HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 277, Issue 10, 8166-8171, March 8, 2002

This Article Abstract Full Text (PDF) All Versions of this Article: 277/10/8166    most recent M110470200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Gardner, A. M. Articles by Gardner, P. R. Search for Related Content PubMed PubMed Citation Articles by Gardner, A. M. Articles by Gardner, P. R. Social Bookmarking                      What's this?

Flavohemoglobin Detoxifies Nitric Oxide in Aerobic, but Not Anaerobic, Escherichia coli

EVIDENCE FOR A NOVEL INDUCIBLE ANAEROBIC NITRIC OXIDE-SCAVENGING ACTIVITY^*

Anne M. Gardner and Paul R. Gardner

From the Division of Critical Care Medicine, Children's Hospital Medical Center, Cincinnati, Ohio 45229

Received for publication, October 31, 2001, and in revised form, December 11, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Nitric-oxide dioxygenase (NOD) and reductase (NOR) activities of flavohemoglobin (flavoHb) have been suggested as mechanisms for NO metabolism and detoxification in a variety of microbes. Mechanisms of NO detoxification were tested in Escherichia coli using flavoHb-deficient mutants and overexpressors. flavoHb showed negligible anaerobic NOR activity and afforded no protection to the NO-sensitive aconitase or the growth of anoxic E. coli, whereas the NOD activity and the protection afforded with O₂ were substantial. A NO-inducible, O₂-sensitive, and cyanide-resistant NOR activity efficiently metabolized NO and protected anaerobic cells from NO toxicity independent of the NOR activity of flavoHb. flavoHb possesses nitrosoglutathione and nitrite reductase activities that may account for the protection it affords against these agents. NO detoxification by flavoHb occurs most effectively via O₂-dependent NO dioxygenation.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Nitric oxide (NO) is a water-soluble gas commonly produced during the combustion of nitrogenous compounds and during the biological decay of organic matter. NO is also an important by-product of microbial denitrification (1, 2) and is produced by NO synthases of animals and plants, where it functions as a broad-spectrum antibiotic and signaling molecule (2-5).

Nanomolar NO concentrations inactivate or inhibit critical enzymes, including the citric acid cycle enzyme aconitase (6-8) and terminal respiratory oxidases (9, 10). NO also has the potential to damage a variety of biomolecules by forming the more indiscriminate toxin and oxidizing agent peroxynitrite (11).

Organisms have evolved mechanisms for NO metabolism and detoxification. Inducible nitric-oxide reductases (NORs)¹ are produced by denitrifying bacteria, nitrogen-dissimilating fungi, and pathogenic microorganisms. NORs reduce NO to N₂O and are essential for denitrification by various bacteria and for the viability of gonococci (1, 12, 13). N₂O is further reduced to N₂ by [4Cu-S]-containing N₂O reductases in denitrifying microorganisms to generate energy through membrane-linked processes analogous to those utilized for O₂ respiration (1, 14). In addition, NO-inducible flavohemoglobins (flavoHbs) dioxygenate NO to form NO (10, 15-21) and reduce NO to form N₂O (19, 22).

Growing evidence supports both aerobic and anaerobic NO detoxification functions for flavoHb. flavoHb protects aerobic Salmonella typhimurium against the growth inhibitory effects of acidified nitrite, S-nitrosoglutathione (GSNO), and the NO-releasing nitroso compound spermine 2,2'-(hydroxynitrosohydrazono)bisethanamine, and flavoHb prevents anaerobic growth inhibition of S. typhimurium by GSNO (23, 24). flavoHb also protects Saccharomyces cerevisiae from aerobic and anaerobic growth inhibition by 2,2'-(hydroxynitrosohydrazono)bisethanamine (20). Pure NO gas (15) and nitroso compounds (19, 25) potently inhibit the growth of Escherichia coli flavoHb-deficient mutants under aerobic growth conditions in contrast to flavoHb-containing parent strains, where growth inhibition is minimal. flavoHb-deficient Dictyostelium discoideum is also sensitive to GSNO and nitroprusside during aerobic growth (26). Modest protective effects of flavoHb against NO-mediated growth inhibition have also been observed in anoxic E. coli exposed to an atmosphere containing 240 ppm NO (15). In addition, anaerobic denitrification and N₂O formation are compromised in flavoHb-deficient mutants of Alcaligenes eutrophus (27). In the plant pathogen Erwinia chrysanthemi, flavoHb provides resistance to the immune response of tobacco leaves (28). Nitric-oxide dioxygenase (NOD) or NOR functions of flavoHbs have been suggested as mechanisms for these varied protections.

We examine here mechanisms for NO detoxification in aerobic and anaerobic E. coli. The results demonstrate a highly efficient function for flavoHb as an O₂-dependent NOD, but they do not support a role for flavoHb as an anaerobic NOR. Our investigations have revealed a novel NO-inducible and O₂-sensitive NOR activity that serves E. coli in this capacity. In addition, we report reductase activities of flavoHb for GSNO and nitrite that may explain the anaerobic protection flavoHb affords against these and other nitroso compounds.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Reagents-- Glucose oxidase from Aspergillus niger, cytochrome c from S. cerevisiae, FAD, MOPS, MnCl₂, paraquat, 2-heptyl-4-hydroxyquinoline-N-oxide, reduced glutathione, sodium nitrite, and sodium cyanide were obtained from Sigma. Bovine liver catalase (260,000 units/ml) was purchased from Roche Molecular Biochemicals. GSNO was prepared by incubating 400 mM reduced glutathione and 400 mM sodium nitrite in 1 M HCl and by neutralizing the reaction with 1 M NaOH (29). S-Nitroso-N-acetylpenicillamine was purchased from Calbiochem and was freshly prepared in dimethyl sulfoxide. flavoHb was purified from anaerobic nitrate-induced RB9060 containing pAlterhmp (17). Manganese-containing superoxide dismutase (2300 units/mg) was induced with paraquat in strain DH5 bearing plasmid pD11c and was isolated from extracts (45, 46). P1 phage was obtained from Dr. Jim Imlay (University of Illinois). Bacto-Tryptone and yeast extracts were obtained from Difco. Gas cylinders containing 1200 ppm NO in ultrapure N₂, 99.999% N₂, and 99.993% O₂ were obtained from Praxair (Bethlehem, PA). NO-saturated water (2 mM) was prepared as previously described (7).

Bacterial Strains-- E. coli K12 strain RB9060 (glyA-hmp-glnB) was provided by Dr. Alex Ninfa (University of Michigan) (30). E. coli K12 strain AB1157 (F thr-1 ara-C14 leuB6 DE(gpt-proA)62 lacY1 tsx-33 gsr'-0 glnV44 galK2 Rac-0 hisG4 rfbD1 mgl-51 rpoS396 rpsL31(strR) kdgK51 xyl-A5 mtl-1 argE3 thi-1) was provided by Dr. Bruce Demple (Harvard University). Wild-type E. coli K12 (ATCC 23716) was obtained from the American Type Culture Collection (Manassas, VA). Strain JC7623 (F thr-1 ara-C14 leuB6 DE(gpt-proA)62 lacY1 sbcC201 tsx-33 gsr'-0 glnV44 galK2 Rac-0 sbcB15 hisG4 rfbD1 rpoS396 recB21 recC22 rpsL31(strR) kdgK51 xyl-A5 mtl-1 argE3 thi-1) was obtained from the E. coli Genetic Stock Center of Yale University. Plasmid pAlterhmp was constructed as previously described (15). An ~20-kb EcoRI fragment from plasmid pGS26 (31) containing the Tn5 kan^R element inserted in the hmp gene (hmp::Tn5) after isoleucine 81 was isolated and used to transform JC7623. Stable insertion of hmp::Tn5 was selected for on LB agarose containing kanamycin (30 µg/ml). Loss of NO-inducible NOD activity was verified in kanamycin-resistant clones. The hmp::Tn5 mutation was transduced to strain AB1157 and wild-type E. coli K12 (ATCC 23716) using P1 phage. flavoHb-deficient strains of AB1157 and wild-type E. coli K12 were designated AG103 and AG102, respectively.

Growth of Bacteria-- Aerobic cultures were routinely grown in phosphate-buffered LB medium prepared with 10 g of Tryptone, 5 g of yeast extract, and 10 g of NaCl in 1 liter of water containing 66 mM K₂HPO₄ and 33 mM KH₂PO₄ (pH 7.0). Aeration was achieved with a rotary water bath set at 275 rpm and 37 °C. Glucose (20 mM) was added to phosphate-buffered LB medium for anaerobic cultures. Tetracycline (12 µg/ml) was added to the growth medium for strains harboring pAlter or pAlterhmp. Minimal salts medium (pH 7.0) contained 60 mM K₂HPO₄, 33 mM KH₂PO₄, 7.6 mM (NH₄)₂SO₄, 1.7 mM sodium citrate, 1 mM MgSO₄, 10 µM MnCl₂, 10 µg/ml thiamin, and 20 mM glucose. Overnight aerobic cultures were grown at 37 °C in 15-ml tubes in 5 ml of medium with vigorous shaking for aeration. Overnight anaerobic cultures were grown static at 37 °C in 15-ml tubes containing 10 ml of medium. Cell growth was monitored by turbidity at 550 nm. Cell density was determined by dilution, plating, and counting. A log phase absorbance of 1.0 at 550 nm corresponds to 3 × 10⁸ bacteria/ml.

Gas Exposures-- Three-way stainless steel gas proportioners (Cole-Parmer Instrument Co.) were used to mix O₂, N₂, and NO and to deliver gases at a constant flow rate. Gas mixtures were passed through a trap containing NaOH pellets to remove higher oxides of nitrogen. Thick wall (3.2 mm) Tygon R3603 tubing was used throughout the system to limit gas exchange with the atmosphere. To achieve rapid gas equilibration, cultures were shaken at 275 rpm at 37 °C in rubber stopper-sealed 50-ml Erlenmeyer flasks flushed at 30 ml/min with gas mixtures at a culture/flask volume ratio of at most 1:5. To minimize the disturbance of gases, samples were removed using a 1-ml tuberculin syringe connected to the culture via narrow tubing.

