Purification and Properties of Nitrogenase from the Cyanobacterium, Anabaena cylindrica

Abstract

The nitrogenase [EC 1.7.99.2] complex was isolated from N-starved cultures of A. cylindrica. Sodium dithionite, photochemically reduced ferredoxin, and NADPH were effective electron donors to nitrogenase in crude extracts whereas H2 and pyruvate were not. The Km for acetylene in vivo is 10-fold higher than the Km in vitro, whereas this pattern does not hold for the non-heterocystous cyanobacterium, Plectonema boryanum. At least 1 mechanism of O2 protection in vivo involves a gas diffusion barrier presented by the heterocyst cell wall. The Mo-Fe component was purified to homogeneity. Its MW (220,000), subunit composition, isoelectric point (4.8), Mo, Fe, and S2- content (2, 20 and 20 mol/mol component), and amino acid composition indicate that this component has similar properties to Mo-Fe-containing components isolated from other bacterial sources. The isolated components from A. cylindrica cross-reacted, to varying degrees, with components isolated from Azotobacter vinelandii, Rhodospirillum rubrum, and P. boryanum.