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Measurement of the Potential Rates of Dissimilatory Nitrate Reduction to Ammonium Based on 14NH4+/15NH4+ Analyses via Sequential Conversion to N2O
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Protocols for cofactor isolation of nitrogenase.

Aaron W Fay1, Chi-Chung Lee, Jared A Wiig

  • 1Department of Molecular Biology and Biochemistry, University of California, 92697 Irvine, CA, USA. awfay@uci.edu

Methods in Molecular Biology (Clifton, N.J.)
|August 12, 2011
PubMed
Summary
This summary is machine-generated.

Researchers detail a method for isolating the iron-molybdenum cofactor (FeMoco) from nitrogenase MoFe protein. This technique yields catalytically active FeMoco for studying its unique structure and function in dinitrogen reduction.

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Area of Science:

  • Bioinorganic Chemistry
  • Biochemistry
  • Enzyme Catalysis

Background:

  • The iron-molybdenum cofactor (FeMoco) is central to nitrogenase MoFe protein function.
  • FeMoco is the proposed active site for biological dinitrogen reduction.
  • Understanding FeMoco's properties requires its intact isolation.

Purpose of the Study:

  • To describe a robust method for isolating FeMoco from the MoFe protein.
  • To provide a protocol for obtaining catalytically active and spectrally interesting FeMoco.
  • To facilitate future structure-function analyses of this unique cofactor.

Main Methods:

  • Acid treatment of the MoFe protein.
  • Extraction of FeMoco into an organic solvent.
  • Refinements to an established isolation protocol over 30 years.

Main Results:

  • Successful isolation of intact FeMoco.
  • Obtained FeMoco exhibits catalytic activity.
  • Isolated FeMoco is spectrally interesting for analysis.

Conclusions:

  • The described acid-extraction method is effective for FeMoco isolation.
  • Isolated FeMoco serves as a valuable tool for biochemical studies.
  • This protocol supports ongoing research into nitrogenase function.