Paul R. Gardner

Pseudomonas aeruginosa Anaerobic Respiration in Biofilms: Relationships to Cystic Fibrosis Pathogenesis

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.

Nitric Oxide Sensitivity of the Aconitases

Aconitases are important cellular targets of nitric oxide (NO⋅) toxicity, and NO⋅-derived species, rather than NO⋅ per se, have been proposed to mediate their inactivation. NO⋅-mediated inactivation of theEscherichia coli aconitase and the porcine mitochondrial aconitase was investigated. In E. coli, aconitase activity decreased by ∼70% during a 2-h exposure to an atmosphere containing 120 ppm NO⋅ in N2. The NO⋅-inactivated aconitase reactivated poorly in E. coli under anaerobic or aerobic conditions. Elevated superoxide dismutase activity did not affect the aerobic inactivation of aconitase by NO⋅, thus indicating a limited role of the NO⋅- and superoxide-derived species peroxynitrite. Glutathione-deficient and glutathione-containingE. coli were comparably sensitive to NO⋅-mediated aconitase inactivation, thus excluding the participation of S-nitrosoglutathione or more oxidizing NO⋅-derived species. NO⋅ progressively decreased aconitase activity in extracts in the presence of substrates, and inactivation was greatest at an acidic pH withcis-aconitate. The porcine mitochondrial aconitase was sensitive to NO⋅ when exposed at pH 6.5, but not at pH 7.5, and irreversible inactivation occurred during catalysis. The requirement of an acidic pH or substrates for sensitivity may explain the reported resistance of aconitases to NO⋅ in vitro (Castro, L., Rodriguez, M., and Radi, R. (1994) J. Biol. Chem.269, 29409–29415; Hausladen, A., and Fridovich, I. (1994)J. Biol. Chem. 269, 29405–29408). AnS-nitrosation of the aconitase [4Fe-4S] center catalyzed by the solvent-exposed electron withdrawing iron atom (Fea) is proposed.

Nitric-oxide Dioxygenase Activity and Function of Flavohemoglobins SENSITIVITY TO NITRIC OXIDE AND CARBON MONOXIDE INHIBITION

Widely distributed flavohemoglobins (flavoHbs) function as NO dioxygenases and confer upon cells a resistance to NO toxicity. FlavoHbs from Saccharomyces cerevisiae,Alcaligenes eutrophus, and Escherichia colishare similar spectra, O2, NO, and CO binding kinetics, and steady-state NO dioxygenation kinetics. Turnover numbers (V max) for S. cerevisiae, A. eutrophus, and E. coli flavoHbs are 112, 290, and 365 NO heme−1 s−1, respectively, at 37 °C with 200 μm O2. The K M values for NO are low and range from 0.1 to 0.25 μm.V max/K M (NO) ratios of 900–2900 μm −1 s−1 indicate an extremely efficient dioxygenation mechanism. ApproximateK M values for O2 range from 60 to 90 μm. NO inhibits the dioxygenases at NO:O2ratios of ≥1:100 and makes true K M (O2) values difficult to determine. High and roughly equal second order rate constants for O2 and NO association with the reduced flavoHbs (17–50 μm −1 s−1) and small NO dissociation rate constants suggest that NO inhibits the dioxygenase reaction by forming inactive flavoHbNO complexes. Carbon monoxide also binds reduced flavoHbs with high affinity and competitively inhibits NO dioxygenases with respect to O2(K I (CO) = ∼1 μm). These results suggest that flavoHbs and related hemoglobins evolved as NO detoxifying components of nitrogen metabolism capable of discriminating O2 from inhibitory NO and CO.

Nitric oxide scavenging and detoxification by the Mycobacterium tuberculosis haemoglobin, HbN in Escherichia coli.

