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Crystallization of Nitrogenase Proteins

  • Belinda B. Wenke
  • Renee J. Arias
  • Thomas Spatzal
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1876)

Abstract

Nitrogenase is the only known enzymatic system capable of reducing atmospheric dinitrogen to ammonia. This unique reaction requires tightly choreographed interactions between the nitrogenase component proteins, the molybdenum–iron (MoFe)- and iron (Fe)-proteins, as well as regulation of electron transfer between multiple metal centers that are only found in these components. Several decades of research beginning in the 1950s yielded substantial information of how nitrogenase manages the task of N2 fixation. However, key mechanistic steps in this highly oxygen-sensitive and ATP-intensive reaction have only recently been identified at an atomic level. A critical part in any mechanistic elucidation is the necessity to connect spectroscopic and functional properties of the component proteins to the detailed three-dimensional structures. Structural information derived from X-ray diffraction (XRD) methods has provided detailed atomic insights into the enzyme system and, in particular, its active site FeMo-cofactor. The following chapter outlines the general protocols for the crystallization of Azotobacter vinelandii (Av) nitrogenase component proteins, with a special emphasis on different applications, such as high-resolution XRD, single-crystal spectroscopy, and the structural characterization of bound inhibitors.

Key words

Nitrogenase MoFe protein (Av1) Fe protein (Av2) Crystallization X-ray diffraction (XRD) Single-crystal spectroscopy 

Notes

Acknowledgments

The authors thank Douglas C. Rees and James B. Howard for their support. The authors are supported by the National Institute of Health grant GM45162 and NIH/NRSA training grant 5 T32 GM07616.

References

  1. 1.
    Burgess BK, Lowe DJ (1996) Mechanism of molybdenum nitrogenase. Chem Rev 96:2983–3012CrossRefGoogle Scholar
  2. 2.
    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 U S A 103:17088–17093CrossRefGoogle Scholar
  3. 3.
    Howard JB, Rees DC (1996) Structural basis of biological nitrogen fixation. Chem Rev 96:2965–2982CrossRefGoogle Scholar
  4. 4.
    Kim J, Rees D (1992) Structural models for the metal centers in the nitrogenase molybdenum-iron protein. Science 257:1677–1683CrossRefGoogle Scholar
  5. 5.
    Spatzal T, Aksoyoglu M, Zhang L et al (2011) Evidence for interstitial carbon in nitrogenase FeMo cofactor. Science 334:940CrossRefGoogle Scholar
  6. 6.
    Georgiadis MM, Komiya H, Chakrabarti P et al (1992) Crystallographic structure of the nitrogenase iron protein from Azotobacter vinelandii. Science 257:1653–1659CrossRefGoogle Scholar
  7. 7.
    Schindelin H, Kisker C, Schlessman JL et al (1997) Structure of ADP·AlF4-stabilized nitrogenase complex and its implications for signal transduction. Nature 387:370–376CrossRefGoogle Scholar
  8. 8.
    Tezcan FA, Kaiser JT, Howard JB et al (2015) Structural evidence for asymmetrical nucleotide interactions in nitrogenase. J Am Chem Soc 137:146–149CrossRefGoogle Scholar
  9. 9.
    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 297:1696–1700CrossRefGoogle Scholar
  10. 10.
    Spatzal T, Perez KA, Einsle O et al (2014) Ligand binding to the FeMo-cofactor: structures of CO-bound and reactivated nitrogenase. Science 345:1620–1623CrossRefGoogle Scholar
  11. 11.
    Spatzal T, Perez KA, Howard JB et al (2015) Catalysis-dependent selenium incorporation and migration in the nitrogenase active site iron-molybdenum cofactor. elife 4:e11620CrossRefGoogle Scholar
  12. 12.
    Drenth J, Hol W, Wierenga R (1975) Crystallization and preliminary x-ray investigation of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens. J Biol Chem 250:5268–5269PubMedGoogle Scholar
  13. 13.
    Schmid B, Einsle O, Chiu HJ et al (2002) Biochemical and structural characterization of the cross-linked complex of nitrogenase: comparison to the ADP-AlF4(−)-stabilized structure. Biochemistry 41:15557–15565CrossRefGoogle Scholar
  14. 14.
    Rees D, Howard JB (1983) Crystallization of the Azotobacter vinelandii nitrogenase iron protein. J Biol Chem 258:12733–12734PubMedGoogle Scholar
  15. 15.
    Schlessman JL, Woo D, Joshua-Tor L et al (1998) Conformational variability in structures of the nitrogenase iron proteins from Azotobacter vinelandii and Clostridium pasteurianum 1. J Mol Biol 280:669–685CrossRefGoogle Scholar
  16. 16.
    Strop P, Takahara PM, Chiu H et al (2001) Crystal structure of the all-ferrous [4Fe-4S]0 form of the nitrogenase iron protein from Azotobacter vinelandii. Biochemistry 40:651–656CrossRefGoogle Scholar
  17. 17.
    Yang KY, Haynes CA, Spatzal T et al (2014) Turnover-dependent inactivation of the nitrogenase MoFe-protein at high pH. Biochemistry 53:333–343CrossRefGoogle Scholar
  18. 18.
    Wolle D, Kim C, Dean D et al (1992) Ionic interactions in the nitrogenase complex. Properties of Fe-protein containing substitutions for Arg-100. J Biol Chem 267:3667–3673PubMedGoogle Scholar
  19. 19.
    Renner KA, Howard JB (1996) Aluminum fluoride inhibition of nitrogenase: stabilization of a nucleotide·Fe-protein·MoFe-protein complex. Biochemistry 35:5353–5358CrossRefGoogle Scholar
  20. 20.
    Martin RB (1988) Ternary hydroxide complexes in neutral solutions of Al3+ and F. Biochem Biophys Res Commun 155:1194–1200CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Belinda B. Wenke
    • 1
  • Renee J. Arias
    • 1
  • Thomas Spatzal
    • 1
  1. 1.Division of Chemistry and Chemical EngineeringCalifornia Institute of TechnologyPasadenaUSA

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