Expression, Isolation, and Characterization of Vanadium Nitrogenase from Azotobacter vinelandii

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.