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
. 1972 Nov;69(11):3142–3145. doi: 10.1073/pnas.69.11.3142

Electron Paramagnetic Resonance of Nitrogenase and Nitrogenase Components from Clostridium pasteurianum W5 and Azotobacter vinelandii OP

W H Orme-Johnson *,†, W D Hamilton *, T L Jones †, M-Y W Tso †, R H Burris †, V K Shah ‡, W J Brill ‡
PMCID: PMC389722  PMID: 4343957

Abstract

The electron paramagnetic resonance of nitrogenase components, separately and together with the other reactants in the nitrogenase system (namely, reductant and Mg·ATP), have been examined at low temperatures (<20°K). The MoFe protein, component I or molybdoferredoxin, in the oxidized (but not oxygen-inactivated) state yields signals with g-values of 4.3, 3.7, and 2.01, and when reduced has no observable electron paramagnetic resonance. The Fe protein, component II, or azoferredoxin, yields a signal with g-values of 2.05, 1.94, and 1.89 in the reduced state that is converted by Mg·ATP into an axial signal with g-values near 2.05 and 1.94, and a second split signal near g = 4.3. The Fe protein has no definite electron paramagnetic resonance in the oxidized (not oxygen-denatured) state under these conditions. The Mg·ATP complex of reduced Fe protein reduces the MoFe protein, whereas dithionite alone does not reduce the MoFe protein. Reoxidation of the system by substrate leads to disappearance of the Fe protein signal and the reappearance of the MoFe protein signal. Thus Mg·ATP, which is hydrolyzed during substrate reduction, converts the Fe protein to a reductant capable of transferring electrons to MoFe protein, after which substrate reduction occurs.

Keywords: iron-sulfur proteins, ferredoxins, electron transfer, nitrogen fixation, Mg·ATP

Full text

PDF
3143

Selected References

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

  1. Bui P. T., Mortenson L. E. Mechanism of the enzymic reduction of N2: the binding of adenosine 5'-triphosphate and cyanide to the N2-reducing system. Proc Natl Acad Sci U S A. 1968 Nov;61(3):1021–1027. doi: 10.1073/pnas.61.3.1021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bulen W. A., LeComte J. R. The nitrogenase system from Azotobacter: two-enzyme requirement for N2 reduction, ATP-dependent H2 evolution, and ATP hydrolysis. Proc Natl Acad Sci U S A. 1966 Sep;56(3):979–986. doi: 10.1073/pnas.56.3.979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Burns R. C., Holsten R. D., Hardy R. W. Isolation by crystallization of the Mo-Fe protein of Azotobacter nitrogenase. Biochem Biophys Res Commun. 1970 Apr 8;39(1):90–99. doi: 10.1016/0006-291x(70)90762-x. [DOI] [PubMed] [Google Scholar]
  4. Dalton H., Mortenson L. E. Dinitrogen (N 2 ) fixation (with a biochemical emphasis). Bacteriol Rev. 1972 Jun;36(2):231–260. doi: 10.1128/br.36.2.231-260.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Davis L. C., Shah V. K., Brill W. J., Orme-Johnson W. H. Nitrogenase. II. Changes in the EPR signal of component I (iron-molybdenum protein) of Azotobacter vinelandii nitrogenase during repression and derepression. Biochim Biophys Acta. 1972 Feb 28;256(2):512–523. doi: 10.1016/0005-2728(72)90079-5. [DOI] [PubMed] [Google Scholar]
  6. Eady R. R., Smith B. E., Cook K. A., Postgate J. R. Nitrogenase of Klebsiella pneumoniae. Purification and properties of the component proteins. Biochem J. 1972 Jul;128(3):655–675. doi: 10.1042/bj1280655. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. GOA J. A micro biuret method for protein determination; determination of total protein in cerebrospinal fluid. Scand J Clin Lab Invest. 1953;5(3):218–222. doi: 10.3109/00365515309094189. [DOI] [PubMed] [Google Scholar]
  8. Hansen R. E., Van Gelder B. F., Beinert H. Attachment to split-beam spectrophotometer for absorbance recording in round tubes of small diameter. Anal Biochem. 1970 May;35(1):287–292. doi: 10.1016/0003-2697(70)90036-9. [DOI] [PubMed] [Google Scholar]
  9. Kelly M. Some properties of purified nitrogenase of Azotobacter chroococcum. Biochim Biophys Acta. 1969 Jan 7;171(1):9–22. doi: 10.1016/0005-2744(69)90101-6. [DOI] [PubMed] [Google Scholar]
  10. Ljones T., Burris R. H. ATP hydrolysis and electron transfer in the nitrogenase reaction with different combinations of the iron protein and the molybdenum-iron protein. Biochim Biophys Acta. 1972 Jul 12;275(1):93–101. doi: 10.1016/0005-2728(72)90027-8. [DOI] [PubMed] [Google Scholar]
  11. Mortenson L. E. Components of cell-free extracts of Clostridium pasteurianum required for ATP-dependent H2 evolution from dithionite and for N2 fixation. Biochim Biophys Acta. 1966 Sep 26;127(1):18–25. doi: 10.1016/0304-4165(66)90470-3. [DOI] [PubMed] [Google Scholar]
  12. Mortenson L. E., Morris J. A., Jeng D. Y. Purification, metal composition and properties of molybdoferredoxin and azoferredoxin, two of the components of the nitrogen-fixing system of Clostridium pasteurianum. Biochim Biophys Acta. 1967 Aug 29;141(3):516–522. doi: 10.1016/0304-4165(67)90180-8. [DOI] [PubMed] [Google Scholar]
  13. Palmer G., Sands R. H. On the magnetic resonance of spinach ferredoxin. J Biol Chem. 1966 Jan 10;241(1):253–253. [PubMed] [Google Scholar]
  14. 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]
  15. Tso M. Y., Ljones T., Burris R. H. Purification of the nitrogenase proteins from Clostridium pasteurianum. Biochim Biophys Acta. 1972 Jun 23;267(3):600–604. doi: 10.1016/0005-2728(72)90193-4. [DOI] [PubMed] [Google Scholar]
  16. Van de Bogart M., Beinert H. Micro methods for the quantitative determination of iron and copper in biological material. Anal Biochem. 1967 Aug;20(2):325–334. doi: 10.1016/0003-2697(67)90038-3. [DOI] [PubMed] [Google Scholar]
  17. Vandecasteele J. P., Burris R. H. Purification and properties of the constituents of the nitrogenase complex from Clostridium pasteurianum. J Bacteriol. 1970 Mar;101(3):794–801. doi: 10.1128/jb.101.3.794-801.1970. [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