Flavohemoglobin detoxifies nitric oxide in aerobic, but not anaerobic, Escherichia coli. Evidence for a novel inducible anaerobic nitric oxide-scavenging activity.

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 O(2) were substantial. A NO-inducible, O(2)-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 O(2)-dependent NO dioxygenation.

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 O 2 were substantial. A NOinducible, O 2 -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 O 2 -dependent NO dioxygenation.
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)(3)(4)(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).
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 NOreleasing nitroso compound spermine 2,2Ј-(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 2,2Ј-(hydroxynitrosohydrazono)bisethanamine (20). Pure NO gas (15) and nitroso compounds (19,25) potently inhibit the growth of Escherichia coli flavoHbdeficient 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 N 2 O formation are compromised in flavoHb-deficient mutants of Alcaligenes eutrophus (27). In the plant pathogen Erwinia chrysanthemi, fla-voHb 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 O 2 -dependent NOD, but they do not support a role for flavoHb as an anaerobic NOR. Our investigations have revealed a novel NO-inducible and O 2 -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.
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 K 2 HPO 4 and 33 mM KH 2 PO 4 (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 g/ml) was added to the growth medium for strains harboring pAlter or pAlterhmp. Minimal salts medium (pH 7.0) contained 60 mM K 2 HPO 4 , 33 mM KH 2 PO 4 , 7.6 mM (NH 4 ) 2 SO 4 , 1.7 mM sodium citrate, 1 mM MgSO 4 , 10 M MnCl 2 , 10 g/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 ϫ 10 8 bacteria/ml.
Gas Exposures-Three-way stainless steel gas proportioners (Cole-Parmer Instrument Co.) were used to mix O 2 , N 2 , 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 (Ն3.2 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 stoppersealed 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 K 2 HPO 4 , 33 mM KH 2 PO 4 , 7.6 mM (NH 4 ) 2 SO 4 , 1.7 mM sodium citrate, 10 mM glucose, 200 M O 2 , and 200 g/ml chloramphenicol and thermostatted at 37°C. NO (2 M) was injected into the closed chamber through the narrow port of the glass stopper with a 10-l Hamilton syringe, and the electrode signal was recorded on chart paper. Cells (0.2-2 ϫ 10 7 ) 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 M NO or a half-maximal signal amplitude. Aerobic NO consumption rates were corrected for relatively small background rates of NO decomposition at 1.0 M NO of Յ0.5 nmol of NO/min. Anaerobic NO consumption activities of cells were measured as described for aerobic activity measurements, except that O 2 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. O 2 depletion under these conditions was verified with a Clark-type O 2 electrode (YSI Inc.). Anaerobic NO consumption activities were determined for 1.5 M NO and were corrected for the background rates of NO decomposition of Յ0.3 nmol of NO/min. Culture aliquots (1 ml) were quickly centrifuged for 20 s at 20,000 ϫ 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 ϫ g for 20 s. Cell pellets were resuspended at 1 ϫ 10 7 cells/l 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 ϫ 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 l of ice-cold 50 mM Tris-Cl (pH 7.4) containing 20 M fluorocitrate and 0.6 mM MnCl 2 . Lysates were clarified by centrifugation at 20,000 ϫ 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).

RESULTS
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,  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% O 2 balanced with N 2 or N 2 only, and aconitase activity was determined after various cell exposure times. In the presence of O 2 , elevated flavoHb afforded complete protection of aconitase activity in gas mixtures containing 960 ppm NO, an exposure producing Յ2 M NO in the medium ( Fig. 2A, compare lines 1 and 2). However, in the absence of O 2 , overexpressed flavoHb failed to protect aconitase from inactivation during exposures to 240 ppm NO gas (Յ0.5 M) (Fig. 2B, compare lines 1 and 2). These results clearly demonstrate that flavoHb requires O 2 to protect the sensitive aconitase and to detoxify NO within cells.
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 (Յ0.5 M) 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.
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 fla-voHb-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 (Յ2 M) 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 M O 2 .
Dioxygen and Cyanide Sensitivity of Anaerobic NO Consumption-The O 2 sensitivity of the anaerobic NO consumption activity was tested by exposing anaerobically induced cells to air. Under N 2 , 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 (t1 ⁄2 ϭ 5 min) upon exposure of cells to normoxia (line 2). The inactivation kinetics suggest a bimolecular reaction between O 2 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.
To further characterize the anaerobic NO consumption activity, we measured sensitivity to cyanide. The cytochrome bcand P450-type NORs are 50% inhibited by ϳ0.3 and ϳ1 mM cyanide, respectively (36,37). The E. coli activity was relatively resistant to cyanide; 2 mM NaCN was required to inhibit the activity by ϳ50% (Table I). By comparison, half-maximal inhibition of NOD activity was observed with ϳ2.5 M NaCN. In addition, 2-heptyl-4-hydroxyquinoline-N-oxide (100 M), 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 O 2 . 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 fla-  1 and 2) and flavoHb-deficient strain AG103 (lines 3 and 4) were exposed to 960 ppm NO in N 2 . Cultures were incubated either with chloramphenicol (200 g/ml) (lines 2 and 4) or no addition (lines 1 and 3) for 15 min before the NO or N 2 exposure. Aconitase activity in NO-exposed cells was measured at intervals. Percent aconitase activity was calculated relative to aconitase activity measured in N 2 -exposed control cells. Cultures were initiated at an A 550 of ϳ0.8 from overnight static cultures grown in minimal salts medium with 20 mM glucose and were incubated for 45 min under N 2 before exposure to NO. Results are representative of two independent experiments.
voHb-deficient strains to 960 ppm NO gas (Յ2 M) 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 (Յ1 M) (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.
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 s Ϫ1 was recently reported by others (38), it is important to note that rates were measured at NO:O 2 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 heme Ϫ1 s Ϫ1 ) (Table II). In these reductive reactions, flavoHb generated NO from GSNO with a turnover number of 0.08 NO heme Ϫ1 s Ϫ1 . flavoHb also catalyzed NO release from S-nitrosopenicillamine (1 mM), another S-nitrosothiol, at a rate of 0.05 NO heme Ϫ1 s Ϫ1 . However, we were unable to detect NO NO-treated AB1157 cells were maintained under N 2 (line 1) or were exposed to 21% O 2 in N 2 (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 N 2 . At an A 550 of ϳ0.3, cultures were exposed to 960 ppm NO in N 2 for 30 min to induce the anaerobic activity and were treated with chloramphenicol (200 g/ml) for 15 min prior to O 2 or N 2 exposures. Cells were harvested, and anaerobic NO consumption activity was assayed as described under "Materials and Methods." Results are representative of three independent trials.  ) were grown under an atmosphere of N 2 to an A 550 of ϳ0.4 in phosphate-buffered LB medium with 20 mM glucose and were exposed to 960 ppm NO in N 2 for 30 min. Chloramphenicol (200 g/ml) was added, and the incubation with NO gas was continued for an additional 15 min. Cells were harvested, washed, and assayed for anaerobic and aerobic NO consumption activities in the presence of sodium cyanide (NaCN) or 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO). Relative percentages are given in parentheses. Data are representative of two experiments. ND, not determined. NO 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% O 2 balanced with N 2 or N 2 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. 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 (O 2 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)(24)(25). DISCUSSION The maximal turnover rate for NO dioxygenation by isolated E. coli flavoHb (670 s Ϫ1 ) is several orders of magnitude greater than that which we measured for NO reduction (ϳ0.02 s Ϫ1 ) (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 O 2 (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 O 2 -sensitive and cyanideresistant 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 O 2 , 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.