Advertisement

Assembly of Nitrogenase MoFe Protein

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

Abstract

Biosynthesis of MoFe protein and, particularly, that of its associated P-cluster and FeMoco has raised a significant amount of interest because of the biological importance and chemical exclusiveness of these unique clusters. Following a brief introduction to the properties of Azotobacter vinelandii MoFe protein, this chapter will focus on the recent progress toward understanding the assembly mechanism of MoFe protein, with an emphasis on studies that provide important structural or spectroscopic insights into this process.

Key words

Nitrogenase MoFe protein NifEN FeMoco P-cluster assembly 

Notes

Acknowledgments

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.).

References

  1. 1.
    Burgess BK, Lowe DJ (1996) Mechanism of molybdenum nitrogenase. Chem Rev 96:2983–3012PubMedCrossRefGoogle Scholar
  2. 2.
    Howard JB, Rees DC (1996) Structural basis of biological nitrogen fixation. Chem Rev 96:2965–2982PubMedCrossRefGoogle Scholar
  3. 3.
    Smith BE (1999) Structure, function, and biosynthesis of the metallosulfur clusters in nitrogenases. Adv Inorg Chem 47:159–218CrossRefGoogle Scholar
  4. 4.
    Rees DC, Tezcan FA, Haynes CA et al (2005) Structural basis of biological nitrogen fixation. Philos Trans R Soc London, Ser A 363:971–984CrossRefGoogle Scholar
  5. 5.
    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
  6. 6.
    Schwarz G, Mendel RR, Ribbe MW (2009) Molybdenum cofactors, enzymes and pathways. Nature 460:839–847PubMedCrossRefGoogle Scholar
  7. 7.
    Hu Y, Fay AW, Lee CC et al (2008) Assembly of nitrogenase MoFe protein. Biochemistry 47:3973–3981PubMedCrossRefGoogle Scholar
  8. 8.
    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
  9. 9.
    Kim J, Rees DC (1992) Crystallographic structure and functional implications of the nitrogenase molybdenum iron protein from Azotobacter vinelandii. Nature 360:553–560CrossRefGoogle Scholar
  10. 10.
    Chan MK, Kim J, Rees DC (1993) The nitrogenase FeMo-cofactor and P-cluster pair: 2.2 Å resolution structures. Science 260:792–794PubMedCrossRefGoogle Scholar
  11. 11.
    Peters JW, Stowell MH, Soltis SM et al (1997) Redox-dependent structural changes in the nitrogenase P-cluster. Biochemistry 36:1181–1187PubMedCrossRefGoogle Scholar
  12. 12.
    Zimmermann R, Orme-Johnson WH, Münck E et al (1978) Nitrogenase-X: Mössbauer and EPR studies on reversibly oxidized MoFe protein from Azotobacter vinelandii OP: Nature of iron centers. Biochim Biophys Acta 537:185–207PubMedGoogle Scholar
  13. 13.
    Surerus KK, Hendrich MP, Christie PD et al (1992) Mössbauer and integer-spin EPR of the oxidized P-clusters of nitrogenase: Pox is a non-Kramers system with a nearly degenerate ground doublet. J Am Chem Soc 114:8579–8590CrossRefGoogle Scholar
  14. 14.
    Watt GD, Burns A, Lough S et al (1980) Redox and spectroscopic properties of oxidized MoFe protein from Azotobacter vinelandii. Biochemistry 19:4926–4932PubMedCrossRefGoogle Scholar
  15. 15.
    Pierik AJ, Wassink H, Haaker H et al (1993) Redox properties and EPR spectroscopy of the P-clusters of Azotobacter vinelandii MoFe Protein. Eur J Biochem 212:51–61PubMedCrossRefGoogle Scholar
  16. 16.
    Shah VK, Brill WJ (1977) Isolation of an iron-molybdenum cofactor from nitrogenase. Proc Natl Acad Sci USA 74:3249–3253PubMedCrossRefGoogle Scholar
  17. 17.
    Burgess BK (1990) The iron molybdenum cofactor of nitrogenase. Chem Rev 90:1377–1406CrossRefGoogle Scholar
  18. 18.
    Dos Santos PC, Dean DR, Hu YL et al (2004) Formation and insertion of the nitrogenase iron-molybdenum cofactor. Chem Rev 104:1159–1173PubMedCrossRefGoogle Scholar
  19. 19.
    Frazzon J, Dean DR (2002) Biosynthesis of the nitrogenase iron-molybdenum-cofactor from Azotobacter vinelandii. Met Ions Biol Syst 39:163–186PubMedGoogle Scholar
