Recent data indicate that cystic fibrosis (CF) airway mucus is anaerobic. This suggests that Pseudomonas aeruginosa infection in CF reflects biofilm formation and persistence in an anaerobic environment. P. aeruginosa formed robust anaerobic biofilms, the viability of which requires rhl quorum sensing and nitric oxide (NO) reductase to modulate or prevent accumulation of toxic NO, a byproduct of anaerobic respiration. Proteomic analyses identified an outer membrane protein, OprF, that was upregulated approximately 40-fold under anaerobic versus aerobic conditions. Further, OprF exists in CF mucus, and CF patients raise antisera to OprF. An oprF mutant formed poor anaerobic biofilms, due, in part, to defects in anaerobic respiration. Thus, future investigations of CF pathogenesis and therapy should include a better understanding of anaerobic metabolism and biofilm development by P. aeruginosa.

ABSTRACT

Aconitase is a member of a family of iron-sulfur-containing (de)hydratases whose activities are modulated in bacteria by superoxide radical (O2-.)-mediated inactivation and iron-dependent reactivation. The inactivation-reactivation of aconitase(s) in cultured mammalian cells was explored since these reactions may impact important and diverse aconitase functions in the cytoplasm and mitochondria. Conditions which increase O2-. production including exposure to the redox-cycling agent phenazine methosulfate (PMS), inhibitors of mitochondrial ubiquinol-cytochrome c oxidoreductase, or hyperoxia inactivated aconitase in mammalian cells. Overproduction of mitochondrial Mn-superoxide dismutase protected aconitase from inactivation by PMS or inhibitors of ubiquinol-cytochrome c oxidoreductase, but not from normobaric hyperoxia. Aconitase activity was reactivated (t1/2 of 12 +/- 3 min) upon removal of PMS. The iron chelator deferoxamine impaired reactivation and increased net inactivation of aconitase by O2-.. The ability of ubiquinol-cytochrome c oxidoreductase-generated O2-. to inactivate aconitase in several cell types correlated with the fraction of the aconitase activity localized in mitochondria. Extracellular O2-. generated with xanthine oxidase did not affect aconitase activity nor did exogenous superoxide dismutase decrease aconitase inactivation by PMS. The results demonstrate a dynamic and cyclical O2-.-mediated inactivation and iron-dependent reactivation of the mammalian [4Fe-4S] aconitases under normal and stress conditions and provide further evidence for the membrane compartmentalization of O2-..

Nitric oxide (NO) is a poison, and organisms employ diverse systems to protect against its harmful effects. In Escherichia coli, ygaA encodes a transcription regulator (b2709) controlling anaerobic NO reduction and detoxification. Adjacent to ygaA and oppositely transcribed are ygaK (encoding a flavorubredoxin (flavoRb) (b2710) with a NO-binding non-heme diiron center) and ygbD (encoding a NADH:(flavo)Rb oxidoreductase (b2711)), which function in NO reduction and detoxification. Mutation of either ygaA or ygaK eliminated inducible anaerobic NO metabolism, whereas ygbD disruption partly impaired the activity. NO-sensitive [4Fe-4S] (de)hydratases, including the Krebs cycle aconitase and the Entner-Doudoroff pathway 6-phosphogluconate dehydratase, were more susceptible to inactivation in ygaK or ygaA mutants than in the parental strain, and these metabolic poisonings were associated with conditional growth inhibitions. flavoRb (NO reductase) and flavohemoglobin (NO dioxygenase) maximally metabolized and detoxified NO in anaerobic and aerobic E. coli, respectively, whereas both enzymes scavenged NO under microaerobic conditions. We suggest designation of the ygaA-ygaK-ygbD gene cluster as the norRVW modulon for NO reduction and detoxification.Nitric oxide (NO) is present throughout the biosphere (1-3). In humans, tightly regulated NO synthases produce sufficient NO to poison pathogens, opportunistic organisms, and neoplastic tissue (4, 5). Nanomolar NO potently inhibits terminal oxidases and aerobic respiration (6, 7) and alters the amphibolic and regulatory reactions of the citric acid cycle enzyme aconitase by destroying its labile [4Fe-4S] center (7-10). In addition, significant secondary toxicity of NO can occur via reactions of NO 2 , ONOO Ϫ , NO Ϫ , dinitrosyl iron, and nitrosothiols (11-14). It has become increasingly evident that most organisms metabolize and detoxify NO. Enzymes decompose NO in microorganisms (1, 3, 15-18) and humans (7) and prevent the accumulation of toxic NO levels. Nitric-oxide reductases (NORs) 1 metabolize NO to N 2 O in anaerobic denitrifying bacteria and fungi and likely serve an additional role in minimizing NO toxicity (1,3,19). Nitric-oxide dioxygenases (NODs) convert NO to NO 3 Ϫ in organisms as diverse as bacteria and mammals and have been shown to protect aerobic cells from NO damage (7, 20 -26). In microorganisms, (flavo)hemoglobins catalyze NO dioxygenation (20 -24, 27, 28).In the accompanying article (17), we provide evidence for an inducible and robust NO-metabolizing and -detoxifying activity in anaerobic Escherichia coli. Attempts to biochemically identify the NO reduction system have been complicated by its instability. Moreover, the E. coli genome lacks a NOR belonging to either the cytochrome bc complex or cytochrome P450 families (1). The list of proteins displaying a reductase activity for NO in vitro with potential for function in E. coli is long and includes flavohemoglobin (flavoHb) (27, 28), cytochrome c or cЈ (29), multi-heme nitrite reductase (2, 30), copper-n...

