Historic Overview of Nitrogenase Research

  • Yilin Hu
  • Markus W. RibbeEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 766)


The history of nitrogenase research dates all the way back to the 1800s. This chapter provides a brief account of the advances in this particular research area over the past few hundred years, which include such events as the initial discovery of biological nitrogen fixation, the preparation of active cell-free extracts, the purification of nitrogenase enzyme, the proposal of the Thorneley–Lowe model, and the report of x-ray crystallographic structures of the component proteins of nitrogenase.

Key words

Biological nitrogen fixation nitrogenase cell-free extracts purification x-ray crystallography 



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


  1. 1.
    Hellriegel H, Wilfarth H (1888) Untersuchungen über die Stickstoffnährung der Gramineen und Leguminosen. Beil Z Ver dt ZuchInd, 1–234Google Scholar
  2. 2.
    Beijerinck MW (1888) Die Bakterien der Papilionaceen-Knölchen. Bot Ztg 46:725–735; 741–750; 757–771; 781–790; 797–804Google Scholar
  3. 3.
    Winogradsky S (1893) Sur l’assimilation de l’azote gaseux de l’atmosphère par les microbes. C r hedb Séanc Acad Sci Paris 116:1385–1388Google Scholar
  4. 4.
    Beijerinck MW (1901) Über oligonitrophile Mikroben. ZentBl Bakt ParasitKde 7:561–582Google Scholar
  5. 5.
    Meyerhof O, Burk D (1928) On the fixation of air nitrogen through Azotobacter. Z Phys Chem 139:117–142Google Scholar
  6. 6.
    Bortels H (1930) Molybdän als Katalysator bei der biologischen Stickstoffbindung. Arch Mikrobiol 1:333–342CrossRefGoogle Scholar
  7. 7.
    Burris H (1942) Distribution of isotopic nitrogen in Azotobacter vinelandii. J Biol Chem 143:509–517Google Scholar
  8. 8.
    Carnahan JE, Mortenson LE, Mower HF et al (1960) Nitrogen fixation in cell-free extracts of Clostridium pasteurianum. Biochim Biophys Acta 38:188–189PubMedCrossRefGoogle Scholar
  9. 9.
    Burris RH (1969) Progress in the biochemistry of nitrogen fixation. Proc R Soc Lond B Biol Sci 172:339–354PubMedCrossRefGoogle Scholar
  10. 10.
    Bulen WA, Burns RC, LeComte JR (1964) Nitrogen fixation: cell-free system with extracts of Azotobacter. Biochem Biophys Res Commun 17:265–271PubMedCrossRefGoogle Scholar
  11. 11.
    Bulen WA, Burns RC, Lecomte JR (1965) Nitrogen fixation: hydrogensulfite as electron donor with cell-free preparations of Azotobacter vinelandii and Rhodospirillum rubrum. Proc Natl Acad Sci USA 53:532–539PubMedCrossRefGoogle Scholar
  12. 12.
    Bulen WA, LeComte JR (1966) The nitrogenase system from Azotobacter: two-enzyme requirement for N2 reduction, ATP-dependent H2 evolution, and ATP hydrolysis. Proc Natl Acad Sci USA 56:979–986PubMedCrossRefGoogle Scholar
  13. 13.
    Mortenson LE, Morris JA, Jeng DY (1967) Purification, metal composition and properties of molybdoferredoxin and azoferredoxin, two of the components of the nitrogen-fixing system of Clostridium pasteurianum. Biochim Biophys Acta 141:516–522PubMedCrossRefGoogle Scholar
  14. 14.
    Thorneley RNF, Lowe DJ (1985) Kinetics and mechanism of the nitrogenase enzyme system. In: Spiro, TG (ed) Molybdenum Enzymes, pp. 221–284. Wiley, New York, NYGoogle Scholar
  15. 15.
