Protocols for Cofactor Isolation of Nitrogenase

  • Aaron W. Fay
  • Chi-Chung Lee
  • Jared A. Wiig
  • Yilin Hu
  • Markus W. RibbeEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 766)


The iron-molybdenum cofactor (FeMoco) of the nitrogenase MoFe protein has remained a focal point in the field of bioinorganic chemistry for decades. This unique metal cluster has long been regarded as the actual site of dinitrogen reduction, and it is structurally complex and chemically unprecedented. A detailed characterization of the isolated FeMoco is crucial for elucidating the physiochemical properties of this biologically important cofactor. Such a study requires an effective technique to extract FeMoco intact, and in high yield, from the MoFe protein. A method involving the acid treatment of the MoFe protein and the subsequent extraction of FeMoco into an organic solvent was developed over 30 years ago and has been improved upon ever since. FeMoco isolated by this strategy is catalytically active and spectrally interesting, which provides a useful platform for future structure–function analyses of this unique cofactor. A general working protocol for FeMoco isolation is described in this chapter, along with some of the major modifications reported in the past years.

Key words

Nitrogenase FeMoco NMF DMF extraction 



The authors are supported by National Institutes of Health grant GM 67626 (M.W.R.) and Herman Frasch Foundation grant 617-HF07 (M.W.R.).


  1. 1.
    Burgess BK, Lowe DJ (1996) Mechanism of molybdenum nitrogenase. Chem Rev 96:2983–3012PubMedCrossRefGoogle Scholar
  2. 2.
    Smith BE (1999) Structure, function, and biosynthesis of the metallosulfur clusters in nitrogenases. Adv Inorg Chem 47:159–218CrossRefGoogle Scholar
  3. 3.
    Rees DC, Tezcan FA, Haynes CA et al (2005) Structural basis of biological nitrogen fixation. Philos Trans R Soc Lond Ser A363:971–984CrossRefGoogle Scholar
  4. 4.
    Howard JB, Rees DC (2006) How many metals does it take to fix N2? A mechanistic overview of biological nitrogen fixation. Proc Natl Acad Sci USA 103:17088–17093PubMedCrossRefGoogle Scholar
  5. 5.
    Einsle O, Tezcan FA, Andrade SL et al (2002) Nitrogenase MoFe-protein at 1.16 Å resolution: A central ligand in the FeMo-cofactor. Science 297:1696–1700PubMedCrossRefGoogle Scholar
  6. 6.
    Kim J, Rees DC (1992) Crystallographic structure and functional implications of the nitrogenase molybdenum iron protein from Azotobacter vinelandii. Nature 360:553–560CrossRefGoogle Scholar
  7. 7.
    Shah VK, Brill WJ (1977) Isolation of an iron-molybdenum cofactor from nitrogenase. Proc Natl Acad Sci USA 74:3249–3253PubMedCrossRefGoogle Scholar
  8. 8.
    Frank P, Ghellar SF, Newton WE et al (1990) Purification and spectroscopic characteristics in N-methylformamide of the Azotobacter vinelandii Fe-Mo cofactor. Biochem Biophys Res Commun 163:746–754CrossRefGoogle Scholar
  9. 9.
    Schultz FA, Gheller SF, Burgess BK et al (1985) Electrochemical characterization of the iron-molybdenum cofactor from Azotobacter vinelandii nitrogenase. J Am Chem Soc 107:5364–5368CrossRefGoogle Scholar
  10. 10.
    Rewlings J, Shah VK, Chisnell JR et al (1978) Novel metal cluster in the iron-molybdenum cofactor of nitrogenase. Spectroscopic evidence. J Biol Chem 253:1001–1004Google Scholar
  11. 11.
    Lindahl PA, Papaefthymiou V, Orme-Johnson WH et al (1988) Mössbauer studies of solid thionin-oxidized MoFe protein of nitrogenase. J Biol Chem 263:19412–19418PubMedGoogle Scholar
  12. 12.
    Conradkn SD, Burgess BK, Newton WE et al (1987) Structural studies of the molybdenum site in the MoFe protein and its FeMo cofactor by EXAFS. J Am Chem Soc 109:7507–7515CrossRefGoogle Scholar
  13. 13.
    Hu Y, Fay AW, Lee CC et al (2008) Assembly of nitrogenase MoFe protein. Biochemistry 47:3973–3981PubMedCrossRefGoogle Scholar
  14. 14.
    Burgess BK (1990) The iron-molybdenum cofactor of nitrogenase. Chem Rev 90:1377–1406CrossRefGoogle Scholar
  15. 15.
    Walters MA, Chapman SK, Orme-Johnson WH (1986) The nature of amide ligation to the metal sites of FeMoco. Polyhedron 5:561–565CrossRefGoogle Scholar
  16. 16.
    Yang SS, Pan WH, Friesen GD et al (1982) Iron-molybdenum cofactor from nitrogenase. Modified extraction methods as probes for composition. J Biol Chem 257:8042–8048PubMedGoogle Scholar
  17. 17.
    McLean PA, Wink DA, Chapman SK et al (1989) A new method for extraction of iron-molybdenum cofactor (FeMoco) from nitrogenase adsorbed to DEAE-cellulose. 1. Effects of anions, cations, and pre-extraction treatments. Biochemistry 28:9402–9406PubMedCrossRefGoogle Scholar
  18. 18.
    Wink DA, McLean PA, Hickman AB et al (1989) A new method for extraction of iron-molybdenum cofactor (FeMoco) from nitrogenase adsorbed to DEAE-cellulose. 2. Solubilization of FeMoco in a wide range of organic solvents. Biochemistry 28:9407–9412PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Aaron W. Fay
    • 1
  • Chi-Chung Lee
    • 1
  • Jared A. Wiig
    • 1
  • Yilin Hu
    • 1
  • Markus W. Ribbe
    • 1
    Email author
  1. 1.Department of Molecular Biology and BiochemistryUniversity of CaliforniaIrvineUSA

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