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The Flavohemoglobin of Escherichia coli Confers Resistance to a Nitrosating Agent, a “Nitric Oxide Releaser,” and Paraquat and Is Essential for Transcriptional Responses to Oxidative Stress*

  • Jorge Membrillo-Hernández
    Footnotes
    Affiliations
    The Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, The University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, United Kingdom, the
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  • Malini D. Coopamah
    Footnotes
    Affiliations
    The Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, The University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, United Kingdom, the
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  • Muna F. Anjum
    Affiliations
    The Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, The University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, United Kingdom, the
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  • Tania M. Stevanin
    Affiliations
    The Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, The University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, United Kingdom, the
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  • Andrew Kelly
    Affiliations
    Life Sciences Division, King's College London, Campden Hill Road, London W8 7AH, United Kingdom, and the
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  • Martin N. Hughes
    Affiliations
    Chemistry Department, King's College London, Strand, London WC2R 2LS, United Kingdom
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  • Robert K. Poole
    Correspondence
    To whom correspondence should be addressed: The Krebs Institute for Biomolecular Research, Dept. of Molecular Biology & Biotechnology, The University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, UK. Tel.: 44-114-222-4447; Fax: 44-114-272-8697;
    Affiliations
    The Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, The University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, United Kingdom, the
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  • Author Footnotes
    * This work was supported by Biotechnology and Biological Sciences Research Council (BBSRC) Grant P05184 and by a BBSRC Studentship (to T. M. S.).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.
    § Present address: Dept. of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Ave., Boston, MA 02115.
    ¶ Recipient of Biochemical Society Krebs Memorial Scholarship and an Overseas Research Student Award.
Open AccessPublished:January 08, 1999DOI:https://doi.org/10.1074/jbc.274.2.748
      Escherichia coli possesses a flavohemoglobin (Hmp), product of hmp, the first microbial globin gene to be sequenced and characterized at the molecular level. Although related proteins occur in numerous prokaryotes and eukaryotic microorganisms, the function(s) of these proteins have been elusive. Here we report construction of a defined hmp mutation and its use to probe Hmp function. As anticipated from up-regulation ofhmp expression by nitric oxide (NO),S-nitrosoglutathione (GSNO) or sodium nitroprusside (SNP), the hmp mutant is hypersensitive to these agents. Thehmp promoter is more sensitive to SNP andS-nitroso-N-penicillamine (SNAP) than is thesoxS promoter, consistent with the role of Hmp in protection from reactive nitrogen species. Additional functions for Hmp are indicated by (a) parallel sensitivity of thehmp mutant to the redox-cycling agent, paraquat, (b) inability of the mutant to up-regulate fully thesoxS and sodA promoters in response to oxidative stress caused by paraquat, GSNO and SNP, and (c) failure of the mutant to accumulate reduced paraquat radical after anoxic growth. We conclude that Hmp plays a role in protection from nitrosating agents and NO-related species and oxidative stress. This protective role probably involves direct detoxification of those species and sensing of NO-related and oxidative stress.
      The best known members of the ancient globin superfamily are the hemoglobins of vertebrate blood and intramuscular myoglobin (
      • Riggs A.F.
      ), which are primarily responsible for oxygen delivery and storage in animals, although the circulating hemoglobin has also been implicated in transport of NO
      The abbreviations used are: NO, nitric oxide; SNP, sodium nitroprusside; GSNO, S-nitrosoglutathione; SNAP, S-nitroso-N-penicillamine; MOPS, 4-morpholinepropanesulfonic acid; kb, kilobase pair(s).
      1The abbreviations used are: NO, nitric oxide; SNP, sodium nitroprusside; GSNO, S-nitrosoglutathione; SNAP, S-nitroso-N-penicillamine; MOPS, 4-morpholinepropanesulfonic acid; kb, kilobase pair(s).
      (
      • Gow A.J.
      • Stamler J.S.
      ). It is now clear that homologous hemoglobins also occur in many bacteria and yeast as well as in invertebrates and higher plants (
      • Hardison R.
