Crystallization of Nitrogenase Proteins

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


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 



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.


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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
    Email author
  1. 1.Division of Chemistry and Chemical EngineeringCalifornia Institute of TechnologyPasadenaUSA

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