i>: A mini review

404-409Giardia intestinalis is the etiological agent of giardiasis, a common human intestinal disease with 280 million cases per year. Giardiasis is typically treated with the broad-range antibiotic metronidazole; however, the emergence of drug-resistant strains calls for the development of new anti-parasitic drugs. Very little is known regarding the molecular mechanisms adopted by Giardia to cope with the oxidative/nitrosative environmental stress, encountered by the parasite during colonization of the human intestine. Giardia is particularly sensitive to oxidative stress, as it lacks some of the most common ROS-detoxifying enzymes and it is endowed with O2-labile key metabolic enzymes. Surprisingly, it colonizes a fairly aerobic (up to 50 M O2) tract of the human gut (the upper part of the small intestine). Accordingly, survival of the parasite relies on antioxidant systems, though, as yet, the only two H2O-forming and O2-consuming enzymes described in Giardia are NADH oxidase and flavodiiron protein (FDP). Nitric oxide (NO) is an antimicrobial agent produced, together with ROS, by the host immune system to fight pathogens. In vitro NO-stress has been reported to have cytostatic, rather than cytotoxic, effects on Giardia. This effect leads to the suggestion that Giardia is endowed with defense mechanisms against NO and, very recently, the NO-detoxifying flavohemoglobin from it has been characterized

New Evidence for Cross Talk between Melatonin and Mitochondria Mediated by a Circadian-Compatible Interaction with Nitric Oxide

Abstract: Extending our previous observations, we have shown on HaCat cells that melatonin, at ~10 −9 M concentration, transiently raises not only the expression of the neuronal nitric oxide synthase (nNOS) mRNA, but also the nNOS protein synthesis and the nitric oxide oxidation products, nitrite and nitrate. Interestingly, from the cell bioenergetic point of view, the activated NO-related chemistry induces a mild decrease of the oxidative phosphorylation (OXPHOS) efficiency, paralleled by a depression of the mitochondrial membrane potential. The OXPHOS depression is apparently balanced by glycolysis. The mitochondrial effects described have been detected only at nanomolar concentration of melatonin and within a time window of a few hours ’ incubation; both findings compatible with the melatonin circadian cycle