      ). Microbial hemoglobins are divisible into two groups: dimeric hemoproteins comprising two polypeptides each having one heme, as inVitreoscilla VGB (
      • Wakabayashi S.
      • Matsubara H.
      • Webster D.A.
      ), and monomeric, chimeric flavohemoproteins composed of a single polypeptide having both a single heme and FAD. The sequence of the hmp gene (
      • Vasudevan S.G.
      • Armarego W.L.F.
      • Shaw D.C.
      • Lilley P.E.
      • Dixon N.E.
      • Poole R.K.
      ), encoding the prototype of the latter class, Escherichia coli Hmp, has revealed an N-terminal domain homologous to vertebrate, plant, andVitreoscilla globins, whereas a C-terminal domain has FAD- and NAD(P)H-binding sites as in proteins in the ferredoxin-NADP reductase family (
      • Andrews S.C.
      • Shipley D.
      • Keen J.N.
      • Findlay J.B.C.
      • Harrison P.M.
      • Guest J.R.
      ). Closely related flavohemoglobins occur in the yeasts Saccharomyces cerevisiae (
      • Zhu H.
      • Riggs A.F.
      ) and Candida norvegensis (
      • Iwaasa H.
      • Takagi T.
      • Shikama K.
      ) and in the bacteria Alcaligenes eutrophus (
      • Cramm R.
      • Siddiqui R.A.
      • Friedrich B.
      ), Erwinia chrysanthemi (
      • Favey S.
      • Labesse G.
      • Vouille V.
      • Boccara M.
      ), andBacillus subtilis (
      • LaCelle M.
      • Kumano M.
      • Kurita K.
      • Yamane K.
      • Zuber P.
      • Nakano M.M.
      ). On the basis of polymerase chain reaction experiments (
      • Membrillo-Hernández J.
      • Poole R.K.
      ) and genome sequencing projects,e.g. that on Mycobacterium tuberculosis (
      • Cole S.T.
      • et al.
      ), related hemoglobins are predicted to be also present in many other bacteria.
      The functions of microbial globins have been elusive. Based on up-regulation of the Vitreoscilla globin at low oxygen tensions (
      • Dikshit K.L.
      • Spaulding D.
      • Braun A.
      • Webster D.A.
      ) and the ability of this protein to restore aerobic respiration when expressed in oxidase-deficient E. colimutants (
      • Dikshit R.P.
      • Dikshit K.L.
      • Liu Y.X.
      • Webster D.A.
      ), VGB has been implicated in oxygen storage, delivery, or reduction (
      • Khosla C.
      • Bailey J.E.
      ). The E. coli Hmp protein also consumes oxygen (
      • Poole R.K.
      • Ioannidis N.
      • Orii Y.
      ) and reduces various acceptors, including cytochrome c(
      • Poole R.K.
      • Rogers N.J.
      • D'mello R.A.M.
      • Hughes M.N.
      • Orii Y.
      ), Fe(III) (
      • Andrews S.C.
      • Shipley D.
      • Keen J.N.
      • Findlay J.B.C.
      • Harrison P.M.
      • Guest J.R.
      ,
      • Eschenbrenner M.
      • Coves J.
      • Fontecave M.
      ,
      • Membrillo-Hernández J.
      • Ioannidis N.
      • Poole R.K.
      ), and the Azotobacter regulatory flavoprotein NifL (
      • Macheroux P.
      • Hill S.
      • Austin S.
      • Eydmann T.
      • Jones T.
      • Kim S.O.
      • Poole R.K.
      • Dixon R.
      ). This dual ability might allow Hmp to act as an oxygen sensor (
      • Poole R.K.
      • Ioannidis N.
      • Orii Y.
      ,
      • Poole R.K.
      ).
      The first evidence that Hmp might function in responses to NO came from the discovery that its expression is markedly up-regulated by NO, both aerobically and anaerobically (
      • Poole R.K.
      • Anjum M.F.
      • Membrillo-Hernández J.
      • Kim S.O.
      • Hughes M.N.
      • Stewart V.
      ). Bacillus subtilis hmp B is also induced by nitrite (
      • LaCelle M.
      • Kumano M.
      • Kurita K.
