Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1986 Mar;83(6):1636–1640. doi: 10.1073/pnas.83.6.1636

In vitro synthesis of the iron-molybdenum cofactor of nitrogenase.

V K Shah, J Imperial, R A Ugalde, P W Ludden, W J Brill
PMCID: PMC323138  PMID: 3006060

Abstract

Molybdate- and ATP-dependent in vitro synthesis of the iron-molybdenum cofactor (FeMo-co) of nitrogenase requires the protein products of at least the nifB, nifN, and nifE genes. Extracts of FeMo-co-negative mutants of Klebsiella pneumoniae and Azotobacter vinelandii with lesions in different genes can be complemented for FeMo-co synthesis. Both K. pneumoniae and A. vinelandii dinitrogenase (component I) deficient in FeMo-co can be activated by FeMo-co synthesized in vitro. Properties of the partially purified dinitrogenase activated by FeMo-co synthesized in vitro were comparable to those of dinitrogenase from the wild-type organism; e.g., ratios of acetylene- to nitrogen-reduction activities, as well as those of acetylene reduction activities to EPR spectrum peak height at g = 3.65, were very similar. A. vinelandii mutants UW45 and CA30 have mutations in a gene functionally equivalent to nifB of K. pneumoniae.

Full text

PDF
1636

Selected References

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

  1. Benemann J. R., Smith G. M., Kostel P. J., McKenna C. E. Tungsten incorporation into Azotobacter vinelandii nitrogenase. FEBS Lett. 1973 Feb 1;29(3):219–221. doi: 10.1016/0014-5793(73)80023-7. [DOI] [PubMed] [Google Scholar]
  2. Bishop P. E., Brill W. J. Genetic analysis of Azotobacter vinelandii mutant strains unable to fix nitrogen. J Bacteriol. 1977 May;130(2):954–956. doi: 10.1128/jb.130.2.954-956.1977. [DOI] [PMC free article] [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: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  4. Brill W. J. Biochemical genetics of nitrogen fixation. Microbiol Rev. 1980 Sep;44(3):449–467. doi: 10.1128/mr.44.3.449-467.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cardenas J., Mortenson L. E. Role of molybdenum in dinitrogen fixation by Clostridium pasteurianum. J Bacteriol. 1975 Sep;123(3):978–984. doi: 10.1128/jb.123.3.978-984.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Hawkes T. R., McLean P. A., Smith B. E. Nitrogenase from nifV mutants of Klebsiella pneumoniae contains an altered form of the iron-molybdenum cofactor. Biochem J. 1984 Jan 1;217(1):317–321. doi: 10.1042/bj2170317. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Imperial J., Ugalde R. A., Shah V. K., Brill W. J. Mol- mutants of Klebsiella pneumoniae requiring high levels of molybdate for nitrogenase activity. J Bacteriol. 1985 Sep;163(3):1285–1287. doi: 10.1128/jb.163.3.1285-1287.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Imperial J., Ugalde R. A., Shah V. K., Brill W. J. Role of the nifQ gene product in the incorporation of molybdenum into nitrogenase in Klebsiella pneumoniae. J Bacteriol. 1984 Apr;158(1):187–194. doi: 10.1128/jb.158.1.187-194.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. KEELER R. F., VARNER J. E. Tungstate as an antagonist of molybdate in Azotobacter vinelandii. Arch Biochem Biophys. 1957 Aug;70(2):585–590. doi: 10.1016/0003-9861(57)90146-7. [DOI] [PubMed] [Google Scholar]
  10. Kennedy C., Postgate J. R. Expression of Klebsiella pneumoniae nitrogen fixation genes in nitrate reductase mutants of Escherichia coli. J Gen Microbiol. 1977 Feb;98(2):551–557. doi: 10.1099/00221287-98-2-551. [DOI] [PubMed] [Google Scholar]
  11. MacNeil T., MacNeil D., Roberts G. P., Supiano M. A., Brill W. J. Fine-structure mapping and complementation analysis of nif (nitrogen fixation) genes in Klebsiella pneumoniae. J Bacteriol. 1978 Oct;136(1):253–266. doi: 10.1128/jb.136.1.253-266.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. McLean P. A., Dixon R. A. Requirement of nifV gene for production of wild-type nitrogenase enzyme in Klebsiella pneumoniae. Nature. 1981 Aug 13;292(5824):655–656. doi: 10.1038/292655a0. [DOI] [PubMed] [Google Scholar]
  13. McLean P. A., Smith B. E., Dixon R. A. Nitrogenase of Klebsiella pneumoniae nifV mutants. Biochem J. 1983 Jun 1;211(3):589–597. doi: 10.1042/bj2110589. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Nagatani H. H., Brill W. J. Nitrogenase V. The effect of Mo, W and V on the synthesis of nitrogenase components in Azotobacter vinelandii. Biochim Biophys Acta. 1974 Aug 7;362(1):160–166. doi: 10.1016/0304-4165(74)90037-3. [DOI] [PubMed] [Google Scholar]