NO Consumption Assays-- Aerobic NO consumption was measured amperometrically with a NO meter and a 2-mm ISO-NOP NO electrode (World Precision Instruments, Sarasota, FL). The electrode was fixed and sealed into a 2-ml zero-head space glass stopper-closed magnetically-stirred reaction chamber containing 60 mM K₂HPO₄, 33 mM KH₂PO₄, 7.6 mM (NH₄)₂SO₄, 1.7 mM sodium citrate, 10 mM glucose, 200 µM O₂, and 200 µg/ml chloramphenicol and thermostatted at 37 °C. NO (2 µM) was injected into the closed chamber through the narrow port of the glass stopper with a 10-µl Hamilton syringe, and the electrode signal was recorded on chart paper. Cells (0.2-2 × 10⁷) were injected into the chamber at the apex of the signal response. Rates of aerobic NO consumption were determined from instantaneous rates of NO removal at 1.0 µM NO or a half-maximal signal amplitude. Aerobic NO consumption rates were corrected for relatively small background rates of NO decomposition at 1.0 µM NO of 0.5 nmol of NO/min. Anaerobic NO consumption activities of cells were measured as described for aerobic activity measurements, except that O₂ was removed by incubating the reaction for 5 min with 2 units/ml glucose oxidase and 130 units/ml catalase prior to adding NO and cells. O₂ depletion under these conditions was verified with a Clark-type O₂ electrode (YSI Inc.). Anaerobic NO consumption activities were determined for 1.5 µM NO and were corrected for the background rates of NO decomposition of 0.3 nmol of NO/min. Culture aliquots (1 ml) were quickly centrifuged for 20 s at 20,000 × g, and supernatants were aspirated. Cell pellets were overlaid and washed with 1 ml of ice-cold assay medium and were centrifuged at 20,000 × g for 20 s. Cell pellets were resuspended at 1 × 10⁷ cells/µl of ice-cold assay medium and placed on ice. Anaerobic NO consumption activities were assayed within 2 min of cell harvests to minimize the loss of activity.

Aconitase Activity Assays-- Culture samples were centrifuged for 20 s at 20,000 × g, and supernatants were aspirated. Cell pellets were overlaid and washed with 1 ml of ice-cold assay medium and were recentrifuged. Supernatants were removed, and cells were placed on dry ice. Cells were lysed with a 10-s sonic burst with a microprobe in 100 µl of ice-cold 50 mM Tris-Cl (pH 7.4) containing 20 µM fluorocitrate and 0.6 mM MnCl₂. Lysates were clarified by centrifugation at 20,000 × g for 30 s, and clarified extracts were kept on ice. Aconitase activity was assayed in 96-well microplates as described previously (7, 32). Protein was determined using the Coomassie Blue dye-binding assay with bovine serum albumin as the standard (33).

flavoHb Activity Assays-- All measurements were at 37 °C with 100 µM NADH and 1 µM FAD. The NADH oxidase activity of flavoHb was assayed by following the oxidation of NADH in an air-saturated (200 µM O₂) 1-ml reaction in 100 mM potassium phosphate buffer (pH 7.0). The NO, GSNO, and nitrite reductase activities of flavoHb were determined at 37 °C by following NADH oxidation at 340 nm in an anoxic N₂-scrubbed 1-ml reaction containing 100 mM sodium phosphate buffer (pH 7.0), 0.3 mM EDTA, and NO (20 µM), GSNO (1 mM), or sodium nitrite (1 mM). Cytochrome c reduction was followed at 550 nm in a 1-ml reaction at 37 °C containing 20 µM cytochrome c, 8 units of glucose oxidase, 10 mM glucose, 130 units of catalase, 230 units of manganese-containing superoxide dismutase, and flavoHb in 50 mM MOPS (pH 7.0) and 50 mM NaCl. Reactions were preincubated for 2 min to allow for O₂ depletion by glucose oxidase prior to the addition of flavoHb and cytochrome c. Manganese-containing superoxide dismutase was included to prevent superoxide-mediated cytochrome c reduction. An E_550 nm value of 21.0 mM¹ cm¹ was applied for the reduced form of cytochrome c (34).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

flavoHb Is an Inefficient Catalyst for NO Reduction in E. coli-- We measured the ability of flavoHb to metabolize NO under aerobic and anaerobic conditions in an E. coli strain engineered to express flavoHb at high levels from plasmid pAlterhmp. Cells containing pAlter grown under aerobic conditions expressed low constitutive aerobic and anaerobic NO consumption activities (Fig. 1, A and B, white bars, respectively), whereas cells bearing pAlterhmp expressed ~25- and ~6-fold higher aerobic and anaerobic consumption activities, respectively (black bars). Dioxygen (200 µM) increased the NO consumption rate of flavoHb-overproducing cells by >400-fold (compare A and B, black bars). The results demonstrate the importance of O₂ for NO metabolism via flavoHb within cells.