      • Yamane K.
      • Zuber P.
      • Nakano M.M.
      ). Furthermore, inA. eutrophus, mutation of the hmp homologue,fhp, results in failure to detect nitrous oxide as an intermediate during denitrification (
      • Cramm R.
      • Siddiqui R.A.
      • Friedrich B.
      ). Recently the Salmonella typhimurium flavohemoglobin has been shown to confer resistance to acidified nitrite (and thus presumably NO) andS-nitrosothiols (
      • Crawford M.J.
      • Goldberg D.E.
      ), and E. coli Hmp has been shown to have NO dioxygenase activity (
      • Gardner P.R.
      • Gardner A.M.
      • Martin L.A.
      • Salzman A.L.
      ). These findings implicate bacterial flavohemoglobins in detoxification or utilization of NO.
      However, other evidence suggests that microbial flavohemoglobins are involved in responses to oxidative stress. Paraquat (1,1′-dimethyl-4–4′-bipyridinium dichloride; methyl viologen) is a strong inducer of the hmp gene, independently of the SoxRS regulatory system (
      • Membrillo-Hernández J.
      • Kim S.O.
      • Cook G.M.
      • Poole R.K.
      ), and Hmp itself generates superoxide anion, detectable using a superoxide-sensitive Φ(sodA-lacZ) fusion or with the purified protein (
      • Membrillo-Hernández J.
      • Ioannidis N.
      • Poole R.K.
      ). The yeast flavohemoglobin encoded by the YHB1 gene is also induced by agents that promote oxidative stress and antimycin A (
      • Zhao X.-J.
      • Raitt D.
      • Burke P.V.
      • Clewell A.S.
      • Kwast K.E.
      • Poyton R.O.
      ), but a subsequent re-examination has produced conflicting views (
      • Buisson N.
      • Labbe-Bois R.
      ).
      To resolve whether E. coli Hmp is important in responses to oxidative stress or NO, or both, we have constructed the first defined null allele of hmp and used this mutant to test responses to paraquat, sodium nitroprusside (SNP, a nitrosating agent) andS-nitrosoglutathione (GSNO) andS-nitroso-N-penicillamine (SNAP), the last two being widely used as NO-releasing agents. In addition, since the SoxRS system has been shown to respond to both oxidative stress and NO (
      • Nunoshiba T.
      • de Rojas-Walker T.
      • Tannenbaum S.R.
      • Demple B.
      ,
      • Nunoshiba T.
      • DeRojas T.
      • Wishnok J.S.
      • Tannenbaum S.R.
      • Demple B.
      ), we have compared the response of the hmp andsoxS promoters to challenge with “NO-releasing” agents. These data suggest that Hmp is pre-eminently involved in responses to NO and related reactive nitrogen species.

      DISCUSSION

      Several possible functions have been proposed for bacterial hemoglobins, including oxygen storage and delivery (
      • Wakabayashi S.
      • Matsubara H.
      • Webster D.A.
      ), as terminal oxidase (
      • Dikshit R.P.
      • Dikshit K.L.
      • Liu Y.X.
      • Webster D.A.
      ), in denitrification (
      • Cramm R.
      • Siddiqui R.A.
      • Friedrich B.
      ) and as oxygen sensor (
      • Poole R.K.
      • Ioannidis N.
      • Orii Y.
      ). While this work was being reviewed, Gardner et al. (
      • Gardner P.R.
      • Gardner A.M.
      • Martin L.A.
      • Salzman A.L.
      ) reported that Hmp has NO dioxygenase activity and that a mutant carrying an undefined deletion that extends into hmp is more sensitive to growth inhibition by NO and lacks the NO-consuming activity of the parent strain. In this paper we directly addressed the function ofE. coli Hmp by constructing a genetically marked null allele of hmp and characterizing the hmp mutant strain.
      No microbial globin described to date appears to be essential for either aerobic or anaerobic growth under normal laboratory conditions. For example, destruction of the S. cerevisiae globin by ethyl hydrogen peroxide (
      • Oshino R.
      • Oshino N.
      • Chance B.
