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. 2001 Jan 15;353(Pt 2):207–213. doi: 10.1042/0264-6021:3530207

Escherichia coli flavohaemoglobin (Hmp) with equistoichiometric FAD and haem contents has a low affinity for dioxygen in the absence or presence of nitric oxide.

C E Mills 1, S Sedelnikova 1, B Søballe 1, M N Hughes 1, R K Poole 1
PMCID: PMC1221560  PMID: 11139382

Abstract

A purification procedure for flavohaemoglobin Hmp (NO oxygenase) is described that gives high yields of protein with equistoichiometric haem and FAD contents. H(2)O(2) accumulated on NADH oxidation by the purified protein and in cell extracts with elevated Hmp contents. H(2)O(2) probably arose by dismutation from superoxide, which was also detectable during oxygen reduction; water was not a product. In the absence of agents that scavenge superoxide and peroxide, the mean K(m) for oxygen was 80 microM; the addition of 15 microM FAD decreased the K(m) for oxygen to 15 microM without a change in V(max) but catalysed cyanide-insensitive oxygen consumption, attributed to electron transfer from flavins to O(2). Purified Hmp consumed NO in the absence of added FAD (approx. 1 O(2) per NO), which is consistent with NO oxygenation. However, half-maximal rates of NO-stimulated O(2) consumption required approx. 47 microM O(2); NO removal was ineffective at physiologically relevant O(2) concentrations (below approx. 30 microM O(2)). On exhaustion of O(2), NO was removed by a cyanide-sensitive process attributed to NO reduction, with a turnover number approx. 1% of that for oxygenase activity. These results suggest that the ability of Hmp to detoxify NO might be compromised in hypoxic environments.

