A facile method of isolation for the iron molybdenum cofactor of nitrogenase and bacterial ferritin from extracts of azotobacter vinelandii

An alternative method has been developed for the isolation of both the iron molybdenum cofactor of nitrogenase (FeMoco), a small molecular weight FeMoS cluster which is the putative nitrogen- reducing site of the enzyme, and bacterioferritin, an iron storage protein similar to other ferritins, but containing heme prosthetic groups. Previously the isolation of these two species, the characterization of which is of significant current interest, has been dependent on the purification of the nitrogenase enzyme from Azotobacter vinelandii. Out new procedure eliminates the use of the anaerobic column chromatography necessary to obtain pure nitrogenase components, involving instead the heat and RNAase/ DNAase treatment of crude extracts of ruptured cells followed by sedimentation (150000 × g for 18 h) of both the 'nitrogenase complex' and bacterioferritin. The redissolved pellet from this centrifugation yields the pure crystalline bacterioferritin on addition of Mg²⁺. and cooling, the iron content of the protein being higher by this method than in previous reports. Likewise, denaturation by acid/base treatment of this protein mixture yields a precipitate which can be extracted with either N-methylformamide or N,N-dimethylformamide containing dithionite ion to yield solutions of FeMoco, as evidenced by UW 45 reconstitution and EPR spectral criteria. Unfortunately, preparations of FeMoco obtained by this method have a variable, but consistently low, Fe/Mo ratio and additional visible spectral features, indicating that they are significantly less pure than that those generated from purified nitrogenase. The aqueous supernatant from the denaturation also yields bacterioferritin, but with a lower iron content than that from the direct crystallization method.