View larger version (17K): [in this window] [in a new window]   Fig. 1.   flavoHb-catalyzed NO consumption is O2-dependent. AB1157 cells containing pAlter or pAlterhmp were assayed for NO consumption activity with 200 µM O2 (A) or ~0 µM O2 (B). Aerobic and anaerobic NO consumption activities were measured as described under "Materials and Methods." Cells were grown aerobically in phosphate-buffered LB medium and harvested in log phase. Results are means ± S.D. of three independent trials. Note that there is a 400-fold difference in the ordinate scales of A and B.

flavoHb Protects Aconitase from NO-mediated Inactivation in Aerobic, but Not Anoxic, E. coli-- The ability of flavoHb to protect aconitase from NO-mediated inactivation was tested in the flavoHb-deficient strain RB9060 expressing flavoHb from pAlterhmp and in control RB9060 containing pAlter only. In these experiments, cells were grown to early log phase and treated with chloramphenicol to block nascent protein synthesis. Cultures were then exposed to NO mixed with an atmosphere containing either 21% O₂ balanced with N₂ or N₂ only, and aconitase activity was determined after various cell exposure times. In the presence of O₂, elevated flavoHb afforded complete protection of aconitase activity in gas mixtures containing 960 ppm NO, an exposure producing 2 µM NO in the medium (Fig. 2A, compare lines 1 and 2). However, in the absence of O₂, overexpressed flavoHb failed to protect aconitase from inactivation during exposures to 240 ppm NO gas (0.5 µM) (Fig. 2B, compare lines 1 and 2). These results clearly demonstrate that flavoHb requires O₂ to protect the sensitive aconitase and to detoxify NO within cells.

View larger version (14K): [in this window] [in a new window]   Fig. 2.   flavoHb protects aconitase from NO-mediated inactivation in aerated E. coli. Aerobic cultures of flavoHb-deficient RB9060 containing pAlter (lines 1) and flavoHb-overproducing RB9060 containing pAlterhmp (lines 2) were grown to an A550 of ~0.5 and treated with chloramphenicol (200 µg/ml) for 15 min prior to exposure to an atmosphere containing 960 ppm NO in 21% O2 balanced with N2 (A) or containing 240 ppm NO in N2 only (B). Cells were harvested and assayed for aconitase activity at intervals as described under "Materials and Methods." Percent aconitase activity is expressed relative to aconitase activity at t = 0. 100% aconitase activity equaled 63 ± 2 and 51 ± 9 milliunits/mg of extract protein for RB9060 bearing pAlter and pAlterhmp, respectively. Under these growth conditions, RB9060 bearing pAlter and pAlterhmp consumed NO at <0.1 and 60 nmol/min/108 cells, respectively. Results are representative of two independent experiments.

E. coli Adaptively Protects Aconitase against Anaerobic NO-- We previously demonstrated that anaerobic E. coli protects aconitase from NO-mediated inactivation and that this protection is dependent upon active protein synthesis (16). The importance of flavoHb for anaerobic protection required examination. Aconitase showed similar sensitivity to 240 ppm NO gas (0.5 µM) in flavoHb-deficient and flavoHb-proficient cells in the presence of chloramphenicol (Fig. 3, compare lines 2 and 4). Moreover, mutant and parental cells showed a similar capacity for protection of aconitase against NO during active protein synthesis (compare lines 1 and 3). Clearly, flavoHb has no role in inducible anaerobic protection of aconitase. These data also suggest the existence of an alternate anaerobic NO detoxification mechanism requiring active protein synthesis for its function.

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Anaerobically induced protein(s) protect aconitase from NO-mediated inactivation independent of flavoHb expression. Anaerobic cultures of AB1157 (lines 1 and 2) and flavoHb-deficient strain AG103 (lines 3 and 4) were exposed to 960 ppm NO in N2. Cultures were incubated either with chloramphenicol (200 µg/ml) (lines 2 and 4) or no addition (lines 1 and 3) for 15 min before the NO or N2 exposure. Aconitase activity in NO-exposed cells was measured at intervals. Percent aconitase activity was calculated relative to aconitase activity measured in N2-exposed control cells. Cultures were initiated at an A550 of ~0.8 from overnight static cultures grown in minimal salts medium with 20 mM glucose and were incubated for 45 min under N2 before exposure to NO. Results are representative of two independent experiments.

E. coli Produces a Unique NO-metabolizing Activity in Response to NO-- In the absence of NO exposure, anaerobic E. coli expresses negligible anaerobic NO consumption activity (data not shown) (35). To determine whether E. coli produces a flavoHb-independent NO consumption activity, anoxic cultures of wild-type E. coli K12 (ATCC 23716) and the corresponding flavoHb-deficient mutant (AG102) were exposed to 960 ppm NO (2 µM) for 45 min, and their anaerobic and aerobic NO consumption activities were measured. Under these conditions, both wild-type and flavoHb-deficient cells produced similar high levels of an anaerobic NO consumption activity (Fig. 4A). Anaerobic cells clearly induced an aerobic NO consumption activity attributable to flavoHb because this activity was not expressed by flavoHb-deficient AG102 (Fig. 4B). Furthermore, the flavoHb-independent activity was not detectable in cells assayed in the presence of 200 µM O₂.