Nitric oxide (NO), generated in large amounts within the macrophages, controls and restricts the growth of internalized human pathogen, Mycobacterium tuberculosis H37Rv. The molecular mechanism by which tubercle bacilli survive within macrophages is currently of intense interest. In this work, we have demonstrated that dimeric haemoglobin, HbN, from M. tuberculosis exhibits distinct nitric oxide dioxygenase (NOD) activity and protects growth and cellular respiration of heterologous hosts, Escherichia coli and Mycobacterium smegmatis, from the toxic effect of exogenous NO and the NO‐releasing compounds. A flavohaemoglobin (HMP)‐deficient mutant of E. coli, unable to metabolize NO, acquired an oxygen‐dependent NO consumption activity in the presence of HbN. On the basis of cellular haem content, the specific NOD activity of HbN was nearly 35‐fold higher than the single‐domain Vitreoscilla haemoglobin (VHb) but was sevenfold lower than the two‐domain flavohaemoglobin. HbN‐dependent NO consumption was sustained with repeated addition of NO, demonstrating that HbN is catalytically reduced within E. coli. Aerobic growth and respiration of a flavohaemoglobin (HMP) mutant of E. coli was inhibited in the presence of exogenous NO but remained insensitive to NO inhibition when these cells produced HbN, VHb or flavohaemoglobin. M. smegmatis, carrying a native HbN very similar to M. tuberculosis HbN, exhibited a 7.5‐fold increase in NO uptake when exposed to gaseous NO, suggesting NO‐induced NOD activity in these cells. In addition, expression of plasmid‐encoded HbN of M. tuberculosis in M. smegmatis resulted in 100‐fold higher NO consumption activity than the isogenic control cells. These results provide strong experimental evidence in support of NO scavenging and detoxification function for the M. tuberculosis HbN. The catalytic NO scavenging by HbN may be highly advantageous for the survival of tubercle bacilli during infection and pathogenesis.

CONSTITUTIVE AND ADAPTIVE DETOXIFICATION OF NITRIC OXIDE IN ESCHERICHIA COLI ROLE OF NITRIC-OXIDE DIOXYGENASE IN THE PROTECTION OF ACONITASE

Nitric oxide (NO⋅) is a naturally occurring toxin that some organisms adaptively resist. In aerobic or anaerobic Escherichia coli, low levels of NO⋅exposure inactivated the NO⋅-sensitive citric acid cycle enzyme aconitase, and inactivation was more effective when the adaptive synthesis of NO⋅-defensive proteins was blocked with chloramphenicol. Protection of aconitase in aerobically grown E. coli was dependent upon O2, was potently inhibited by cyanide, and was correlated with an induced rate of cellular NO⋅consumption. Constitutive and adaptive cellular NO⋅ consumption in aerobic cells was also dependent upon O2 and inhibited by cyanide. Exposure of aerobic cells to NO⋅ accordingly elevated the activity of the O2-dependent and cyanide-sensitive NO⋅ dioxygenase (NOD). Anaerobic E. coli exposed to NO⋅ or nitrate induced a modest O2-independent and cyanide-resistant NO⋅-metabolizing activity and a more robust O2-stimulated cyanide-sensitive activity. The latter activity was attributed to NOD. The results support a role for NOD in the aerobic detoxification of NO⋅ and suggest functions for NOD and a cyanide-resistant NO⋅ scavenging activity in anaerobic cells.

Dioxygen-dependent metabolism of nitric oxide in mammalian cells.

Steady-state gradients of NO within tissues and cells are controlled by rates of NO synthesis, diffusion, and decomposition. Mammalian cells and tissues actively decompose NO. Of several cell lines examined, the human colon CaCo-2 cell produces the most robust NO consumption activity. Cellular NO metabolism is mostly O2-dependent, produces near stoichiometric NO3-, and is inhibited by the heme poisons CN-, CO (K(I) approximately 3 microM), phenylhydrazine, and NO and the flavoenzyme inhibitor diphenylene iodonium. NO consumption is saturable by O2 and NO and shows apparent K(M) values for O2 and NO of 17 and 0.2 microM, respectively. Mitochondrial respiration, O2*-, and H2O2 are neither sufficient nor necessary for O2-dependent NO metabolism by cells. The existence of an efficient mammalian heme and flavin-dependent NO dioxygenase is suggested. NO dioxygenation protects the NO-sensitive aconitases, cytochrome c oxidase, and cellular respiration from inhibition, and may serve a dual function in cells by limiting NO toxicity and by spatially coupling NO and O2 gradients.

Nitric-oxide Dioxygenase Function of Human Cytoglobin with Cellular Reductants and in Rat Hepatocytes

Cytoglobin (Cygb) was investigated for its capacity to function as a NO dioxygenase (NOD) in vitro and in hepatocytes. Ascorbate and cytochrome b5 were found to support a high NOD activity. Cygb-NOD activity shows respective Km values for ascorbate, cytochrome b5, NO, and O2 of 0.25 mm, 0.3 μm, 40 nm, and ∼20 μm and achieves a kcat of 0.5 s−1. Ascorbate and cytochrome b5 reduce the oxidized Cygb-NOD intermediate with apparent second order rate constants of 1000 m−1 s−1 and 3 × 106 m−1 s−1, respectively. In rat hepatocytes engineered to express human Cygb, Cygb-NOD activity shows a similar kcat of 1.2 s−1, a Km(NO) of 40 nm, and a kcat/Km(NO) (k′NOD) value of 3 × 107 m−1 s−1, demonstrating the efficiency of catalysis. NO inhibits the activity at [NO]/[O2] ratios >1:500 and limits catalytic turnover. The activity is competitively inhibited by CO, is slowly inactivated by cyanide, and is distinct from the microsomal NOD activity. Cygb-NOD provides protection to the NO-sensitive aconitase. The results define the NOD function of Cygb and demonstrate roles for ascorbate and cytochrome b5 as reductants.

