Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1995 Aug;177(16):4587–4592. doi: 10.1128/jb.177.16.4587-4592.1995

Aerobic inactivation of fumarate reductase from Escherichia coli by mutation of the [3Fe-4S]-quinone binding domain.

G Cecchini 1, H Sices 1, I Schröder 1, R P Gunsalus 1
PMCID: PMC177221  PMID: 7642483

Abstract

Fumarate reductase from Escherichia coli functions both as an anaerobic fumarate reductase and as an aerobic succinate dehydrogenase. A site-directed mutation of E. coli fumarate reductase in which FrdB Pro-159 was replaced with a glutamine or histidine residue was constructed and overexpressed in a strain of E. coli lacking a functional copy of the fumarate reductase or succinate dehydrogenase complex. The consequences of these mutations on bacterial growth, assembly of the enzyme complex, and enzymatic activity were investigated. Both mutations were found to have no effect on anaerobic bacterial growth or on the ability of the enzyme to reduce fumarate compared with the wild-type enzyme. The FrdB Pro-159-to-histidine substitution was normal in its ability to oxidize succinate. In contrast, however, the FrdB Pro-159-to-Gln substitution was found to inhibit aerobic growth of E. coli under conditions requiring a functional succinate dehydrogenase, and furthermore, the aerobic activity of the enzyme was severely inhibited upon incubation in the presence of its substrate, succinate. This inactivation could be prevented by incubating the mutant enzyme complex in an anaerobic environment, separating the catalytic subunits of the fumarate reductase complex from their membrane anchors, or blocking the transfer of electrons from the enzyme to quinones. The results of these studies suggest that the succinate-induced inactivation occurs by the production of hydroxyl radicals generated by a Fenton-type reaction following introduction of this mutation into the [3Fe-4S] binding domain. Additional evidence shows that the substrate-induced inactivation requires quinones, which are the membrane-bound electron acceptors and donors for the succinate dehydrogenase and fumarate reductase activities. These data suggest that the [3Fe-4S] cluster is intimately associated with one of the quinone binding sites found n fumarate reductase and succinate dehydrogenase.