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Selected References

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  1. Andrews S. C., Shipley D., Keen J. N., Findlay J. B., Harrison P. M., Guest J. R. The haemoglobin-like protein (HMP) of Escherichia coli has ferrisiderophore reductase activity and its C-terminal domain shares homology with ferredoxin NADP+ reductases. FEBS Lett. 1992 May 18;302(3):247–252. doi: 10.1016/0014-5793(92)80452-m. [DOI] [PubMed] [Google Scholar]
  2. Anjum M. F., Ioannidis N., Poole R. K. Response of the NAD(P)H-oxidising flavohaemoglobin (Hmp) to prolonged oxidative stress and implications for its physiological role in Escherichia coli. FEMS Microbiol Lett. 1998 Sep 15;166(2):219–223. doi: 10.1111/j.1574-6968.1998.tb13893.x. [DOI] [PubMed] [Google Scholar]
  3. Cramm R., Siddiqui R. A., Friedrich B. Primary sequence and evidence for a physiological function of the flavohemoprotein of Alcaligenes eutrophus. J Biol Chem. 1994 Mar 11;269(10):7349–7354. [PubMed] [Google Scholar]
  4. Crawford M. J., Goldberg D. E. Role for the Salmonella flavohemoglobin in protection from nitric oxide. J Biol Chem. 1998 May 15;273(20):12543–12547. doi: 10.1074/jbc.273.20.12543. [DOI] [PubMed] [Google Scholar]
  5. Gardner A. M., Martin L. A., Gardner P. R., Dou Y., Olson J. S. Steady-state and transient kinetics of Escherichia coli nitric-oxide dioxygenase (flavohemoglobin). The B10 tyrosine hydroxyl is essential for dioxygen binding and catalysis. J Biol Chem. 2000 Apr 28;275(17):12581–12589. doi: 10.1074/jbc.275.17.12581. [DOI] [PubMed] [Google Scholar]
  6. Gardner P. R., Costantino G., Salzman A. L. Constitutive and adaptive detoxification of nitric oxide in Escherichia coli. Role of nitric-oxide dioxygenase in the protection of aconitase. J Biol Chem. 1998 Oct 9;273(41):26528–26533. doi: 10.1074/jbc.273.41.26528. [DOI] [PubMed] [Google Scholar]
  7. Gardner P. R., Gardner A. M., Martin L. A., Salzman A. L. Nitric oxide dioxygenase: an enzymic function for flavohemoglobin. Proc Natl Acad Sci U S A. 1998 Sep 1;95(18):10378–10383. doi: 10.1073/pnas.95.18.10378. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hausladen A., Gow A. J., Stamler J. S. Nitrosative stress: metabolic pathway involving the flavohemoglobin. Proc Natl Acad Sci U S A. 1998 Nov 24;95(24):14100–14105. doi: 10.1073/pnas.95.24.14100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Ingledew W. J., Poole R. K. The respiratory chains of Escherichia coli. Microbiol Rev. 1984 Sep;48(3):222–271. doi: 10.1128/mr.48.3.222-271.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Ioannidis N., Cooper C. E., Poole R. K. Spectroscopic studies on an oxygen-binding haemoglobin-like flavohaemoprotein from Escherichia coli. Biochem J. 1992 Dec 1;288(Pt 2):649–655. doi: 10.1042/bj2880649. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kim S. O., Orii Y., Lloyd D., Hughes M. N., Poole R. K. Anoxic function for the Escherichia coli flavohaemoglobin (Hmp): reversible binding of nitric oxide and reduction to nitrous oxide. FEBS Lett. 1999 Feb 26;445(2-3):389–394. doi: 10.1016/s0014-5793(99)00157-x. [DOI] [PubMed] [Google Scholar]
  12. Love C. A., Lilley P. E., Dixon N. E. Stable high-copy-number bacteriophage lambda promoter vectors for overproduction of proteins in Escherichia coli. Gene. 1996 Oct 17;176(1-2):49–53. doi: 10.1016/0378-1119(96)00208-9. [DOI] [PubMed] [Google Scholar]
  13. Markwell M. A., Haas S. M., Bieber L. L., Tolbert N. E. A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal Biochem. 1978 Jun 15;87(1):206–210. doi: 10.1016/0003-2697(78)90586-9. [DOI] [PubMed] [Google Scholar]
  14. Membrillo-Hernández J., Coopamah M. D., Anjum M. F., Stevanin T. M., Kelly A., Hughes M. N., Poole R. K. 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. J Biol Chem. 1999 Jan 8;274(2):748–754. doi: 10.1074/jbc.274.2.748. [DOI] [PubMed] [Google Scholar]
  15. Membrillo-Hernández J., Coopamah M. D., Channa A., Hughes M. N., Poole R. K. A novel mechanism for upregulation of the Escherichia coli K-12 hmp (flavohaemoglobin) gene by the 'NO releaser', S-nitrosoglutathione: nitrosation of homocysteine and modulation of MetR binding to the glyA-hmp intergenic region. Mol Microbiol. 1998 Aug;29(4):1101–1112. doi: 10.1046/j.1365-2958.1998.01000.x. [DOI] [PubMed] [Google Scholar]
  16. Membrillo-Hernández J., Ioannidis N., Poole R. K. The flavohaemoglobin (HMP) of Escherichia coli generates superoxide in vitro and causes oxidative stress in vivo. FEBS Lett. 1996 Mar 11;382(1-2):141–144. doi: 10.1016/0014-5793(96)00154-8. [DOI] [PubMed] [Google Scholar]
