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. 1996 Jun 15;316(Pt 3):887–892. doi: 10.1042/bj3160887

Reconstitution of the [4Fe-4S] cluster in FNR and demonstration of the aerobic-anaerobic transcription switch in vitro.

J Green 1, B Bennett 1, P Jordan 1, E T Ralph 1, A J Thomson 1, J R Guest 1
PMCID: PMC1217433  PMID: 8670167

Abstract

The FNR protein of Escherichia coli is a redox-responsive transcription regulator that activates and represses a family of genes required for anaerobic and aerobic metabolism. Reconstitution of wild-type FNR by anaerobic treatment with ferrous ions, cysteine and the NifS protein of Azotobacter vinelandii leads to the incorporation of two [4Fe-4S]2+ clusters per FNR dimer. The UV-visible spectrum of reconstituted FNR has a broad absorbance at 420 nm. The clusters are EPR silent under anaerobic conditions but are degraded to [3Fe-4S]+ by limited oxidation with air, and completely lost on prolonged air exposure. The association of FNR with the iron-sulphur clusters is confirmed by CD spectroscopy. Incorporation of the [4Fe-4S]2+ clusters increases site-specific DNA binding about 7-fold compared with apo-FNR. Anaerobic transcription activation and repression in vitro likewise depends on the presence of the iron-sulphur cluster, and its inactivation under aerobic conditions provides a demonstration in vitro of the FNR-mediated aerobic-anaerobic transcriptional switch.

