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

2002; Elsevier BV; Volume: 277; Issue: 10 Linguagem: Inglês

10.1074/jbc.m110470200

ISSN

1083-351X

Autores

Anne M. Gardner, Paul R. Gardner,

Tópico(s)

Neuroscience of respiration and sleep

Resumo

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 inEscherichia 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. 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 inEscherichia 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. nitric-oxide reductases flavohemoglobin S-nitrosoglutathione nitric-oxide dioxygenase 4-morpholinepropanesulfonic acid 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 (1Zumft W. Microbiol. Mol. Biol. Rev. 1997; 61: 533-616Crossref PubMed Scopus (2897) Google Scholar, 2Watmough N.J. Butland G. Cheesman M.R. Moir J.W.B. Richardson D.J. Spiro S. Biochim. Biophys. Acta. 1999; 1411: 456-474Crossref PubMed Scopus (118) Google Scholar) and is produced by NO synthases of animals and plants, where it functions as a broad-spectrum antibiotic and signaling molecule (2Watmough N.J. Butland G. Cheesman M.R. Moir J.W.B. Richardson D.J. Spiro S. Biochim. Biophys. Acta. 1999; 1411: 456-474Crossref PubMed Scopus (118) Google Scholar, 3Fang F.C. J. Clin. Invest. 1997; 99: 2818-2825Crossref PubMed Google Scholar, 4Nathan C. FASEB J. 1992; 6: 3051-3064Crossref PubMed Scopus (4158) Google Scholar, 5Durner J. Wendehenne D. Klessig D.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10328-10333Crossref PubMed Scopus (1061) Google Scholar). Nanomolar NO concentrations inactivate or inhibit critical enzymes, including the citric acid cycle enzyme aconitase (6Drapier J.-C. Hibbs Jr., J.B. Methods Enzymol. 1996; 269: 26-36Crossref PubMed Google Scholar, 7Gardner P.R. Costantino G. Szabó C. Salzman A.L. J. Biol. Chem. 1997; 272: 25071-25076Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar, 8Kennedy M.C. Antholine W.E. Beinert H. J. Biol. Chem. 1997; 272: 20340-20347Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar) and terminal respiratory oxidases (9Brown G.C. Biochim. Biophys. Acta. 1999; 1411: 351-369Crossref PubMed Scopus (621) Google Scholar, 10Stevanin T.M. Ioannidis N. Mills C.E. Kim S.O. Hughes M.N. Poole R.K. J. Biol. Chem. 2000; 275: 35868-35875Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). NO also has the potential to damage a variety of biomolecules by forming the more indiscriminate toxin and oxidizing agent peroxynitrite (11Beckman J.S. Beckman T.W. Chen J. Marshall P.A. Freeman B.A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1620-1624Crossref PubMed Scopus (6718) Google Scholar). 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 (1Zumft W. Microbiol. Mol. Biol. Rev. 1997; 61: 533-616Crossref PubMed Scopus (2897) Google Scholar, 12Kudo T. Takaya N. Park S.-Y. Shiro Y. Shoun H. J. Biol. Chem. 2001; 276: 5020-5026Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 13Householder T.C. Fozo E.M. Cardinale J.A. Clark V.L. Infect. Immun. 2000; 68: 5241-5246Crossref PubMed Scopus (85) Google Scholar). 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 (1Zumft W. Microbiol. Mol. Biol. Rev. 1997; 61: 533-616Crossref PubMed Scopus (2897) Google Scholar, 14Brown K. Djinovic-Carugo K. Haltia T. Cabrito I. Saraste M. Moura J.J.G. Moura I. Tegoni M. Cambillau C. J. Biol. Chem. 2000; 275: 41133-41136Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). In addition, NO-inducible flavohemoglobins (flavoHbs) dioxygenate NO to form NO3− (10Stevanin T.M. Ioannidis N. Mills C.E. Kim S.O. Hughes M.N. Poole R.K. J. Biol. Chem. 2000; 275: 35868-35875Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 15Gardner P.R. Gardner A.M. Martin L.A. Salzman A.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10378-10383Crossref PubMed Scopus (497) Google Scholar, 16Gardner P.R. Costantino G. Salzman A.L. J. Biol. Chem. 1998; 273: 26528-26533Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 17Gardner A.M. Martin L.A. Gardner P.R. Dou Y. Olson J.S. J. Biol. Chem. 2000; 275: 12581-12589Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 18Gardner P.R. Gardner A.M. Martin L.A. Dou Y. Li T. Olson J.S. Zhu H. Riggs A.F. J. Biol. Chem. 2000; 275: 31581-31587Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 19Hausladen A. Gow A.J. Stamler J.