The effect of hyperoxia on activity of the superoxide-sensitive citric acid cycle enzyme aconitase was measured in cultured human epithelial-like A549 cells and in rat lungs. Rapid and progressive loss of >80% of the aconitase activity in A549 cells was seen during a 24-hr exposure to a Po2 of 600 mmHg (1 mmHg = 133 Pa). Inhibition of mitochondrial respiratory capacity correlated with loss of aconitase activity in A549 cells exposed to hyperoxia, and this effect could be mimicked by fluoroacetate (or fluorocitrate), a metabolic poison of aconitase. Exposure ofrats to an atmospheric Po2 of 760 mmHg or 635 mmHg for 24 hr caused respective 73% and 61% decreases in total lung aconitase activity. We propose that early inactivation of aconitase and inhibition ofthe energy-producing and biosynthetic reactions of the citric acid cycle contribute to the sequelae of lung damage and edema seen during exposure to hyperoxia. (1,4,(7)(8)(9)(10)(11)(12)(13). Morphological and biochemical alterations in the lung ultimately cause morbidity resulting from decreased blood oxygenation (4). Edema of the interstitial space and increased permeability of pulmonary microvasculature are early signs of pathology in the lungs of rats exposed to lethal levels of dioxygen (Po2 of 760 mmHg). This initial damage is followed by and amplified by the activation and infiltration of platelets, macrophages, and neutrophils and precedes the death of the animal or individual (3, 12). After sublethal exposures of rats to hyperoxia (Po2 of 456-650 mmHg), damage is marked by fibrosis, alveolar type II cell proliferation (11, 12), and mitochondrial structural deformities (7,8,11,12). The contribution of individual reactive oxygen intermediates and dioxygen to the morphological and biochemical alterations in the lungs, however, remains only vaguely defined (5, 6, 12).Mitochondrial respiration and energy production have been identified as sensitive and critical sites of hyperoxic damage in lung tissue (9, 10) and in cell culture models (14). In rats exposed to a Po2 of 760 mmHg for 24 hr, lung mitochondrial respiratory capacity and citric acid cycle activity are impaired (9, 10). Potential enzymatic sites for the poisoning action of hyperoxia have been described. The mitochondrial enzymes NADH dehydrogenase (14), succinate dehydrogenase (14, 15), and a-ketoglutarate dehydrogenase (14) have various sensitivities to hyperoxic exposure. However, evidence is lacking for a loss of dehydrogenase activities during the early impairment of rat lung oxidative metabolism by normobaric hyperoxia.The citric acid cycle enzyme aconitase is a member of a growing family of 02--sensitive [4Fe-4S]-containing (de)-hydratases that have been implicated to be important sites of 02 -/02 toxicity (16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26). The activity ofaconitase is sensitive to inactivation by 02- (16,22) and is modulated by changes in 02-levels in bacteria and mammalian cells (23, 24, 26).Thus, the ability of elevated levels of 02 to exacerbate mitochondrial productio...

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The flavoHbs 1 belong to a 1.8 billion-year-old family of globin molecules that includes O 2 -binding Hbs and Mbs isolated from animals, plants, fungi, protozoa, bacteria, and worms (1-6). FlavoHbs have a unique two-domain structure containing linked Hb and reductase domains with extensive homology to the mammalian Hbs and metHb reductases (1, 7). Other Hbs appear to be co-expressed with associated metHb reductases (8). An O 2 transport or storage function, like that of the erythrocytic Hbs and muscle Mbs, has been suggested for some microbial and plant (flavo)Hbs (4, 9); however, other functions including the catalysis of oxidations have long seemed more likely (10 -12).Recently, we described an NO dioxygenase (NOD) produced by Escherichia coli that utilizes O 2 and NAD(P)H to convert NO to nitrate (Equation 1) and identified it as a flavoHb (13,14). Subsequent studies have shown that related bacterial and yeast flavoHbs display a similar NOD activity.A role for flavoHbs in NO detoxification is supported by the ability of flavoHbs to protect bacteria against NO or nitroso compounds (13, 14, 16 -18) and by their induction in bacteria exposed to NO, nitrate, nitrite, or nitroso compounds (13, 14, 17, 19 -22). However, the mechanism of NO detoxification, and thus the function of the flavoHbs, is obscured by the possibility of multiple reaction mechanisms involving NO. Other NO detoxifying activities for flavoHbs, including denitrosylation of nitrosothiols (17), NO reduction (17, 23), and NO sequestration (16, 23), have been offered to explain the protection flavoHbs afford to bacteria against NO and nitrosoglutathione. Thus, an understanding of the biological function(s) of the flavoHbs demands a greater knowledge of their various activities.In this report, steady-state, reduction, and ligand binding kinetics of the E. coli NOD (flavoHb) were measured in order to define its function and the mechanism of NO dioxygenation. We also examined the effects of amino acid substitutions at the highly conserved Tyr(B10) position on NOD activity, reduction, and ligand binding kinetics (7,24). Key differences between flavoHb and other Hbs are discussed in light of this specialized but perhaps ancient NO dioxygenation and detoxification function of hemoglobin. MATERIALS AND METHODSCells and Reagents-The flavoHb-deficient E. coli strain RB9060 (25) was generously provided by Alex Ninfa (University of Michigan). Plasmid pAlter containing the E. coli hmp gene was prepared as described previously (13). FAD, NADPH, and bovine hemin were purchased from Sigma. NADH, bovine liver catalase (260,000 units/ml), and deoxyribonuclease were obtained from Roche Molecular Biochemicals. Saturated NO was prepared as described previously (26). Saturated O 2 (1.14 mM) was prepared by vigorously scrubbing a solution of 50 mM potassium phosphate, pH 7.8, containing 0.1 mM EDTA (buffer A) at 25°C and atmospheric pressure with 99.993% O 2 (Praxair, Bethlehem, PA) in a rubber septum-sealed glass vial vented with a syringe needle. Manganese-containing...