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Anaerobic exposure of E. coli to NO induces expression of an anaerobic NO consumption activity independent of flavoHb. Wild-type E. coli K12 (ATCC 23716) and the flavoHb-deficient mutant AG102 were grown in phosphate-buffered LB medium with 20 mM glucose under N2. Following NO exposures, cells were harvested at intervals and assayed for NO consumption activity in the absence (A) or presence (B) of 200 µM O2. Cultures were initiated with 4% inocula from static overnight anoxic cultures. At an A550 of ~0.3, cultures were either exposed to an atmosphere of 960 ppm NO in N2 or maintained under N2 for 45 min. Cultures were then treated with chloramphenicol (200 µg/ml) for an additional 15 min to limit protein synthesis during harvesting and assays. Results are means ± S.D. of three independent experiments.

Dioxygen and Cyanide Sensitivity of Anaerobic NO Consumption-- The O₂ sensitivity of the anaerobic NO consumption activity was tested by exposing anaerobically induced cells to air. Under N₂, the activity was stable in both wild-type (Fig. 5, line 1) and flavoHb-deficient (data not shown) cells. However, the activity was rapidly and irreversibly lost with a single exponential inactivation rate (t_1/2 = 5 min) upon exposure of cells to normoxia (line 2). The inactivation kinetics suggest a bimolecular reaction between O₂ and the enzymatic activity. Similar, albeit slower, losses of the activity were observed during sample processing and incubation of cells on ice (data not shown). For these reasons, rapid cell harvest and immediate assay of NO consumption were absolutely imperative.

View larger version (19K): [in this window] [in a new window]   Fig. 5.   O2 sensitivity of the induced anaerobic NO consumption activity. NO-treated AB1157 cells were maintained under N2 (line 1) or were exposed to 21% O2 in N2 (line 2). Cultures were initiated with 4% inocula from static overnight anaerobic cultures and grown in phosphate-buffered LB medium with 20 mM glucose under N2. At an A550 of ~0.3, cultures were exposed to 960 ppm NO in N2 for 30 min to induce the anaerobic activity and were treated with chloramphenicol (200 µg/ml) for 15 min prior to O2 or N2 exposures. Cells were harvested, and anaerobic NO consumption activity was assayed as described under "Materials and Methods." Results are representative of three independent trials.

To further characterize the anaerobic NO consumption activity, we measured sensitivity to cyanide. The cytochrome bc- and P450-type NORs are 50% inhibited by ~0.3 and ~1 mM cyanide, respectively (36, 37). The E. coli activity was relatively resistant to cyanide; 2 mM NaCN was required to inhibit the activity by ~50% (Table I). By comparison, half-maximal inhibition of NOD activity was observed with ~2.5 µM NaCN. In addition, 2-heptyl-4-hydroxyquinoline-N-oxide (100 µM), an inhibitor of menaquinone-dependent membrane-bound electron transfer reactions, had no effect on the anaerobic activity. Although these cyanide sensitivity data are suggestive of a cytochrome bc- or P450-type NOR, there is no indication that cytochrome bc- and P450-type NOR activities are sensitive to inactivation by O₂. These data, together with the absence of a recognizable homolog of a cytochrome bc- or P450-type NOR in E. coli genome searches, provide strong evidence for a novel NOR activity in E. coli.

flavoHb Protects Aerobic, but Not Anaerobic, E. coli from NO-mediated Growth Inhibition-- flavoHb-deficient strains exhibited aerobic growth rates that were very similar to their parental strains (Fig. 6, A and B, compare lines 1 and 2). As previously reported for strain RB9060 (15), exposure of flavoHb-deficient strains to 960 ppm NO gas (2 µM) arrested aerobic growth (line 4), whereas the parental strains were far less sensitive to NO under these conditions (line 3). In contrast, under anaerobic growth conditions, flavoHb mutants divided somewhat slower even in the absence of a NO stress (C and D, compare lines 1 and 2). Furthermore, the anaerobic growth deficiency was comparable to that observed with exposure to 480 ppm NO gas (1 µM) (C and D, compare lines 3 and 4). Similar growth differentials between flavoHb-deficient and parental strains were also observed with lower NO exposures (data not shown). It should be noted that the flavoHb-deficient strain RB9060 also shows a small, but reproducible, anaerobic growth defect in the absence of NO stress (15). However, RB9060 contains a deletion encompassing the glyA-hmp-glnB region (30), thus making interpretations of growth differences more complicated.

View larger version (27K): [in this window] [in a new window]   Fig. 6.   Effects of flavoHb expression on the resistance of E. coli to NO-mediated growth inhibition. Aerobic growth (A) and anaerobic growth (C) of AB1157 (lines 1 and 3) and the flavoHb-deficient mutant AG103 (lines 2 and 4) were followed with or without NO exposure. Aerobic cultures were exposed to 0 ppm NO (lines 1 and 2) or 960 ppm NO gas (lines 3 and 4) at the times indicated by the arrows. Anaerobic cultures were exposed to 0 ppm NO (lines 1 and 2) or 480 ppm NO gas (lines 3 and 4) at the times indicated by the arrows. Aerobic growth (B) and anaerobic growth (D) of wild-type E. coli K12 (ATCC 23716) (lines 1 and 3) and the flavoHb-deficient mutant AG102 (lines 2 and 4) were measured as described above. Cultures were grown in phosphate-buffered LB medium at 37 °C under an atmosphere containing 21% O2 balanced with N2 or N2 only. Aerobic and anaerobic cultures were initiated with 2 and 4% inocula from aerobic and static overnight cultures, respectively. Results are representative of two independent experiments.