  20. 20.
    Yuvaniyama P, Agar JN, Cash VL et al (2000) NifS-directed assembly of a transient [2Fe-2S] cluster within the NifU protein. Proc Natl Acad Sci USA 97:599–604PubMedCrossRefGoogle Scholar
  21. 21.
    Agar JN, Yuvaniyama P, Jack RF et al (2000) Modular organization and identification of a mononuclear iron-binding site within the NifU protein. J Biol Inorg Chem 5:167–177PubMedCrossRefGoogle Scholar
  22. 22.
    Dos Santos PC, Smith AD, Frazzon J et al (2004) Iron-sulfur cluster assembly: NifU-directed activation of the nitrogenase Fe protein. J Biol Chem 279:19705–19711PubMedCrossRefGoogle Scholar
  23. 23.
    Smith AD, Jameson GNL, Dos Santos PC et al (2005) NifS-mediated assembly of [4Fe-4S] clusters in the N- and C-terminal domains of the NifU scaffold protein. Biochemistry 44:12955–12969PubMedCrossRefGoogle Scholar
  24. 24.
    Allen RM, Chatterjee R, Ludden PW et al (1995) Incorporation of iron and sulfur from NifB cofactor into the iron-molybdenum cofactor of dinitrogenase. J Biol Chem 270:26890–26896PubMedCrossRefGoogle Scholar
  25. 25.
    Dean DR, Jacobson MR (1992) Biochemical genetics of nitrogenase. In: Stacey G, Burris RH, Evans HJ (eds) Biological Nitrogen Fixation, pp. 763–834. Chapman & Hall, New York, NYGoogle Scholar
  26. 26.
    Curatti L, Hernandez JA, Igarashi RY et al (2007) In vitro synthesis of the iron-molybdenum cofactor of nitrogenase from iron, sulfur, molybdenum, and homocitrate using purified proteins. Proc Natl Acad Sci USA 104:17626–17631PubMedCrossRefGoogle Scholar
  27. 27.
    Curatti L, Ludden PW, Rubio LM (2006) NifB-dependent in vitro synthesis of the iron-molybdenum cofactor of nitrogenase. Proc Natl Acad Sci USA 103:5297–5301PubMedCrossRefGoogle Scholar
  28. 28.
    Roll JT, Shah VK, Dean DR et al (1995) Characteristics of NifNE in Azotobacter vinelandii strains: Implications for the synthesis of the iron-molybdenum cofactor of dinitrogenase. J Biol Chem 270:4432–4437PubMedCrossRefGoogle Scholar
  29. 29.
    Goodwin PJ, Agar JN, Roll JT et al (1998) The Azotobacter vinelandii NifEN complex contains two identical [4Fe-4S] clusters. Biochemistry 37:10420–10428PubMedCrossRefGoogle Scholar
  30. 30.
    Brigle KE, Weiss MC, Newton WE et al (1987) Products of the iron-molybdenum cofactor-specific biosynthetic genes, nifE and nifN, are structurally homologous to the products of the nitrogenase molybdenum-iron protein genes, nifD and nifK. J Bacteriol 169:1547–1553PubMedGoogle Scholar
  31. 31.
    Hu Y, Fay AW, Ribbe MW (2005) Identification of a nitrogenase iron-molybdenum cofactor precursor on NifEN complex. Proc Natl Acad Sci USA 102:3236–3241PubMedCrossRefGoogle Scholar
  32. 32.
    Corbett MC, Hu Y, Fay AW et al (2006) Structural insights into a protein-bound iron-molybdenum cofactor precursor. Proc Natl Acad Sci USA 103:1238–1243PubMedCrossRefGoogle Scholar
  33. 33.
    Hu Y, Corbett MC, Fay AW et al (2006) FeMo cofactor maturation on NifEN. Proc Natl Acad Sci USA 103:17119–17124PubMedCrossRefGoogle Scholar
  34. 34.
    Yoshizawa JM, Blank MA, Fay AW et al (2009) Optimization of FeMoco maturation on NifEN. J Am Chem Soc 131:9321–9325PubMedCrossRefGoogle Scholar
  35. 35.
    Hu Y, Corbett MC, Fay AW et al (2006) Nitrogenase Fe protein: A molybdate/homocitrate insertase. Proc Natl Acad Sci USA 103:17125–17130PubMedCrossRefGoogle Scholar
  36. 36.
    Homer MJ, Dean DR, Roberts GP (1995) Characterization of the gamma-protein and its involvement in the metallocluster assembly and maturation of dinitrogenase from Azotobacter vinelandii. J Biol Chem 270:24745–24752PubMedCrossRefGoogle Scholar
  37. 37.
    White TC, Harris GS, Orme-Johnson W (1992) Electrophoretic studies on the assembly of the nitrogenase molybdenum-iron protein from the Klebsiella pneumoniae nifD and nifK gene-products. J Biol Chem 267:24007–24016PubMedGoogle Scholar