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14100-14105Crossref PubMed Scopus (255) Google Scholar, 20Liu L. Zeng M. Hausladen A. Heitman J. Stamler J.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4672-4676Crossref PubMed Scopus (166) Google Scholar, 21Gardner P.R. Martin L.A. Hall D. Gardner A.M. Free Radic. Biol. Med. 2001; 31: 191-204Crossref PubMed Scopus (85) Google Scholar) and reduce NO to form N2O (19Hausladen A. Gow A.J. Stamler J.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14100-14105Crossref PubMed Scopus (255) Google Scholar, 22Kim S.O. Orii Y. Lloyd D. Hughes M.N. Poole R.K. FEBS Lett. 1999; 445: 389-394Crossref PubMed Scopus (142) Google Scholar). 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 (23Crawford M.J. Goldberg D.E. J. Biol. Chem. 1998; 273: 12543-12547Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar,24Crawford M.J. Goldberg D.E. J. Biol. Chem. 1998; 273: 34028-34032Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). flavoHb also protects Saccharomyces cerevisiae from aerobic and anaerobic growth inhibition by 2,2′-(hydroxynitrosohydrazono)bisethanamine (20Liu L. Zeng M. Hausladen A. Heitman J. Stamler J.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4672-4676Crossref PubMed Scopus (166) Google Scholar). Pure NO gas (15Gardner P.R. Gardner A.M. Martin L.A. Salzman A.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10378-10383Crossref PubMed Scopus (497) Google Scholar) and nitroso compounds (19Hausladen A. Gow A.J. Stamler J.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14100-14105Crossref PubMed Scopus (255) Google Scholar, 25Membrillo-Hernández J. Coopamah M.D. Anjum M.F. Stevanin T.M. Kelly A. Hughes M.N. Poole R.K. J. Biol. Chem. 1999; 274: 748-754Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar) potently inhibit the growth ofEscherichia coli flavoHb-deficient mutants under aerobic growth conditions in contrast to flavoHb-containing parent strains, where growth inhibition is minimal. flavoHb-deficientDictyostelium discoideum is also sensitive to GSNO and nitroprusside during aerobic growth (26Iijima M. Shimizu H. Tanaka Y. Urushihara H. Cell Struct. Funct. 2000; 25: 47-55Crossref PubMed Scopus (29) Google Scholar). 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 (15Gardner P.R. Gardner A.M. Martin L.A. Salzman A.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10378-10383Crossref PubMed Scopus (497) Google Scholar). In addition, anaerobic denitrification and N2O formation are compromised in flavoHb-deficient mutants ofAlcaligenes eutrophus (27Cramm R. Siddiqui R.A. Friedrich B. J. Biol. Chem. 1994; 269: 7349-7354Abstract Full Text PDF PubMed Google Scholar). In the plant pathogenErwinia chrysanthemi, flavoHb provides resistance to the immune response of tobacco leaves (28Favey S. Labesse G. Vouille V. Boccara M. Microbiology. 1995; 141: 863-871Crossref PubMed Scopus (66) Google Scholar). 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. 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 mmreduced glutathione and 400 mm sodium nitrite in 1m HCl and by neutralizing the reaction with 1 mNaOH (29Stamler J.S. Simon D.I. Osborne J.A. Mullins M.E. Jaraki O. Michel T. Singel D.J. Loscalzo J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 444-448Crossref PubMed Scopus (1305) Google Scholar). 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 (17Gardner A.M. Martin L.A. Gardner P.R. Dou Y. Olson J.S. J. Biol. Chem. 2000; 275: 12581-12589Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). Manganese-containing superoxide dismutase (2300 units/mg) was induced with paraquat in strain DH5α bearing plasmid pD11c and was isolated from extracts (45Keele Jr., B.B. McCord J.M. Fridovich I. J. Biol. Chem. 1970; 245: 6176-6181Abstract Full Text PDF PubMed Google Scholar, 46Gardner P.R. Fridovich I. J. Biol. Chem. 1993; 268: 12958-12963Abstract Full Text PDF PubMed Google Scholar). 