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Nitrogenases are the sole enzymes known to mediate biological nitrogen fixation, an essential process for sustaining life on earth. Among the three known variants, molybdenum nitrogenase is the best-studied to date. Recent work on the alternative vanadium nitrogenase provided important insights into the mechanism of nitrogen fixation since this enzyme differs from its molybdenum counterpart in some important aspects. Here, we present a protocol to obtain unmodified vanadium nitrogenase in high yield and purity from the paradigmatic diazotroph Azotobacter vinelandii, including procedures for cell cultivation, purification, and protein characterization., (© 2021. Springer Science+Business Media, LLC, part of Springer Nature.)

A major hurdle in the studies of nitrogenase, one of the most complicated metalloenzymes known to date, is to obtain large amounts of intact, active proteins. Nitrogenase and related proteins are often multimeric and consist of metal centers that are critical for their activities. Most notably, the well-studied MoFe protein of Mo-nitrogenase is a heterotetramer that houses two of the most complicated metal clusters found in nature, the P-cluster and the FeMoco (or M-cluster). The structural complexity of these proteins and the oxygen sensitivity of their associated metal clusters, along with the demand for large amounts of high-quality proteins in most downstream analyses, make large-scale, high-yield purification of fully competent nitrogenase proteins a formidable task and yet, at the same time, a prerequisite for the success of nitrogenase research. This chapter highlights several methods that have been developed over the past few decades chiefly for the purification of naturally expressed nitrogenase in the diazotroph Azotobacter vinelandii. In addition, purification and Fe-S reconstitution strategies are also outlined for the heterologously expressed nitrogenase proteins in Escherichia coli.

Biological nitrogen fixation, the conversion of dinitrogen (N 2 ) into ammonia (NH 3 ), stands as a particularly challenging chemical process. As the entry point into a bioavailable form of nitrogen, biological nitrogen fixation is a critical step in the global nitrogen cycle. In Nature, only one enzyme, nitrogenase, is competent in performing this reaction. Study of this complex metalloenzyme has revealed a potent substrate reduction system that utilizes some of the most sophisticated metalloclusters known. This chapter discusses the structure and function of nitrogenase, covers methods that have proven useful in the elucidation of enzyme properties, and provides an overview of the three known nitrogenase variants.