  38. 38.
    Homer MJ, Paustian TD, Shah VK et al (1993) The NifY product of Klebsiella pneumoniae is associated with apodinitrogenase and dissociates upon activation with the iron-molybdenum cofactor. J Bacteriol 175:4907–4910PubMedGoogle Scholar
  39. 39.
    Hernandez JA, Igarashi RY, Soboh B et al (2007) NifX and NifEN exchange NifB cofactor and the VK-cluster, a newly isolated intermediate of the iron-molybdenum cofactor biosynthetic pathway. Mol Microbiol 63:177–192PubMedCrossRefGoogle Scholar
  40. 40.
    Ribbe MW, Burgess BK (2001) The chaperone GroEL is required for the final assembly of the molybdenum-iron protein of nitrogenase. Proc Natl Acad Sci USA 98:5521–5525PubMedCrossRefGoogle Scholar
  41. 41.
    Fay AW, Blank MA, Yoshizawa JM et al (2010) Formation of a homocitrate-free iron-molybdenum cluster on NifEN: implications for the role of homocitrate in nitrogenase assembly. Dalton Trans 39:3124–3130PubMedCrossRefGoogle Scholar
  42. 42.
    Schmid B, Ribbe MW, Einsle O et al (2002) Structure of a cofactor-deficient nitrogenase MoFe protein. Science 296:352–356PubMedCrossRefGoogle Scholar
  43. 43.
    Hu Y, Fay AW, Schmid B et al (2006) Molecular insights into nitrogenase FeMoco insertion: Trp 444 of MoFe protein α subunit locks FeMoco in its binding site. J Biol Chem 281:30534–30541PubMedCrossRefGoogle Scholar
  44. 44.
    Hu Y, Fay AW, Ribbe MW (2007) Molecular insights into nitrogenase FeMoco insertion: The role of His362 of MoFe protein α subunit in FeMoco incorporation. J Biol Inorg Chem 12:449–460PubMedCrossRefGoogle Scholar
  45. 45.
    Fay AW, Hu Y, Schmid B et al (2007) Molecular insights into nitrogenase FeMoco insertion: The role of His 274 and His 451 of MoFe protein α subunit. J Inorg Biochem 101:1630–1641PubMedCrossRefGoogle Scholar
  46. 46.
    Corbett MC, Hu Y, Fay AW et al (2007) Conformational differences between Azotobacter vinelandii nitrogenase MoFe proteins as studied by small angle X-ray scattering. Biochemistry 46:8066–8074PubMedCrossRefGoogle Scholar
  47. 47.
    Lee SC, Holm RH (2004) The clusters of nitrogenase: Synthetic methodology in the construction of weak-field clusters. Chem Rev 104:1135–1158PubMedCrossRefGoogle Scholar
  48. 48.
    Ribbe MW, Hu Y, Guo M et al (2002) The FeMoco-deficient MoFe protein produced by a nifH-deletion strain of Azotobacter vinelandii shows unusual P-cluster features. J Biol Chem 277:23469–23476PubMedCrossRefGoogle Scholar
  49. 49.
    Corbett MC, Hu Y, Naderi F et al (2004) Comparison of iron-molybdenum cofactor deficient nitrogenase MoFe proteins by X-ray absorption spectroscopy: Implications for P-cluster biosynthesis. J Biol Chem 279:28276–28282PubMedCrossRefGoogle Scholar
  50. 50.
    Broach RB, Rupnik K, Hu Y et al (2006) VTVH-MCD spectroscopic study of the metal clusters in the ΔnifB and ΔnifH MoFe proteins of nitrogenase from Azotobacter vinelandii. Biochemistry 45:15039–15048PubMedCrossRefGoogle Scholar
  51. 51.
    Lee CC, Blank MA, Fay AW et al (2010) Stepwise formation of P-cluster in nitrogenase MoFe protein. Proc Natl Acad Sci USA 106:18474–18478CrossRefGoogle Scholar
  52. 52.
    Hu Y, Fay AW, Dos Santos PC et al (2004) Characterization of Azotobacter vinelandii nifZ-deletion strains: Indication of stepwise MoFe protein assembly. J Biol Chem 279:54963–54971PubMedCrossRefGoogle Scholar
  53. 53.
    Cotton MS, Rupnik K, Broach RB et al (2009) VTVH-MCD study of the ΔnifBΔnifZ MoFe protein from Azotobacter vinelandii. J Am Chem Soc 131:4558–4559PubMedCrossRefGoogle Scholar
  54. 54.
    Hu Y, Fay AW, Lee CC et al (2007) P-Cluster maturation on nitrogenase MoFe protein. Proc Natl Acad Sci USA 104:10424–10429PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

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

Personalised recommendations