      • Hagihara B.
      ) or mutation (
      • Zhao X.-J.
      • Raitt D.
      • Burke P.V.
      • Clewell A.S.
      • Kwast K.E.
      • Poyton R.O.
      ,
      • Crawford M.J.
      • Sherman D.R.
      • Goldberg D.E.
      ) did not alter respiration rates, cell viability, or growth under a variety of oxygen conditions and with various carbon sources. Mutation of hmpXin the plant pathogenic bacterium Erwinia chrysanthemi does not affect growth in either aerobic or microaerobic conditions, buthmpX mutants are compromised in their pathogenic effects (
      • Favey S.
      • Labesse G.
      • Vouille V.
      • Boccara M.
      ). Likewise, we show here that loss of Hmp is not detrimental to respiration or growth under common laboratory conditions. The only growth defect found thus far for the hmp mutant is in the stationary phase of growth where it attained a slightly lower population density; it may be significant that hmp is normally induced in stationary phase (
      • Membrillo-Hernández J.
      • Cook G.M.
      • Poole R.K.
      ). The possible roles for Hmp in stationary phase survival and the possibility of more subtle effects of loss of Hmp on growth under certain conditions remain to be studied.
      An important outcome of this work is that the hmp mutant is more sensitive than its isogenic parent to SNP and GSNO. Although the latter is widely used as an NO-releasing agent, both compounds may act similarly as nitrosating agents (
      • Membrillo-Hernández J.
      • Coopamah M.D.
      • Channa A.
      • Hughes M.N.
      • Poole R.K.
      ). Responses to SNP and GSNO are of special interest, since we have recently elucidated a novel mechanism for hmp up-regulation via nitrosation of homocysteine thus modulating binding of MetR to the glyA-hmp intergenic region (
      • Membrillo-Hernández J.
      • Coopamah M.D.
      • Channa A.
      • Hughes M.N.
      • Poole R.K.
      ). Other mechanisms of toxicity of SNP and GSNO are poorly understood, but such nitrosating agents are expected to be reactive with thiols and may interact with the Fe-S cluster of SoxR preventing full induction of soxS, sodA, and other stress-responsive genes.
      The finding that Hmp is involved in surviving the oxidative stress caused by paraquat or “NO releasers” is consistent with the results of previous studies with the hemoglobins of S. cerevisiaeand E. chrysanthemi (
      • Favey S.
      • Labesse G.
      • Vouille V.
      • Boccara M.
      ,
      • Zhao X.-J.
      • Raitt D.
      • Burke P.V.
      • Clewell A.S.
      • Kwast K.E.
      • Poyton R.O.
      ). Mutations in the S. cerevisiae YHB gene conferred increased sensitivity to oxidative stress from the thiol oxidants diamide and diethylmaleate, but paraquat had only a minor effect on the YHB mutant (
      • Zhao X.-J.
      • Raitt D.
      • Burke P.V.
      • Clewell A.S.
      • Kwast K.E.
      • Poyton R.O.
      ). In E. chrysanthemi, mutations in the gene hmpX conferred loss of plant pathogenicity (
      • Favey S.
      • Labesse G.
      • Vouille V.
      • Boccara M.
      ); it was speculated that this was due to increased sensitivity to oxygen radicals, but the recent discovery (
      • Delledone M.
      • Xia Y.
      • Dixon R.A.
      • Lamb C.
      ) that plants utilize NO to resist pathogenic bacteria suggests thathmpX might also be involved in NO responses inErwinia.
      Following reaction of paraquat with oxygen to generate superoxide, the oxidized divalent paraquat cation formed can be re-reduced leading to redox cycling and sustained superoxide generation. Paraquat reductases (diaphorases) identified in E. coli are ferredoxin:NADP+ oxidoreductase, thioredoxin reductase, and NADPH:sulfite reductase (
      • Bianchi V.
      • Haggård-Ljungquist E.
      • Pontis E.
      • Reichard P.
      ,
      • Liochev S.I.
      • Hausladen A.
      • Beyer W.F.
      • Fridovich I.
      ,
      • Gaudu P.
      • Fontecave M.