    Georgiadis MM, Komiya H, Chakrabarti P et al (1992) Crystallographic structure of the nitrogenase iron protein from Azotobacter vinelandii. Science 257:1653–1659PubMedCrossRefGoogle Scholar
  16. 16.
    Kim J, Rees DC (1992) Structural models for the metal centers in the nitrogenase molybdenum-iron protein. Science 257:1677–1682PubMedCrossRefGoogle Scholar
  17. 17.
    Chan MK, Kim J, Rees DC (1993) The nitrogenase FeMo-cofactor and P-cluster pair: 2.2 A resolution structures. Science 260:792–794PubMedCrossRefGoogle Scholar
  18. 18.
    Lee SC, Holm RH (2003) Speculative synthetic chemistry and the nitrogenase problem. Proc Natl Acad Sci USA 100:3595–3600PubMedCrossRefGoogle Scholar
  19. 19.
    Lee SC, Holm RH (2004) The clusters of nitrogenase: synthetic methodology in the construction of weak-field clusters. Chem Rev 104:1135–1158PubMedCrossRefGoogle Scholar
  20. 20.
    Groysman S, Holm RH (2009) Biomimetic chemistry of iron, nickel, molybdenum, and tungsten in sulfur-ligated protein sites. Biochemistry 48:2310–2320PubMedCrossRefGoogle Scholar
  21. 21.
    Coucouvanis D (1994) Fe/S and Fe/Mo/S clusters as speculative models for the metal centers in uncommon Fe/S proteins and the Fe/Mo protein of the nitrogenases. Adv Inorg Biochem 9:75–122PubMedGoogle Scholar
  22. 22.
    Ohki Y, Sunada Y, Honda M, Katada M, Tatsumi K (2003) Synthesis of the P-cluster inorganic core of nitrogenases. J Am Chem Soc 125:4052–4053PubMedCrossRefGoogle Scholar
  23. 23.
    Holland PL (2008) Electronic structure and reactivity of three-coordinate iron complexes. Acc Chem Res 41:905–914PubMedCrossRefGoogle Scholar
  24. 24.
    Setubal JC, dos Santos P, Goldman BS, Ertesvåg H, Espin G, Rubio LM, Valla S, Almeida NF, Balasubramanian D, Cromes L, Curatti L, Du Z, Godsy E, Goodner B, Hellner-Burris K, Hernandez JA, Houmiel K, Imperial J, Kennedy C, Larson TJ, Latreille P, Ligon LS, Lu J, Maerk M, Miller NM, Norton S, O’Carroll IP, Paulsen I, Raulfs EC, Roemer R, Rosser J, Segura D, Slater S, Stricklin SL, Studholme DJ, Sun J, Viana CJ, Wallin E, Wang B, Wheeler C, Zhu H, Dean DR, Dixon R, Wood D (2009) Genome sequence of Azotobacter vinelandii, an obligate aerobe specialized to support diverse anaerobic metabolic processes. J Bacteriol 191:4534–4545PubMedCrossRefGoogle Scholar
  25. 25.
    Seefeldt LC, Hoffman BM, Dean DR (2009) Mechanism of Mo-dependent nitrogenase. Annu Rev Biochem 78:701–722PubMedCrossRefGoogle Scholar
  26. 26.
    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
  27. 27.
    Burgess BK, Lowe DJ (1996) Mechanism of molybdenum nitrogenase. Chem Rev 96:2983–3012PubMedCrossRefGoogle Scholar
  28. 28.
    Hu Y, Fay AW, Lee CC, Yoshizawa J, Ribbe MW (2008) Assembly of nitrogenase MoFe protein. Biochemistry 47:3973–3981PubMedCrossRefGoogle Scholar
  29. 29.
    Dos Santos PC, Dean DR, Hu Y, Ribbe MW (2004) Formation and insertion of the nitrogenase iron-molybdenum cofactor. Chem Rev 104:1159–1173PubMedCrossRefGoogle Scholar
  30. 30.
    Schwarz G, Mendel RR, Ribbe MW (2009) Molybdenum cofactors, enzymes and pathways. Nature 460:839–847PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Department of Molecular Biology and BiochemistryUniversity of CaliforniaIrvineUSA

Personalised recommendations