Characterization of an oxygen-stable nitrogenase complex isolated from Azotobacter chroococcum

Abstract

In crude cell-free extracts of Azotobacter chroococcum, nitrogenase was much less sensitive to irreversible inactivation by O2 than was the purified enzyme. When nitrogenase was partially purified by anaerobic discontinuous sucrose-density-gradient centrifugation, O2-tolerance was retained. This preparation was considerably enriched in four polypeptides, three of which were derived from the Mo-Fe(molybdenum-iron) protein and Fe (iron) protein of nitrogenase. The fourth was purified to homogeneity and shown to be an iron-sulphur protein (mol.wt. 14000) probably containing a 2Fe–2S centre. When this protein was added to purified nitrogenase, the enzyme was rendered O2-tolerant, through stabilization was Mg2+-dependent. The isolated O2-tolerant nitrogenase was an equimolar stoicheiometric complex between the MO–Fe, Fe and protective proteins. It is likely that the formation of this complex in vivo is the mechanism of ‘conformational protection’ in this organism.