Full Text

The Full Text of this article is available as a PDF (279.9 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. AEvarsson A., Hederstedt L. Ligands to the 2Fe iron-sulfur center in succinate dehydrogenase. FEBS Lett. 1988 May 23;232(2):298–302. doi: 10.1016/0014-5793(88)80757-9. [DOI] [PubMed] [Google Scholar]
  2. Ackrell B. A., Cochran B., Cecchini G. Interactions of oxaloacetate with Escherichia coli fumarate reductase. Arch Biochem Biophys. 1989 Jan;268(1):26–34. doi: 10.1016/0003-9861(89)90561-4. [DOI] [PubMed] [Google Scholar]
  3. Adman E. T., Sieker L. C., Jensen L. H. Structure of a bacterial ferredoxin. J Biol Chem. 1973 Jun 10;248(11):3987–3996. [PubMed] [Google Scholar]
  4. Blaut M., Whittaker K., Valdovinos A., Ackrell B. A., Gunsalus R. P., Cecchini G. Fumarate reductase mutants of Escherichia coli that lack covalently bound flavin. J Biol Chem. 1989 Aug 15;264(23):13599–13604. [PubMed] [Google Scholar]
  5. Broomfield P. L., Hargreaves J. A. A single amino-acid change in the iron-sulphur protein subunit of succinate dehydrogenase confers resistance to carboxin in Ustilago maydis. Curr Genet. 1992 Aug;22(2):117–121. doi: 10.1007/BF00351470. [DOI] [PubMed] [Google Scholar]
  6. Cecchini G., Ackrell B. A., Deshler J. O., Gunsalus R. P. Reconstitution of quinone reduction and characterization of Escherichia coli fumarate reductase activity. J Biol Chem. 1986 Feb 5;261(4):1808–1814. [PubMed] [Google Scholar]
  7. Cecchini G., Thompson C. R., Ackrell B. A., Westenberg D. J., Dean N., Gunsalus R. P. Oxidation of reduced menaquinone by the fumarate reductase complex in Escherichia coli requires the hydrophobic FrdD peptide. Proc Natl Acad Sci U S A. 1986 Dec;83(23):8898–8902. doi: 10.1073/pnas.83.23.8898. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cole S. T., Grundström T., Jaurin B., Robinson J. J., Weiner J. H. Location and nucleotide sequence of frdB, the gene coding for the iron-sulphur protein subunit of the fumarate reductase of Escherichia coli. Eur J Biochem. 1982 Aug;126(1):211–216. doi: 10.1111/j.1432-1033.1982.tb06768.x. [DOI] [PubMed] [Google Scholar]
  9. Darlison M. G., Guest J. R. Nucleotide sequence encoding the iron-sulphur protein subunit of the succinate dehydrogenase of Escherichia coli. Biochem J. 1984 Oct 15;223(2):507–517. doi: 10.1042/bj2230507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dikalov S. I., Rumyantseva G. V., Piskunov A. V., Weiner L. M. Role of quinone-iron(III) interaction in NADPH-dependent enzymatic generation of hydroxyl radicals. Biochemistry. 1992 Sep 22;31(37):8947–8953. doi: 10.1021/bi00152a034. [DOI] [PubMed] [Google Scholar]
  11. Guest J. R. Partial replacement of succinate dehydrogenase function by phage- and plasmid-specified fumarate reductase in Escherichia coli. J Gen Microbiol. 1981 Feb;122(2):171–179. doi: 10.1099/00221287-122-2-171. [DOI] [PubMed] [Google Scholar]
  12. Howard J. B., Lorsbach T. W., Ghosh D., Melis K., Stout C. D. Structure of Azotobacter vinelandii 7Fe ferredoxin. Amino acid sequence and electron density maps of residues. J Biol Chem. 1983 Jan 10;258(1):508–522. [PubMed] [Google Scholar]
  13. Johnson M. K., Kowal A. T., Morningstar J. E., Oliver M. E., Whittaker K., Gunsalus R. P., Ackrell B. A., Cecchini G. Subunit location of the iron-sulfur clusters in fumarate reductase from Escherichia coli. J Biol Chem. 1988 Oct 15;263(29):14732–14738. [PubMed] [Google Scholar]
  14. Kargalioglu Y., Imlay J. A. Importance of anaerobic superoxide dismutase synthesis in facilitating outgrowth of Escherichia coli upon entry into an aerobic habitat. J Bacteriol. 1994 Dec;176(24):7653–7658. doi: 10.1128/jb.176.24.7653-7658.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kröger A. Fumarate as terminal acceptor of phosphorylative electron transport. Biochim Biophys Acta. 1978 Oct 23;505(2):129–145. doi: 10.1016/0304-4173(78)90010-1. [DOI] [PubMed] [Google Scholar]
  16. Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
  17. Lauterbach F., Körtner C., Albracht S. P., Unden G., Kröger A. The fumarate reductase operon of Wolinella succinogenes. Sequence and expression of the frdA and frdB genes. Arch Microbiol. 1990;154(4):386–393. doi: 10.1007/BF00276536. [DOI] [PubMed] [Google Scholar]
  18. Lemire B. D., Robinson J. J., Weiner J. H. Identification of membrane anchor polypeptides of Escherichia coli fumarate reductase. J Bacteriol. 1982 Dec;152(3):1126–1131. doi: 10.1128/jb.152.3.1126-1131.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Manodori A., Cecchini G., Schröder I., Gunsalus R. P., Werth M. T., Johnson M. K. [3Fe-4S] to [4Fe-4S] cluster conversion in Escherichia coli fumarate reductase by site-directed mutagenesis. Biochemistry. 1992 Mar 17;31(10):2703–2712. doi: 10.1021/bi00125a010. [DOI] [PubMed] [Google Scholar]
