Analytical ultracentrifugation of the nitrogenase of Azotobacter vinelandii under anaerobic conditions

Abstract

Nitrogenase (Azotobacter vinelandii) is a high-molecular-mass enzyme complex responsible for the fixation and metabolization of nitrogen. The complex consists of two different moieties, a larger MoFe protein (240 kDa) and a smaller Fe protein (60 kDa). The stoichiometry and affinity of both components were studied in solution by sedimentation equilibrium in an XL-A analytical ultracentrifuge. Because both components are highly sensitive to oxygen, the experiments were carried out in an argon atmosphere. Data analysis performed using the program Polymole yielded a 2:1 stoichiometry of Fe protein to MoFe protein. Assuming two independent binding sites on the MoFe protein, the association constant for the first Fe protein bound was (1.99 ± 0.48) x 10⁷ M^-1