Imidazole Antibiotics Inhibit the Nitric Oxide Dioxygenase Function of Microbial Flavohemoglobin

ABSTRACT Flavohemoglobins metabolize nitric oxide (NO) to nitrate and protect bacteria and fungi from NO-mediated damage, growth inhibition, and killing by NO-releasing immune cells. Antimicrobial imidazoles were tested for their ability to coordinate flavohemoglobin and inhibit its NO dioxygenase (NOD) function. Miconazole, econazole, clotrimazole, and ketoconazole inhibited the NOD activity of Escherichia coli flavohemoglobin with apparent Ki values of 80, 550, 1,300, and 5,000 nM, respectively. Saccharomyces cerevisiae, Candida albicans, and Alcaligenes eutrophus enzymes exhibited similar sensitivities to imidazoles. Imidazoles coordinated the heme iron atom, impaired ferric heme reduction, produced uncompetitive inhibition with respect to O2 and NO, and inhibited NO metabolism by yeasts and bacteria. Nevertheless, these imidazoles were not sufficiently selective to fully mimic the NO-dependent growth stasis seen with NOD-deficient mutants. The results demonstrate a mechanism for NOD inhibition by imidazoles and suggest a target for imidazole engineering.

Dioxygen-Dependent Metabolism of Nitric Oxide

Nitric oxide (NO) serves critical signaling, energetic, and toxic functions throughout the biosphere. NO steady-state levels and functions are controlled in part by NO metabolism or degradation. Dioxygen-dependent NO dioxygenases (EC 1.14.12.17) and dioxygen-independent NO reductases (EC 1.7.99.7) are being identified as major routes for NO metabolism in various life forms. Here we describe the use of the Clark-type NO electrode, mechanistic inhibitors, and nitrate/nitrite assays to measure, characterize, and identify major NO metabolic pathways and enzymes in bacteria, fungi, plants, mammalian cells, and organelles. The methods may prove to be particularly useful for mechanistic investigations and the development of inhibitors, inducers, and other novel NO-modulating therapeutics.

Globins Scavenge Sulfur Trioxide Anion Radical

Background: Sulfite, an intermediate in sulfur metabolism, can be oxidized to the potentially toxic sulfur trioxide anion radical (STAR). Results: Diverse globins efficiently reduced STAR in vitro, and flavohemoglobin protected yeast from STAR toxicity. Conclusion: Globins can function as STAR scavengers. Significance: The data suggest roles for diverse globins in protecting cells against sulfite stress. Ferrous myoglobin was oxidized by sulfur trioxide anion radical (STAR) during the free radical chain oxidation of sulfite. Oxidation was inhibited by the STAR scavenger GSH and by the heme ligand CO. Bimolecular rate constants for the reaction of STAR with several ferrous globins and biomolecules were determined by kinetic competition. Reaction rate constants for myoglobin, hemoglobin, neuroglobin, and flavohemoglobin are large at 38, 120, 2,600, and ≥ 7,500 × 106 m−1 s−1, respectively, and correlate with redox potentials. Measured rate constants for O2, GSH, ascorbate, and NAD(P)H are also large at ∼100, 10, 130, and 30 × 106 m−1 s−1, respectively, but nevertheless allow for favorable competition by globins and a capacity for STAR scavenging in vivo. Saccharomyces cerevisiae lacking sulfite oxidase and deleted of flavohemoglobin showed an O2-dependent growth impairment with nonfermentable substrates that was exacerbated by sulfide, a precursor to mitochondrial sulfite formation. Higher O2 exposures inactivated the superoxide-sensitive mitochondrial aconitase in cells, and hypoxia elicited both aconitase and NADP+-isocitrate dehydrogenase activity losses. Roles for STAR-derived peroxysulfate radical, superoxide radical, and sulfo-NAD(P) in the mechanism of STAR toxicity and flavohemoglobin protection in yeast are suggested.