  17. Membrillo-Hernández J., Kim S. O., Cook G. M., Poole R. K. Paraquat regulation of hmp (flavohemoglobin) gene expression in Escherichia coli K-12 is SoxRS independent but modulated by sigma S. J Bacteriol. 1997 May;179(10):3164–3170. doi: 10.1128/jb.179.10.3164-3170.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Membrillo-Hernández J., Poole R. K. Bacterial flavohaemoglobins: a consensus sequence and identification of a discrete enterobacterial group and of further bacterial globins. FEMS Microbiol Lett. 1997 Oct 15;155(2):179–184. doi: 10.1016/s0378-1097(97)00384-4. [DOI] [PubMed] [Google Scholar]
  19. Poole R. K., Anjum M. F., Membrillo-Hernández J., Kim S. O., Hughes M. N., Stewart V. Nitric oxide, nitrite, and Fnr regulation of hmp (flavohemoglobin) gene expression in Escherichia coli K-12. J Bacteriol. 1996 Sep;178(18):5487–5492. doi: 10.1128/jb.178.18.5487-5492.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Poole R. K., Baines B. S., Appleby C. A. Haemoprotein b-590 (Escherichia coli), a reducible catalase and peroxidase: evidence for its close relationship to hydroperoxidase I and a 'cytochrome a1b' preparation. J Gen Microbiol. 1986 Jun;132(6):1525–1539. doi: 10.1099/00221287-132-6-1525. [DOI] [PubMed] [Google Scholar]
  21. Poole R. K., Cook G. M. Redundancy of aerobic respiratory chains in bacteria? Routes, reasons and regulation. Adv Microb Physiol. 2000;43:165–224. doi: 10.1016/s0065-2911(00)43005-5. [DOI] [PubMed] [Google Scholar]
  22. Poole R. K., Hughes M. N. New functions for the ancient globin family: bacterial responses to nitric oxide and nitrosative stress. Mol Microbiol. 2000 May;36(4):775–783. doi: 10.1046/j.1365-2958.2000.01889.x. [DOI] [PubMed] [Google Scholar]
  23. Poole R. K., Ioannidis N., Orii Y. Reactions of the Escherichia coli flavohaemoglobin (Hmp) with NADH and near-micromolar oxygen: oxygen affinity of NADH oxidase activity. Microbiology. 1996 May;142(Pt 5):1141–1148. doi: 10.1099/13500872-142-5-1141. [DOI] [PubMed] [Google Scholar]
  24. Poole R. K., Ioannidis N., Orii Y. Reactions of the Escherichia coli flavohaemoglobin (Hmp) with oxygen and reduced nicotinamide adenine dinucleotide: evidence for oxygen switching of flavin oxidoreduction and a mechanism for oxygen sensing. Proc Biol Sci. 1994 Mar 22;255(1344):251–258. doi: 10.1098/rspb.1994.0036. [DOI] [PubMed] [Google Scholar]
  25. Poole R. K., Rogers N. J., D'mello R. A., Hughes M. N., Orii Y. Escherichia coli flavohaemoglobin (Hmp) reduces cytochrome c and Fe(III)-hydroxamate K by electron transfer from NADH via FAD: sensitivity of oxidoreductase activity to haem-bound dioxygen. Microbiology. 1997 May;143(Pt 5):1557–1565. doi: 10.1099/00221287-143-5-1557. [DOI] [PubMed] [Google Scholar]
  26. Savage D. C. Microbial ecology of the gastrointestinal tract. Annu Rev Microbiol. 1977;31:107–133. doi: 10.1146/annurev.mi.31.100177.000543. [DOI] [PubMed] [Google Scholar]
  27. Stevanin T. M., Ioannidis N., Mills C. E., Kim S. O., Hughes M. N., Poole R. K. Flavohemoglobin Hmp affords inducible protection for Escherichia coli respiration, catalyzed by cytochromes bo' or bd, from nitric oxide. J Biol Chem. 2000 Nov 17;275(46):35868–35875. doi: 10.1074/jbc.M002471200. [DOI] [PubMed] [Google Scholar]
  28. Søballe B., Poole R. K. Ubiquinone limits oxidative stress in Escherichia coli. Microbiology. 2000 Apr;146(Pt 4):787–796. doi: 10.1099/00221287-146-4-787. [DOI] [PubMed] [Google Scholar]
  29. Vasudevan S. G., Armarego W. L., Shaw D. C., Lilley P. E., Dixon N. E., Poole R. K. Isolation and nucleotide sequence of the hmp gene that encodes a haemoglobin-like protein in Escherichia coli K-12. Mol Gen Genet. 1991 Apr;226(1-2):49–58. doi: 10.1007/BF00273586. [DOI] [PubMed] [Google Scholar]
  30. Wink D. A., Cook J. A., Kim S. Y., Vodovotz Y., Pacelli R., Krishna M. C., Russo A., Mitchell J. B., Jourd'heuil D., Miles A. M. Superoxide modulates the oxidation and nitrosation of thiols by nitric oxide-derived reactive intermediates. Chemical aspects involved in the balance between oxidative and nitrosative stress. J Biol Chem. 1997 Apr 25;272(17):11147–11151. doi: 10.1074/jbc.272.17.11147. [DOI] [PubMed] [Google Scholar]
  31. Yu H., Sato E. F., Nagata K., Nishikawa M., Kashiba M., Arakawa T., Kobayashi K., Tamura T., Inoue M. Oxygen-dependent regulation of the respiration and growth of Escherichia coli by nitric oxide. FEBS Lett. 1997 Jun 9;409(2):161–165. doi: 10.1016/s0014-5793(97)00494-8. [DOI] [PubMed] [Google Scholar]

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