In total, these growth data demonstrate a minor role (if any) for flavoHb in anaerobic NO detoxification. Interestingly, however, the growth stimulatory effects of flavoHb suggest the possibility of anaerobic roles for flavoHb.

Enzymatic Activities of Isolated flavoHb-- E. coli flavoHb has a number of anaerobic and aerobic enzymatic activities. The turnover rate of the NOD activity was orders of magnitude greater than that of any other activity measured (Table II). Although a lower NOD turnover number of 10 s¹ was recently reported by others (38), it is important to note that rates were measured at NO:O₂ ratios that produce significant inhibition of NOD activity by NO (17, 18). flavoHb also showed anaerobic NADH oxidase activities with 1 mM GSNO and 1 mM nitrite as electron acceptors. Moreover, these turnover rates were comparable to those determined for NO under similar conditions (0.02 NO heme¹ s¹) (Table II). In these reductive reactions, flavoHb generated NO from GSNO with a turnover number of 0.08 NO heme¹ s¹. flavoHb also catalyzed NO release from S-nitrosopenicillamine (1 mM), another S-nitrosothiol, at a rate of 0.05 NO heme¹ s¹. However, we were unable to detect NO production or accumulation with nitrite (1 mM) as the anaerobic substrate (data not shown), suggesting a reduction mechanism not involving significant release or accumulation of NO. The reaction of nitrite with reduced flavoHb may be similar to the reaction of nitrite/nitrous acid with human deoxyhemoglobin (39) and may result in slow NO release, Hb oxidation, and nitrosylhemoglobin formation. flavoHb also showed aerobic NADH oxidase (O₂ reductase) and anaerobic cytochrome c reductase activities that were higher than those determined for NO, nitrite, or GSNO reduction (Table II).

Together, our results suggest that flavoHb provides mechanisms, albeit inefficient, for NO, nitrite, or nitroso compound reduction. Furthermore, the reductase activities of flavoHb may explain the growth protection flavoHb affords against various nitrosative and oxidative agents (19, 20, 23-25).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The maximal turnover rate for NO dioxygenation by isolated E. coli flavoHb (670 s¹) is several orders of magnitude greater than that which we measured for NO reduction (~0.02 s¹) (Table II) (17, 18). Yet, it has never been determined whether the NOR activity of inducible flavoHb functions in anaerobic NO detoxification, as recently suggested (19, 20, 22). Our results indicate that flavoHb plays a minor role (if any) in anaerobic NO metabolism and detoxification. flavoHb showed little NO metabolic activity in anaerobic cells (Fig. 1). Furthermore, flavoHb afforded no protection against NO as measured by effects on aconitase activity (Fig. 2) and growth under anaerobic conditions (Fig. 6). Moreover, the reported anaerobic (or aerobic) growth protective effects of flavoHb against "nitrosative stressors," including acidified nitrite, GSNO, and various NO donors (19, 20, 23, 26), are unlikely to be related to NO reduction (or dioxygenation) given the capacity of flavoHb to reduce these compounds directly (Table II). Understanding the role of flavoHb in protection against various nitrosative stressors demands a critical evaluation of the detoxification mechanisms for these agents within cells. Pure NO gas is readily available and is clearly preferable for investigations aimed at understanding NO toxicity and detoxification mechanisms.

Data demonstrating a robust NO-inducible anaerobic NO metabolic activity that is independent of flavoHb (Fig. 4 and Table I) also argue strongly against a functional role for the NOR activity of flavoHb (Table II) (35). This novel NOR activity catalyzed anaerobic NO removal at a rate that was roughly 150-fold higher than that of overexpressed or anaerobically induced flavoHb (Figs. 1 and 4). Anaerobically induced flavoHb may nevertheless serve an important function in cells. The exquisite sensitivity of the anaerobic NO-scavenging activity to O₂ (Fig. 5) may limit the survival value of this NO detoxification system during anaerobic-to-aerobic transitions. Furthermore, growth stimulatory effects of flavoHbs and homologous single domain Hbs have been previously reported for microaerobic growth conditions and have been associated with altered glycolytic and citric acid cycle intermediates and activities (40). Thus, flavoHb may additionally benefit cells in which aconitase, the citric acid cycle, respiration, and energy production are threatened by NO inhibition. Further investigations of the mechanism of anaerobic (Fig. 6) and microaerobic growth stimulation and metabolic alterations promise further insights into anaerobic functions of (flavo)Hbs.

The identity and mechanism of the O₂-sensitive and cyanide-resistant anaerobic NO metabolic activity are of special interest because neither cytochrome bc- nor P450-type NORs have homologs in E. coli. Neither of these NORs is sensitive to O₂, nor are they as resistant to cyanide (1, 37). It is likely that the newly identified NO-scavenging activity (35) plays a critical role in the detoxification of NO and bacterial survival in the anaerobic and microaerobic environments encountered by E. coli and other pathogens.