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Selected References

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  1. Beinert H. Semi-micro methods for analysis of labile sulfide and of labile sulfide plus sulfane sulfur in unusually stable iron-sulfur proteins. Anal Biochem. 1983 Jun;131(2):373–378. doi: 10.1016/0003-2697(83)90186-0. [DOI] [PubMed] [Google Scholar]
  2. Bennett B., Gruer M. J., Guest J. R., Thomson A. J. Spectroscopic characterisation of an aconitase (AcnA) of Escherichia coli. Eur J Biochem. 1995 Oct 1;233(1):317–326. doi: 10.1111/j.1432-1033.1995.317_1.x. [DOI] [PubMed] [Google Scholar]
  3. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  4. Busch J. L., Breton J. L., Bartlett B. M., James R., Hatchikian E. C., Thomson A. J. Expression in Escherichia coli and characterization of a reconstituted recombinant 7Fe ferredoxin from Desulfovibrio africanus. Biochem J. 1996 Feb 15;314(Pt 1):63–71. doi: 10.1042/bj3140063. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cammack R. "Super-reduction" of chromatium high-potential iron-sulphur protein in the presence of dimethyl sulphoxide. Biochem Biophys Res Commun. 1973 Sep 18;54(2):548–554. doi: 10.1016/0006-291x(73)91457-5. [DOI] [PubMed] [Google Scholar]
  6. Flint D. H., Emptage M. H., Guest J. R. Fumarase a from Escherichia coli: purification and characterization as an iron-sulfur cluster containing enzyme. Biochemistry. 1992 Oct 27;31(42):10331–10337. doi: 10.1021/bi00157a022. [DOI] [PubMed] [Google Scholar]
  7. Fried M. G., Crothers D. M. Equilibrium studies of the cyclic AMP receptor protein-DNA interaction. J Mol Biol. 1984 Jan 25;172(3):241–262. doi: 10.1016/s0022-2836(84)80025-x. [DOI] [PubMed] [Google Scholar]
  8. Georgiadis M. M., Komiya H., Chakrabarti P., Woo D., Kornuc J. J., Rees D. C. Crystallographic structure of the nitrogenase iron protein from Azotobacter vinelandii. Science. 1992 Sep 18;257(5077):1653–1659. doi: 10.1126/science.1529353. [DOI] [PubMed] [Google Scholar]
  9. Green J., Guest J. R. A role for iron in transcriptional activation by FNR. FEBS Lett. 1993 Aug 23;329(1-2):55–58. doi: 10.1016/0014-5793(93)80192-w. [DOI] [PubMed] [Google Scholar]
  10. Green J., Guest J. R. Activation of FNR-dependent transcription by iron: an in vitro switch for FNR. FEMS Microbiol Lett. 1993 Oct 15;113(2):219–222. doi: 10.1111/j.1574-6968.1993.tb06517.x. [DOI] [PubMed] [Google Scholar]
  11. Green J., Irvine A. S., Meng W., Guest J. R. FNR-DNA interactions at natural and semi-synthetic promoters. Mol Microbiol. 1996 Jan;19(1):125–137. doi: 10.1046/j.1365-2958.1996.353884.x. [DOI] [PubMed] [Google Scholar]
  12. Green J., Trageser M., Six S., Unden G., Guest J. R. Characterization of the FNR protein of Escherichia coli, an iron-binding transcriptional regulator. Proc Biol Sci. 1991 May 22;244(1310):137–144. doi: 10.1098/rspb.1991.0062. [DOI] [PubMed] [Google Scholar]
  13. Guest J. R. The Leeuwenhoek Lecture, 1995. Adaptation to life without oxygen. Philos Trans R Soc Lond B Biol Sci. 1995 Nov 29;350(1332):189–202. doi: 10.1098/rstb.1995.0152. [DOI] [PubMed] [Google Scholar]
  14. Hudson J. M., Crowe L. G., Fried M. G. A new DNA binding mode for CAP. J Biol Chem. 1990 Feb 25;265(6):3219–3225. [PubMed] [Google Scholar]
  15. Kennedy M. C., Kent T. A., Emptage M., Merkle H., Beinert H., Münck E. Evidence for the formation of a linear [3Fe-4S] cluster in partially unfolded aconitase. J Biol Chem. 1984 Dec 10;259(23):14463–14471. [PubMed] [Google Scholar]
  16. Khoroshilova N., Beinert H., Kiley P. J. Association of a polynuclear iron-sulfur center with a mutant FNR protein enhances DNA binding. Proc Natl Acad Sci U S A. 1995 Mar 28;92(7):2499–2503. doi: 10.1073/pnas.92.7.2499. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kiley P. J., Reznikoff W. S. Fnr mutants that activate gene expression in the presence of oxygen. J Bacteriol. 1991 Jan;173(1):16–22. doi: 10.1128/jb.173.1.16-22.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lazazzera B. A., Bates D. M., Kiley P. J. The activity of the Escherichia coli transcription factor FNR is regulated by a change in oligomeric state. Genes Dev. 1993 Oct;7(10):1993–2005. doi: 10.1101/gad.7.10.1993. [DOI] [PubMed] [Google Scholar]
  19. Melville S. B., Gunsalus R. P. Mutations in fnr that alter anaerobic regulation of electron transport-associated genes in Escherichia coli. J Biol Chem. 1990 Nov 5;265(31):18733–18736. [PubMed] [Google Scholar]
  20. Oñate Y. A., Vollmer S. J., Switzer R. L., Johnson M. K. Spectroscopic characterization of the iron-sulfur cluster in Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase. J Biol Chem. 1989 Nov 5;264(31):18386–18391. [PubMed] [Google Scholar]
  21. Sharrocks A. D., Green J., Guest J. R. FNR activates and represses transcription in vitro. Proc Biol Sci. 1991 Sep 23;245(1314):219–226. doi: 10.1098/rspb.1991.0113. [DOI] [PubMed] [Google Scholar]
  22. Spiro S., Guest J. R. FNR and its role in oxygen-regulated gene expression in Escherichia coli. FEMS Microbiol Rev. 1990 Aug;6(4):399–428. doi: 10.1111/j.1574-6968.1990.tb04109.x. [DOI] [PubMed] [Google Scholar]
  23. Stephens P. J., Thomson A. J., Dunn J. B., Keiderling T. A., Rawlings J., Rao K. K., Hall D. O. Circular dichroism and magnetic circular dichroism of iron-sulfur proteins. Biochemistry. 1978 Oct 31;17(22):4770–4778. doi: 10.1021/bi00615a026. [DOI] [PubMed] [Google Scholar]
  24. Trageser M., Unden G. Role of cysteine residues and of metal ions in the regulatory functioning of FNR, the transcriptional regulator of anaerobic respiration in Escherichia coli. Mol Microbiol. 1989 May;3(5):593–599. doi: 10.1111/j.1365-2958.1989.tb00206.x. [DOI] [PubMed] [Google Scholar]
  25. Unden G., Becker S., Bongaerts J., Holighaus G., Schirawski J., Six S. O2-sensing and O2-dependent gene regulation in facultatively anaerobic bacteria. Arch Microbiol. 1995 Aug;164(2):81–90. [PubMed] [Google Scholar]
  26. Woodland M. P., Dalton H. Purification and characterization of component A of the methane monooxygenase from Methylococcus capsulatus (Bath). J Biol Chem. 1984 Jan 10;259(1):53–59. [PubMed] [Google Scholar]
  27. Zheng L., White R. H., Cash V. L., Jack R. F., Dean D. R. Cysteine desulfurase activity indicates a role for NIFS in metallocluster biosynthesis. Proc Natl Acad Sci U S A. 1993 Apr 1;90(7):2754–2758. doi: 10.1073/pnas.90.7.2754. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Ziegelhoffer E. C., Kiley P. J. In vitro analysis of a constitutively active mutant form of the Escherichia coli global transcription factor FNR. J Mol Biol. 1995 Jan 27;245(4):351–361. doi: 10.1006/jmbi.1994.0029. [DOI] [PubMed] [Google Scholar]
  29. Zumft W. G., Palmer G., Mortenson L. E. Electron paramagnetic resonance studies on nitrogenase. II. Interaction of adenosine 5'-triphosphate with azoferredoxin. Biochim Biophys Acta. 1973 Feb 22;292(2):413–421. doi: 10.1016/0005-2728(73)90047-9. [DOI] [PubMed] [Google Scholar]

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