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. 1991 Oct 1;279(Pt 1):155–158. doi: 10.1042/bj2790155

Purification and partial characterization of a pyruvate oxidoreductase from the photosynthetic bacterium Rhodospirillum rubrum grown under nitrogen-fixing conditions.

E Brostedt 1, S Nordlund 1
PMCID: PMC1151561  PMID: 1930134

Abstract

A pyruvate oxidoreductase with the capacity to support pyruvate-dependent nitrogenase activity in vitro has been purified from the photosynthetic bacterium Rhodospirillum rubrum. The enzyme requires CoA for activity and is irreversibly inactivated by oxygen. The molecular properties and Km values for the substrates have been studied. In supporting nitrogenase activity addition of ferredoxin is required. Overall the enzyme is similar to the nif-specific pyruvate: flavodoxin oxidoreductase purified from Klebsiella pneumoniae.

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Selected References

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  1. Arnold W., Rump A., Klipp W., Priefer U. B., Pühler A. Nucleotide sequence of a 24,206-base-pair DNA fragment carrying the entire nitrogen fixation gene cluster of Klebsiella pneumoniae. J Mol Biol. 1988 Oct 5;203(3):715–738. doi: 10.1016/0022-2836(88)90205-7. [DOI] [PubMed] [Google Scholar]
  2. Benemann J. R., Yoch D. C., Valentine R. C., Arnon D. I. The electron transport system in nitrogen fixation by Azotobacter. I. Azotoflavin as an electron carrier. Proc Natl Acad Sci U S A. 1969 Nov;64(3):1079–1086. doi: 10.1073/pnas.64.3.1079. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Benemann J. R., Yoch D. C., Valentine R. C., Arnon D. I. The electron transport system in nitrogen fixation by azotobacter. 3. Requirements for NADPH-supported nitrogenase activity. Biochim Biophys Acta. 1971 Mar 2;226(2):205–212. doi: 10.1016/0005-2728(71)90087-9. [DOI] [PubMed] [Google Scholar]
  4. Bennett L. T., Jacobson M. R., Dean D. R. Isolation, sequencing, and mutagenesis of the nifF gene encoding flavodoxin from Azotobacter vinelandii. J Biol Chem. 1988 Jan 25;263(3):1364–1369. [PubMed] [Google Scholar]
  5. Bogusz D., Houmard J., Aubert J. P. Electron transport to nitrogenase in Klebsiella pneumoniae: purification and properties of the nifJ protein. Eur J Biochem. 1981 Nov;120(2):421–426. doi: 10.1111/j.1432-1033.1981.tb05719.x. [DOI] [PubMed] [Google Scholar]
  6. 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: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  7. CARNAHAN J. E., MORTENSON L. E., MOWER H. F., CASTLE J. E. Nitrogen fixation in cell-free extracts of Clostridium pasteurianum. Biochim Biophys Acta. 1960 Nov 18;44:520–535. doi: 10.1016/0006-3002(60)91606-1. [DOI] [PubMed] [Google Scholar]
  8. Cusanovich M. A., Edmondson D. E. The isolation and characterization of Rhodospirillum rubrum flavodoxin. Biochem Biophys Res Commun. 1971 Oct 15;45(2):327–336. doi: 10.1016/0006-291x(71)90822-9. [DOI] [PubMed] [Google Scholar]
  9. Deistung J., Cannon F. C., Cannon M. C., Hill S., Thorneley R. N. Electron transfer to nitrogenase in Klebsiella pneumoniae. nifF gene cloned and the gene product, a flavodoxin, purified. Biochem J. 1985 Nov 1;231(3):743–753. doi: 10.1042/bj2310743. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Deistung J., Thorneley R. N. Electron transfer to nitrogenase. Characterization of flavodoxin from Azotobacter chroococcum and comparison of its redox potentials with those of flavodoxins from Azotobacter vinelandii and Klebsiella pneumoniae (nifF-gene product). Biochem J. 1986 Oct 1;239(1):69–75. doi: 10.1042/bj2390069. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fish W. W. Rapid colorimetric micromethod for the quantitation of complexed iron in biological samples. Methods Enzymol. 1988;158:357–364. doi: 10.1016/0076-6879(88)58067-9. [DOI] [PubMed] [Google Scholar]