	ACKNOWLEDGEMENTS

We thank Drs. Alex Ninfa, Boris Magasnik, George Stauffer, Jim Imlay, Bruce Demple, and Mary Berlyn for generously providing E. coli strains, plasmids, and phage used in this study.

	FOOTNOTES

^* This work was supported in part by a grant from the Children's Hospital Research Foundation Trustees, American Heart Association Grant 9730193N, and National Institutes of Health Grant GM65090.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Div. of Critical Care Medicine, MLC7006, Children's Hospital Medical Center, 3333 Burnet Ave., Cincinnati, OH 45229. Tel.: 513-636-4885; Fax: 513-636-4892; E-mail: gardp0@chmcc.org.

Published, JBC Papers in Press, December 18, 2001, DOI 10.1074/jbc.M110470200

	ABBREVIATIONS

The abbreviations used are: NORs, nitric-oxide reductases; flavoHb, flavohemoglobin; GSNO, S-nitrosoglutathione; NOD, nitric-oxide dioxygenase; MOPS, 4-morpholinepropanesulfonic acid.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				I. Gusarov, M. Starodubtseva, Z.-Q. Wang, L. McQuade, S. J. Lippard, D. J. Stuehr, and E. Nudler Bacterial Nitric-oxide Synthases Operate without a Dedicated Redox Partner J. Biol. Chem., May 9, 2008; 283(19): 13140 - 13147. [Abstract] [Full Text] [PDF]

				N. J. Gilberthorpe and R. K. Poole Nitric Oxide Homeostasis in Salmonella typhimurium: ROLES OF RESPIRATORY NITRATE REDUCTASE AND FLAVOHEMOGLOBIN J. Biol. Chem., April 25, 2008; 283(17): 11146 - 11154. [Abstract] [Full Text] [PDF]

				P. C. Mills, G. Rowley, S. Spiro, J. C. D. Hinton, and D. J. Richardson A combination of cytochrome c nitrite reductase (NrfA) and flavorubredoxin (NorV) protects Salmonella enterica serovar Typhimurium against killing by NO in anoxic environments Microbiology, April 1, 2008; 154(4): 1218 - 1228. [Abstract] [Full Text] [PDF]

				T. W. Overton, M. C. Justino, Y. Li, J. M. Baptista, A. M. P. Melo, J. A. Cole, and L. M. Saraiva Widespread Distribution in Pathogenic Bacteria of Di-Iron Proteins That Repair Oxidative and Nitrosative Damage to Iron-Sulfur Centers J. Bacteriol., March 15, 2008; 190(6): 2004 - 2013. [Abstract] [Full Text] [PDF]

				S. M. Sievert, K. M. Scott, M. G. Klotz, P. S. G. Chain, L. J. Hauser, J. Hemp, M. Hugler, M. Land, A. Lapidus, F. W. Larimer, et al. Genome of the Epsilonproteobacterial Chemolithoautotroph Sulfurimonas denitrificans Appl. Envir. Microbiol., February 15, 2008; 74(4): 1145 - 1156. [Abstract] [Full Text] [PDF]

				N. P. Tucker, B. D'Autreaux, F. K. Yousafzai, S. A. Fairhurst, S. Spiro, and R. Dixon Analysis of the Nitric Oxide-sensing Non-heme Iron Center in the NorR Regulatory Protein J. Biol. Chem., January 11, 2008; 283(2): 908 - 918. [Abstract] [Full Text] [PDF]

				S. T. Pullan, M. D. Gidley, R. A. Jones, J. Barrett, T. M. Stevanin, R. C. Read, J. Green, and R. K. Poole Nitric Oxide in Chemostat-Cultured Escherichia coli Is Sensed by Fnr and Other Global Regulators: Unaltered Methionine Biosynthesis Indicates Lack of S Nitrosation J. Bacteriol., March 1, 2007; 189(5): 1845 - 1855. [Abstract] [Full Text] [PDF]

				Y.-Y. Lee, N. Shearer, and S. Spiro Transcription factor NNR from Paracoccus denitrificans is a sensor of both nitric oxide and oxygen: isolation of nnr* alleles encoding effector-independent proteins and evidence for a haem-based sensing mechanism. Microbiology, May 1, 2006; 152(Pt 5): 1461 - 1470. [Abstract] [Full Text] [PDF]

				P. S. Choi, Z. Naal, C. Moore, E. Casado-Rivera, H. D. Abruna, J. D. Helmann, and J. P. Shapleigh Assessing the impact of denitrifier-produced nitric oxide on other bacteria. Appl. Envir. Microbiol., March 1, 2006; 72(3): 2200 - 2205. [Abstract] [Full Text] [PDF]

				D. M. Bodenmiller and S. Spiro The yjeB (nsrR) Gene of Escherichia coli Encodes a Nitric Oxide-Sensitive Transcriptional Regulator J. Bacteriol., February 1, 2006; 188(3): 874 - 881. [Abstract] [Full Text] [PDF]