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 (7Gardner P.R. Costantino G. Szabó C. Salzman A.L. J. Biol. Chem. 1997; 272: 25071-25076Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar). E. coli K12 strain RB9060 (ΔglyA-hmp-glnB) was provided by Dr. Alex Ninfa (University of Michigan) (30Bueno R. Pahel G. Magasanik B. J. Bacteriol. 1985; 164: 816-822Crossref PubMed Google Scholar). 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 (15Gardner P.R. Gardner A.M. Martin L.A. Salzman A.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10378-10383Crossref PubMed Scopus (497) Google Scholar). An ∼20-kb EcoRI fragment from plasmid pGS26 (31Plamann M.D. Stauffer G.V. Gene (Amst.). 1983; 22: 9-18Crossref PubMed Scopus (38) Google Scholar) containing the Tn5 kanRelement inserted in the hmp gene (hmp::Tn5) after isoleucine 81 was isolated and used to transform JC7623. Stable insertion ofhmp::Tn5 was selected for on LB agarose containing kanamycin (30 μg/ml). Loss of NO-inducible NOD activity was verified in kanamycin-resistant clones. Thehmp::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. 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 mmK2HPO4, 33 mmKH2PO4, 7.6 mm(NH4)2SO4, 1.7 mmsodium citrate, 1 mm MgSO4, 10 μmMnCl2, 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 × 108bacteria/ml. 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. 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 mmK2HPO4, 33 mmKH2PO4, 7.6 mm(NH4)2SO4, 1.7 mmsodium citrate, 10 mm glucose, 200 μmO2, 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 × 107cells/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. 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 (7Gardner P.R. Costantino G. Szabó C. Salzman A.L. J. Biol. Chem. 1997; 272: 25071-25076Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar, 32Gardner P.R. Methods Enzymol. 2002; 349: 9-23Crossref PubMed Scopus (247) Google Scholar). Protein was determined using the Coomassie Blue dye-binding assay with bovine serum albumin as the standard (33Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216440) Google Scholar). 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 mmsodium 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 μmcytochrome c, 8 units of glucose oxidase, 10 mmglucose, 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 Ε 550 nmvalue of 21.0 mm−1 cm−1 was applied for the reduced form of cytochrome c (34McCord J.M. Fridovich I. J. Biol. Chem. 1969; 244: 6049-6055Abstract Full Text PDF PubMed Google Scholar). 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 O2for NO metabolism via flavoHb within cells. 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.2 A, compare lines 1and 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.2 B, compare lines 1 and 2). These results clearly demonstrate that flavoHb requires O2 to protect the sensitive aconitase and to detoxify NO within cells. We previously demonstrated that anaerobic E. coli protects aconitase from NO-mediated inactivation and that this protection is dependent upon active protein synthesis (16Gardner P.R. Costantino G. Salzman A.L. J. Biol. Chem. 1998; 273: 26528-26533Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). 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, comparelines 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 and3). 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. In the absence of NO exposure, anaerobic E. coliexpresses negligible anaerobic NO consumption activity (data not shown) (35Gardner A.M. Helmick R.A. Gardner P.R. J. Biol. Chem. 2002; 277: 8172-8177Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). 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.4 A). Anaerobic cells clearly induced an aerobic NO consumption activity attributable to flavoHb because this activity was not expressed by flavoHb-deficient AG102 (Fig. 4 B). Furthermore, the flavoHb-independent activity was not detectable in cells assayed in the presence of 200 μmO2. 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 12 = 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. 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 mmcyanide, respectively (36Fujiwara T. Fukumori Y. J. Bacteriol. 1996; 178: 1866-1871Crossref PubMed Google Scholar, 37Nakahara K. Tanimoto T. Hatano K. Usuda K. Shoun H. J. Biol. Chem. 1993; 268: 8350-8355Abstract Full Text PDF PubMed Google Scholar). The E. coli activity was relatively resistant to cyanide; 2 mm NaCN was required to inhibit the activity by ∼50% (TableI). By comparison, half-maximal inhibition of NOD activity was observed with ∼2.5 μmNaCN. 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 cytochromebc- or P450-type NOR in E. coli genome searches, provide strong evidence for a novel NOR activity in E. coli.Table ICyanide sensitivity of NO consumptionNO consumption activityAnaerobicAerobicnmol/min/108cellsControl34.4 (100)72.0 (100)+2.5 μm NaCNND46.8 (65)+25 μm NaCNND1.2 (2)+1 mm NaCN25.6 (74)ND+2 mm NaCN18.0 (52)ND+100 μmHQNO34.2 (100)NDCultures of E. coli K12 (ATCC 23716) were grown under an atmosphere of N2 to an A 550 of ∼0.4 in phosphate-buffered LB medium with 20 mm glucose and were exposed to 960 ppm NO in N2 for 30 min. Chloramphenicol (200 μg/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. Open table in a new tab Cultures of E. coli K12 (ATCC 23716) were grown under an atmosphere of N2 to an A 550 of ∼0.4 in phosphate-buffered LB medium with 20 mm glucose and were exposed to 960 ppm NO in N2 for 30 min. Chloramphenicol (200 μg/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. 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 (15Gardner P.R. Gardner A.M. Martin L.A. Salzman A.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10378-10383Crossref PubMed Scopus (497) Google Scholar), 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 andD, 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 (15Gardner P.R. Gardner A.M. Martin L.A. Salzman A.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10378-10383Crossref PubMed Scopus (497) Google Scholar). However, RB9060 contains a deletion encompassing theglyA-hmp-glnB region (30Bueno R. Pahel G. Magasanik B. J. Bacteriol. 1985; 164: 816-822Crossref PubMed Google Scholar), 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. E. coliflavoHb 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 (TableII). Although a lower NOD turnover number of 10 s−1 was recently reported by others (38Mills C.E. Sedelnikova S. Søballe B. Hughes M.N. Poole R.K. Biochem. J. 2001; 353: 207-213Crossref PubMed Scopus (59) Google Scholar), it is important to note that rates were measured at NO:O2 ratios that produce significant inhibition of NOD activity by NO (17Gardner A.M. Martin L.A. Gardner P.R. Dou Y. Olson J.S. J. Biol. Chem. 2000; 275: 12581-12589Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 18Gardner P.R. Gardner A.M. Martin L.A. Dou Y. Li T. Olson J.S. Zhu H. Riggs A.F. J. Biol. Chem. 2000; 275: 31581-31587Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). 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−1s−1) (Table II). In these reductive reactions, flavoHb generated NO from GSNO with a turnover number of 0.08 NO heme−1 s−1. flavoHb also catalyzed NO release from S-nitrosopenicillamine (1 mm), anotherS-nitrosothiol, at a rate of 0.05 NO heme−1s−1. 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 (39Doyle M.P. Pickering R.A. DeWeert T.M. Hoekstra J.W. Pater D. J. Biol. Chem. 1981; 256: 12393-12398Abstract Full Text PDF PubMed Google Scholar) 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).Table IIEnzymatic activities of flavoHbActivityTurnover rateRef.s−1NOD10–67017Gardner A.M. Martin L.A. Gardner P.R. Dou Y. Olson J.S. J. Biol. Chem. 2000; 275: 12581-12589Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar,18Gardner P.R. Gardner A.M. Martin L.A. Dou Y. Li T. Olson J.S. Zhu H. Riggs A.F. J. Biol. Chem. 2000; 275: 31581-31587Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 38Mills C.E. Sedelnikova S. Søballe B. Hughes M.N. Poole R.K. Biochem. J. 2001; 353: 207-213Crossref PubMed Scopus (59) Google ScholarNOR0.01–0.2417Gardner A.M. Martin L.A. Gardner P.R. Dou Y. Olson J.S. J. Biol. 