  12. Hill S., Kavanagh E. P. Roles of nifF and nifJ gene products in electron transport to nitrogenase in Klebsiella pneumoniae. J Bacteriol. 1980 Feb;141(2):470–475. doi: 10.1128/jb.141.2.470-475.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Klugkist J., Haaker H., Veeger C. Studies on the mechanism of electron transport to nitrogenase in Azotobacter vinelandii. Eur J Biochem. 1986 Feb 17;155(1):41–46. doi: 10.1111/j.1432-1033.1986.tb09456.x. [DOI] [PubMed] [Google Scholar]
  14. Klugkist J., Voorberg J., Haaker H., Veeger C. Characterization of three different flavodoxins from Azotobacter vinelandii. Eur J Biochem. 1986 Feb 17;155(1):33–40. doi: 10.1111/j.1432-1033.1986.tb09455.x. [DOI] [PubMed] [Google Scholar]
  15. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  16. MORTENSON L. E. FERREDOXIN AND ATP, REQUIREMENTS FOR NITROGEN FIXATION IN CELL-FREE EXTRACTS OF CLOSTRIDIUM PASTEURIANUM. Proc Natl Acad Sci U S A. 1964 Aug;52:272–279. doi: 10.1073/pnas.52.2.272. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Meinecke B., Bertram J., Gottschalk G. Purification and characterization of the pyruvate-ferredoxin oxidoreductase from Clostridium acetobutylicum. Arch Microbiol. 1989;152(3):244–250. doi: 10.1007/BF00409658. [DOI] [PubMed] [Google Scholar]
  18. Nieva-Gómez D., Roberts G. P., Klevickis S., Brill W. J. Electron transport to nitrogenase in Klebsiella pneumoniae. Proc Natl Acad Sci U S A. 1980 May;77(5):2555–2558. doi: 10.1073/pnas.77.5.2555. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. ORMEROD J. G., ORMEROD K. S., GEST H. Light-dependent utilization of organic compounds and photoproduction of molecular hydrogen by photosynthetic bacteria; relationships with nitrogen metabolism. Arch Biochem Biophys. 1961 Sep;94:449–463. doi: 10.1016/0003-9861(61)90073-x. [DOI] [PubMed] [Google Scholar]
  20. Roberts G. P., MacNeil T., MacNeil D., Brill W. J. Regulation and characterization of protein products coded by the nif (nitrogen fixation) genes of Klebsiella pneumoniae. J Bacteriol. 1978 Oct;136(1):267–279. doi: 10.1128/jb.136.1.267-279.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Sauer F. D., Bush R. S., Stevenson I. L. The separation of pyruvate-ferredoxin oxidoreductase from Clostridium pasteurianum into two enzymes catalyzing different reactions. Biochim Biophys Acta. 1976 Sep 14;445(2):518–520. doi: 10.1016/0005-2744(76)90105-4. [DOI] [PubMed] [Google Scholar]
  22. Schatt E., Jouanneau Y., Vignais P. M. Molecular cloning and sequence analysis of the structural gene of ferredoxin I from the photosynthetic bacterium Rhodobacter capsulatus. J Bacteriol. 1989 Nov;171(11):6218–6226. doi: 10.1128/jb.171.11.6218-6226.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Shah V. K., Stacey G., Brill W. J. Electron transport to nitrogenase. Purification and characterization of pyruvate:flavodoxin oxidoreductase. The nifJ gene product. J Biol Chem. 1983 Oct 10;258(19):12064–12068. [PubMed] [Google Scholar]
  24. Uyeda K., Rabinowitz J. C. Pyruvate-ferredoxin oxidoreductase. 3. Purification and properties of the enzyme. J Biol Chem. 1971 May 25;246(10):3111–3119. [PubMed] [Google Scholar]
  25. Wahl R. C., Orme-Johnson W. H. Clostridial pyruvate oxidoreductase and the pyruvate-oxidizing enzyme specific to nitrogen fixation in Klebsiella pneumoniae are similar enzymes. J Biol Chem. 1987 Aug 5;262(22):10489–10496. [PubMed] [Google Scholar]
  26. Yates M. G. Electron transport to nitrogenase in Azotobacter chroococcum: Azotobacter flavodoxin hydroquinone as an electron donor. FEBS Lett. 1972 Oct 15;27(1):63–67. doi: 10.1016/0014-5793(72)80410-1. [DOI] [PubMed] [Google Scholar]
  27. Yoch D. C., Arnon D. I. Comparison of two ferredoxins from Rhodospirillum rubrum as electron carriers for the native nitrogenase. J Bacteriol. 1975 Feb;121(2):743–745. doi: 10.1128/jb.121.2.743-745.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Yoch D. C., Arnon D. I., Sweeney W. V. Characterization of two soluble ferredoxins as distinct from bound iron-sulfur proteins in the photosynthetic bacterium Rhodospirillum rubrum. J Biol Chem. 1975 Nov 10;250(21):8330–8336. [PubMed] [Google Scholar]

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