				H. Arai, M. Hayashi, A. Kuroi, M. Ishii, and Y. Igarashi Transcriptional Regulation of the Flavohemoglobin Gene for Aerobic Nitric Oxide Detoxification by the Second Nitric Oxide-Responsive Regulator of Pseudomonas aeruginosa J. Bacteriol., June 15, 2005; 187(12): 3960 - 3968. [Abstract] [Full Text] [PDF]

				R. A. Helmick, A. E. Fletcher, A. M. Gardner, C. R. Gessner, A. N. Hvitved, M. C. Gustin, and P. R. Gardner Imidazole Antibiotics Inhibit the Nitric Oxide Dioxygenase Function of Microbial Flavohemoglobin Antimicrob. Agents Chemother., May 1, 2005; 49(5): 1837 - 1843. [Abstract] [Full Text] [PDF]

				M. C. Justino, J. B. Vicente, M. Teixeira, and L. M. Saraiva New Genes Implicated in the Protection of Anaerobically Grown Escherichia coli against Nitric Oxide J. Biol. Chem., January 28, 2005; 280(4): 2636 - 2643. [Abstract] [Full Text] [PDF]

				N. P. Tucker, B. D'Autreaux, D. J. Studholme, S. Spiro, and R. Dixon DNA Binding Activity of the Escherichia coli Nitric Oxide Sensor NorR Suggests a Conserved Target Sequence in Diverse Proteobacteria J. Bacteriol., October 1, 2004; 186(19): 6656 - 6660. [Abstract] [Full Text] [PDF]

				C. M. Moore, M. M. Nakano, T. Wang, R. W. Ye, and J. D. Helmann Response of Bacillus subtilis to Nitric Oxide and the Nitrosating Agent Sodium Nitroprusside J. Bacteriol., July 15, 2004; 186(14): 4655 - 4664. [Abstract] [Full Text] [PDF]

				B. D. Ullmann, H. Myers, W. Chiranand, A. L. Lazzell, Q. Zhao, L. A. Vega, J. L. Lopez-Ribot, P. R. Gardner, and M. C. Gustin Inducible Defense Mechanism against Nitric Oxide in Candida albicans Eukaryot. Cell, June 1, 2004; 3(3): 715 - 723. [Abstract] [Full Text] [PDF]

				P. Mukhopadhyay, M. Zheng, L. A. Bedzyk, R. A. LaRossa, and G. Storz Prominent roles of the NorR and Fur regulators in the Escherichia coli transcriptional response to reactive nitrogen species PNAS, January 20, 2004; 101(3): 745 - 750. [Abstract] [Full Text] [PDF]

				H. Corker and R. K. Poole Nitric Oxide Formation by Escherichia coli: DEPENDENCE ON NITRITE REDUCTASE, THE NO-SENSING REGULATOR Fnr, AND FLAVOHEMOGLOBIN Hmp J. Biol. Chem., August 22, 2003; 278(34): 31584 - 31592. [Abstract] [Full Text] [PDF]

				A. M. Gardner, C. R. Gessner, and P. R. Gardner Regulation of the Nitric Oxide Reduction Operon (norRVW) in Escherichia coli. ROLE OF NorR AND sigma 54 IN THE NITRIC OXIDE STRESS RESPONSE J. Biol. Chem., March 14, 2003; 278(12): 10081 - 10086. [Abstract] [Full Text] [PDF]

				S. Van Doorslaer, S. Dewilde, L. Kiger, S. V. Nistor, E. Goovaerts, M. C. Marden, and L. Moens Nitric Oxide Binding Properties of Neuroglobin. A CHARACTERIZATION BY EPR AND FLASH PHOTOLYSIS J. Biol. Chem., February 7, 2003; 278(7): 4919 - 4925. [Abstract] [Full Text] [PDF]

				B. D'Autreaux, D. Touati, B. Bersch, J.-M. Latour, and I. Michaud-Soret Direct inhibition by nitric oxide of the transcriptional ferric uptake regulation protein via nitrosylation of the iron PNAS, December 24, 2002; 99(26): 16619 - 16624. [Abstract] [Full Text] [PDF]

				M. I. Hutchings, N. Mandhana, and S. Spiro The NorR Protein of Escherichia coli Activates Expression of the Flavorubredoxin Gene norV in Response to Reactive Nitrogen Species J. Bacteriol., August 15, 2002; 184(16): 4640 - 4643. [Abstract] [Full Text] [PDF]

				S. R. Poock, E. R. Leach, J. W. B. Moir, J. A. Cole, and D. J. Richardson Respiratory Detoxification of Nitric Oxide by the Cytochrome c Nitrite Reductase of Escherichia coli J. Biol. Chem., June 21, 2002; 277(26): 23664 - 23669. [Abstract] [Full Text] [PDF]

				A. M. Gardner, R. A. Helmick, and P. R. Gardner Flavorubredoxin, an Inducible Catalyst for Nitric Oxide Reduction and Detoxification in Escherichia coli J. Biol. Chem., March 1, 2002; 277(10): 8172 - 8177. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 277/10/8166    most recent M110470200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Gardner, A. M. Articles by Gardner, P. R. Search for Related Content PubMed PubMed Citation Articles by Gardner, A. M. Articles by Gardner, P. R. Social Bookmarking                      What's this?