  20. Messing J. New M13 vectors for cloning. Methods Enzymol. 1983;101:20–78. doi: 10.1016/0076-6879(83)01005-8. [DOI] [PubMed] [Google Scholar]
  21. Mordente A., Martorana G. E., Meucci E., Santini S. A., Littarru G. P. Enzyme inactivation by metal-catalyzed oxidation of coenzyme Q1. Biochim Biophys Acta. 1992 Jun 19;1100(3):235–241. doi: 10.1016/0167-4838(92)90477-u. [DOI] [PubMed] [Google Scholar]
  22. Nohl H. Is redox-cycling ubiquinone involved in mitochondrial oxygen activation? Free Radic Res Commun. 1990;8(4-6):307–315. doi: 10.3109/10715769009053364. [DOI] [PubMed] [Google Scholar]
  23. Nutter L. M., Ngo E. O., Fisher G. R., Gutierrez P. L. DNA strand scission and free radical production in menadione-treated cells. Correlation with cytotoxicity and role of NADPH quinone acceptor oxidoreductase. J Biol Chem. 1992 Feb 5;267(4):2474–2479. [PubMed] [Google Scholar]
  24. Phillips M. K., Hederstedt L., Hasnain S., Rutberg L., Guest J. R. Nucleotide sequence encoding the flavoprotein and iron-sulfur protein subunits of the Bacillus subtilis PY79 succinate dehydrogenase complex. J Bacteriol. 1987 Feb;169(2):864–873. doi: 10.1128/jb.169.2.864-873.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Salach J., Walker W. H., Singer T. P., Ehrenberg A., Hemmerich P., Ghisla S., Hartmann U. Studies on succinate dehydrogenase. Site of attachment of the covalently-bound flavin to the peptide chain. Eur J Biochem. 1972 Mar 27;26(2):267–278. doi: 10.1111/j.1432-1033.1972.tb01765.x. [DOI] [PubMed] [Google Scholar]
  26. Salerno J. C., Ohnishi T. Studies on the stabilized ubisemiquinone species in the succinate-cytochrome c reductase segment of the intact mitochondrial membrane system. Biochem J. 1980 Dec 15;192(3):769–781. doi: 10.1042/bj1920769. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Sato S., Nakazawa K., Hon-Nami K., Oshima T. Purification, some properties and amino acid sequence of Thermus thermophilus HB8 ferredoxin. Biochim Biophys Acta. 1981 Apr 28;668(2):277–289. doi: 10.1016/0005-2795(81)90035-0. [DOI] [PubMed] [Google Scholar]
  29. Schröder I., Gunsalus R. P., Ackrell B. A., Cochran B., Cecchini G. Identification of active site residues of Escherichia coli fumarate reductase by site-directed mutagenesis. J Biol Chem. 1991 Jul 25;266(21):13572–13579. [PubMed] [Google Scholar]
  30. Shigenaga M. K., Hagen T. M., Ames B. N. Oxidative damage and mitochondrial decay in aging. Proc Natl Acad Sci U S A. 1994 Nov 8;91(23):10771–10778. doi: 10.1073/pnas.91.23.10771. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Sinha B. K., Katki A. G., Batist G., Cowan K. H., Myers C. E. Differential formation of hydroxyl radicals by adriamycin in sensitive and resistant MCF-7 human breast tumor cells: implications for the mechanism of action. Biochemistry. 1987 Jun 30;26(13):3776–3781. doi: 10.1021/bi00387a006. [DOI] [PubMed] [Google Scholar]
  32. Stadtman E. R. Oxidation of free amino acids and amino acid residues in proteins by radiolysis and by metal-catalyzed reactions. Annu Rev Biochem. 1993;62:797–821. doi: 10.1146/annurev.bi.62.070193.004053. [DOI] [PubMed] [Google Scholar]
  33. Unden G. Differential roles for menaquinone and demethylmenaquinone in anaerobic electron transport of E. coli and their fnr-independent expression. Arch Microbiol. 1988;150(5):499–503. doi: 10.1007/BF00422294. [DOI] [PubMed] [Google Scholar]
  34. Weiner J. H., Dickie P. Fumarate reductase of Escherichia coli. Elucidation of the covalent-flavin component. J Biol Chem. 1979 Sep 10;254(17):8590–8593. [PubMed] [Google Scholar]
  35. Werth M. T., Cecchini G., Manodori A., Ackrell B. A., Schröder I., Gunsalus R. P., Johnson M. K. Site-directed mutagenesis of conserved cysteine residues in Escherichia coli fumarate reductase: modification of the spectroscopic and electrochemical properties of the [2Fe-2S] cluster. Proc Natl Acad Sci U S A. 1990 Nov;87(22):8965–8969. doi: 10.1073/pnas.87.22.8965. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Westenberg D. J., Gunsalus R. P., Ackrell B. A., Sices H., Cecchini G. Escherichia coli fumarate reductase frdC and frdD mutants. Identification of amino acid residues involved in catalytic activity with quinones. J Biol Chem. 1993 Jan 15;268(2):815–822. [PubMed] [Google Scholar]
  37. Wood D., Darlison M. G., Wilde R. J., Guest J. R. Nucleotide sequence encoding the flavoprotein and hydrophobic subunits of the succinate dehydrogenase of Escherichia coli. Biochem J. 1984 Sep 1;222(2):519–534. doi: 10.1042/bj2220519. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Zhang Y., Marcillat O., Giulivi C., Ernster L., Davies K. J. The oxidative inactivation of mitochondrial electron transport chain components and ATPase. J Biol Chem. 1990 Sep 25;265(27):16330–16336. [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES