On the formation of an oxygen‐tolerant three‐component nitrogenase complex from Azotobacter vinelandii

Abstract

Conditions are defined in which the O2-labile nitrogenase components from A. vinelandii can be protected against O2 inactivation by the Fe/S protein II. O2 protection can be achieved by complex formation of the 3 proteins. Complex formation was studied by gel chromatography. Only when the 3 proteins are in the oxidized state and MgCl2 is present can an O2-tolerant complex be isolated. Quantitative sodium dodecyl sulfate/polyacrylamide gel electrophoresis of such complexes yielded an average ratio of nitrogenase component 2/nitrogenase component 1 (Av2/Av1) of 2.4 .+-. 0.5. Protection by Fe/S protein II was correlated with the amount of [2Fe-2S] clusters present in the protein and not by the amount of protein. Measurements of the amount of Fe and sulfide of Fe/S protein II showed that the Fe and sulfide content of the protein was variable. The maximum values found indicate that Fe/S protein II contains 2 [2 Fe-2S] clusters per dimer of 26 kDa (kilodaltons). Full protection by Fe/S protein II was obtained with a ratio of Fe/S protein iI/Av1 of 1.1 .+-. 0.2, the Fe/S protein II containing 2 [2Fe-2S] clusters per dimer of 26 kDa. When Fe/S protein II contained fewer [2Fe-2S] clusters, more protein was necessary to obtain full protection. The 3-component nitrogenase complex was also O2 stable in the presence of MgATP or mgADP. Analysis of the ultracentrifuge showed that the major fraction of the reconstituted complex has a sedimentation coefficient of .apprx. 34 S. A small fraction (< 30%) sediments at .apprx. 111 S. This suggests an average mass for the O2-stable nitrogenase complex of 1.5 megadaltons. Taking into account the determined stoichiometry of the individual proteins, the molecular composition of the O2-stable nitrogenase complex is presumably 4 molecules of Av1, 8-12 molecules of Av2 and 4-6 molecules of Fe/S protein II containing 2 [2Fe-2S] clusters per dimer of 26 kDa.