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Updated: Feb 23, 2026

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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Nitrogenase Assembly: Strategies and Procedures.

Nathaniel S Sickerman¹, Yilin Hu¹, Markus W Ribbe¹

¹University of California, Irvine, Irvine, CA, United States.

Methods in Enzymology

|September 9, 2017

Summary

This study details methods for growing Azotobacter vinelandii and genetically modifying it to understand nitrogenase assembly. These techniques aid in studying biological nitrogen fixation and ammonia production.

Keywords:

Assembly Azotobacter vinelandii Diazotroph Fe protein Genetics MoFe protein Nitrogen fixation Nitrogenase nif

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Published on: September 17, 2017

Area of Science:

Biochemistry
Microbiology
Enzymology

Background:

Nitrogenase is crucial for biological nitrogen fixation, converting atmospheric dinitrogen to ammonia.
Understanding the assembly of nitrogenase metallocenters is key to its catalytic function.
Azotobacter vinelandii is a model organism for studying nitrogenase due to its culturability and genetic tractability.

Purpose of the Study:

To provide essential protocols for the study of nitrogenase assembly and function.
To outline methods for culturing and genetically manipulating Azotobacter vinelandii.
To describe techniques for nitrogenase protein purification and activity assays.

Main Methods:

Culturing of Azotobacter vinelandii.
Recombinant transformation and protein expression in A. vinelandii.
Purification of nitrogenase proteins.
Enzyme activity assays for substrate reduction.

Main Results:

Established protocols for A. vinelandii manipulation and nitrogenase expression.
Developed methods for purifying nitrogenase components.
Validated assays for assessing nitrogenase catalytic activity.

Conclusions:

The described methods provide a robust framework for investigating nitrogenase assembly and catalysis.
These protocols facilitate research into biological nitrogen fixation mechanisms.
Further studies can utilize these methods to explore nitrogenase function and engineering.