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Turnover numbers are expressed relative to flavoHb heme. Open table in a new tab Activities were measured as described under "Materials and Methods." Turnover numbers are expressed relative to flavoHb heme. 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 (19Hausladen A. Gow A.J. Stamler J.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14100-14105Crossref PubMed Scopus (255) Google Scholar, 20Liu L. Zeng M. Hausladen A. Heitman J. Stamler J.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4672-4676Crossref PubMed Scopus (166) Google Scholar, 23Crawford M.J. Goldberg D.E. J. Biol. Chem. 1998; 273: 12543-12547Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 24Crawford M.J. Goldberg D.E. J. Biol. Chem. 1998; 273: 34028-34032Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 25Membrillo-Hernández J. Coopamah M.D. Anjum M.F. Stevanin T.M. Kelly A. Hughes M.N. Poole R.K. J. Biol. Chem. 1999; 274: 748-754Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). The maximal turnover rate for NO dioxygenation by isolatedE. coli flavoHb (670 s−1) is several orders of magnitude greater than that which we measured for NO reduction (∼0.02 s−1) (Table II) (17Gardner A.M. Martin L.A. Gardner P.R. Dou Y. Olson J.S. J. Biol. Chem. 2000; 275: 12581-12589Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 18Gardner P.R. Gardner A.M. Martin L.A. Dou Y. Li T. Olson J.S. Zhu H. Riggs A.F. J. Biol. Chem. 2000; 275: 31581-31587Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). Yet, it has never been determined whether the NOR activity of inducible flavoHb functions in anaerobic NO detoxification, as recently suggested (19Hausladen A. Gow A.J. Stamler J.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14100-14105Crossref PubMed Scopus (255) Google Scholar, 20Liu L. Zeng M. Hausladen A. Heitman J. Stamler J.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4672-4676Crossref PubMed Scopus (166) Google Scholar, 22Kim S.O. Orii Y. Lloyd D. Hughes M.N. Poole R.K. FEBS Lett. 1999; 445: 389-394Crossref PubMed Scopus (142) Google Scholar). 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 (19Hausladen A. Gow A.J. Stamler J.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14100-14105Crossref PubMed Scopus (255) Google Scholar, 20Liu L. Zeng M. Hausladen A. Heitman J. Stamler J.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4672-4676Crossref PubMed Scopus (166) Google Scholar, 23Crawford M.J. Goldberg D.E. J. Biol. Chem. 1998; 273: 12543-12547Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 26Iijima M. Shimizu H. Tanaka Y. Urushihara H. Cell Struct. Funct. 2000; 25: 47-55Crossref PubMed Scopus (29) Google Scholar), 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) (35Gardner A.M. Helmick R.A. Gardner P.R. J. Biol. Chem. 2002; 277: 8172-8177Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). 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 (40Frey A.D. Fiaux J. Szyperski T. Wuthrich K. Bailey J.E. Kallio P.T. Appl. Environ. Microbiol. 2001; 67: 680-687Crossref PubMed Scopus (30) Google Scholar). 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 (1Zumft W. Microbiol. Mol. Biol. Rev. 1997; 61: 533-616Crossref PubMed Scopus (2897) Google Scholar, 37Nakahara K. Tanimoto T. Hatano K. Usuda K. Shoun H. J. Biol. Chem. 1993; 268: 8350-8355Abstract Full Text PDF PubMed Google Scholar). It is likely that the newly identified NO-scavenging activity (35Gardner A.M. Helmick R.A. Gardner P.R. J. Biol. Chem. 2002; 277: 8172-8177Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar) 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. 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.

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