      ). Although hmp is up-regulated by paraquat, purified Hmp is not itself an effective reductant of paraquat with NADH or NADPH as substrate,
      S. O. Kim and R. K. Poole, unpublished data.
      consistent with the much higher midpoint potential of the flavin (around −150 mV) for the 2-electron reduction from FAD to FADH2 (
      • Cooper C.E.
      • Ioannidis N.
      • D'mello R.
      • Poole R.K.
      ) than of paraquat (E 0′ −446 mV) (
      • Dawson R.M.C.
      • Elliott D.C.
      • Elliott W.H.
      • Jones K.M.
      ). It is therefore unlikely that the accumulation of reduced paraquat in anoxic cell suspensions (Fig. 6) is due to direct reduction by Hmp. More likely, the presence of Hmp is required for full induction of components of the oxidative stress response (Fig. 3). One candidate for the paraquat-reducing enzyme is NADPH:ferredoxin oxidoreductase (
      • Liochev S.I.
      • Hausladen A.
      • Beyer W.F.
      • Fridovich I.
      ), a member of the soxRS regulon.
      A paradoxical aspect of the induction of hmp by paraquat is that Hmp itself generates free superoxide (
      • Membrillo-Hernández J.
      • Ioannidis N.
      • Poole R.K.
      ). This, and the present finding that an hmp mutant fails to elicit full responses to the presence of paraquat (Fig. 3), may be reconciled by considering Hmp as an amplifier of oxidative stress. In this model, paraquat (possibly via superoxide anion) induces synthesis of Hmp, which generates further superoxide, resulting in the activation of SoxR and the cascade of regulatory processes that result in oxidative stress responses. Such a mechanism may explain the increased sensitivity of the hmpmutant to paraquat (Fig. 2). The extreme paraquat sensitivity of thefpr mutant confirms the findings of Bianchi et al. (
      • Bianchi V.
      • Haggård-Ljungquist E.
      • Pontis E.
      • Reichard P.
      ), and we report for the first time the additional sensitivity of the fpr mutant to GSNO and SNP; the mechanism for neither effect is known.
      Interestingly, hmp up-regulation appears to respond to lower concentrations of SNP and SNAP than does transcription ofsoxS. This lends support to the view that Hmp may be an early component of the regulatory cascade. Several possible mechanisms can be envisaged. First, superoxide generation by Hmp might directly facilitate conversion of SoxR to the active form, perhaps by reaction with the FeS cluster (
      • Hidalgo E.
      • Ding H.G.
      • Demple B.
      ). NAD(P)H:flavin oxidoreductase activity (Fre) has also been demonstrated to induce soxRS-regulated genes by superoxide generation (
      • Gaudu P.
      • Touati D.
      • Nivière V.
      • Fontecave M.
      ). Second, Hmp is an NADH (
      • Ioannidis N.
      • Cooper C.E.
      • Poole R.K.
      ) and NADPH oxidase (
      • Anjum M.F.
      • Ioannidis N.
      • Poole R.K.
      ), and its activity will contribute to reducing the anabolic reduction charge ([NADPH]/[NADPH] + [NADP+]) (
      • Gardner P.R.
      • Fridovich I.
      ) that has been regarded as one of the possible signals for SoxR. Such regulatory influences of Hmp on genes involved in stress response would be reinforced by the ability of Hmp to directly convert NO to the relatively innocuous NO3 ion by reaction of NO with oxy-Hmp, recently demonstrated in Ref.
      • Gardner P.R.
      • Gardner A.M.
      • Martin L.A.
      • Salzman A.L.
      , but such a mechanism cannot explain the anaerobic roles of Hmp in protecting cells from NO reported recently (
      • Crawford M.J.
      • Goldberg D.E.
      ,
      • Gardner P.R.
      • Gardner A.M.
      • Martin L.A.
      • Salzman A.L.
      ). Such mechanisms are currently under investigation in this laboratory.

      Acknowledgments

      We thank Drs. Vera Bianchi, Bruce Demple, George Stauffer, and Daniéle Touati for providing the strains and plasmids used in this work.

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