  15. Nagatani H. H., Shah V. K., Brill W. J. Activation of inactive nitrogenase by acid-treated component I. J Bacteriol. 1974 Nov;120(2):697–701. doi: 10.1128/jb.120.2.697-701.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. Orme-Johnson W. H., Hamilton W. D., Jones T. L., Tso M. Y., Burris R. H., Shah V. K., Brill W. J. Electron paramagnetic resonance of nitrogenase and nitrogenase components from Clostridium pasteurianum W5 and Azotobacter vinelandii OP. Proc Natl Acad Sci U S A. 1972 Nov;69(11):3142–3145. doi: 10.1073/pnas.69.11.3142. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Pienkos P. T., Brill W. J. Molybdenum accumulation and storage in Klebsiella pneumoniae and Azotobacter vinelandii. J Bacteriol. 1981 Feb;145(2):743–751. doi: 10.1128/jb.145.2.743-751.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Pienkos P. T., Klevickis S., Brill W. J. In vitro activation of inactive nitrogenase component I with molybdate. J Bacteriol. 1981 Jan;145(1):248–256. doi: 10.1128/jb.145.1.248-256.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Rawlings J., Shah V. K., Chisnell J. R., Brill W. J., Zimmermann R., Münck E., Orme-Johnson W. H. Novel metal cluster in the iron-molybdenum cofactor of nitrogenase. Spectroscopic evidence. J Biol Chem. 1978 Feb 25;253(4):1001–1004. [PubMed] [Google Scholar]
  21. Roberts G. P., Brill W. J. Gene-product relationships of the nif regulon of Klebsiella pneumoniae. J Bacteriol. 1980 Oct;144(1):210–216. doi: 10.1128/jb.144.1.210-216.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Roberts G. P., Brill W. J. Genetics and regulation of nitrogen fixation. Annu Rev Microbiol. 1981;35:207–235. doi: 10.1146/annurev.mi.35.100181.001231. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Shah V. K., Brill W. J. Isolation of an iron-molybdenum cofactor from nitrogenase. Proc Natl Acad Sci U S A. 1977 Aug;74(8):3249–3253. doi: 10.1073/pnas.74.8.3249. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Shah V. K., Brill W. J. Nitrogenase. IV. Simple method of purification to homogeneity of nitrogenase components from Azotobacter vinelandii. Biochim Biophys Acta. 1973 May 30;305(2):445–454. doi: 10.1016/0005-2728(73)90190-4. [DOI] [PubMed] [Google Scholar]
  26. Shah V. K., Chisnell J. R., Brill W. J. Acetylene reduction by the iron-molybdenum cofactor from nitrogenase. Biochem Biophys Res Commun. 1978 Mar 15;81(1):232–236. doi: 10.1016/0006-291x(78)91654-6. [DOI] [PubMed] [Google Scholar]
  27. Shah V. K., Davis I. C., Gordon J. K., Orme-Johnson W. H., Brill W. J. Nitrogenase. 3. Nitrogenaseless mutants of Azotobacter vinelandii: activities, cross-reactions and EPR spectra. Biochim Biophys Acta. 1973 Jan 18;292(1):246–255. doi: 10.1016/0005-2728(73)90269-7. [DOI] [PubMed] [Google Scholar]
  28. Shah V. K., Davis L. C., Brill W. J. Nitrogenase. I. Repression and derepression of the iron-molybdenum and iron proteins of nitrogenase in Azotobacter vinelandii. Biochim Biophys Acta. 1972 Feb 28;256(2):498–511. doi: 10.1016/0005-2728(72)90078-3. [DOI] [PubMed] [Google Scholar]
  29. Shah V. K., Davis L. C., Stieghorst M., Brill W. J. Mutant of Azotobacter vinelandii that hyperproduces nitrogenase component II. J Bacteriol. 1974 Feb;117(2):917–919. doi: 10.1128/jb.117.2.917-919.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. 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]
  31. Shah V. K., Ugalde R. A., Imperial J., Brill W. J. Inhibition of iron-molybdenum cofactor binding to component I of nitrogenase. J Biol Chem. 1985 Apr 10;260(7):3891–3894. [PubMed] [Google Scholar]
  32. Shah V. K., Ugalde R. A., Imperial J., Brill W. J. Molybdenum in nitrogenase. Annu Rev Biochem. 1984;53:231–257. doi: 10.1146/annurev.bi.53.070184.001311. [DOI] [PubMed] [Google Scholar]
  33. Sibold L., Quiviger B., Charpin N., Paquelin A., Elmerich C. Cloning and expression of a DNA fragment carrying a his nifA fusion and the nifBQ operon from a nif constitutive mutant of Klebsiella pneumoniae. Biochimie. 1983 Jan;65(1):53–63. doi: 10.1016/s0300-9084(83)80029-7. [DOI] [PubMed] [Google Scholar]
  34. Ugalde R. A., Imperial J., Shah V. K., Brill W. J. Biosynthesis of iron-molybdenum cofactor in the absence of nitrogenase. J Bacteriol. 1984 Sep;159(3):888–893. doi: 10.1128/jb.159.3.888-893.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES