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

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Abstract
Conditions are defined in which the oxygen‐labile nitrogenase components from Azotobacter vinelandii can be protected against oxygen inactivation by the so‐called Fe/S protein II. It is demonstrated that oxygen protection can be achieved by complex formation of the three proteins. Complex formation was studied by gel chromatography. Only when the three proteins are in the oxidized state and MgCl2 is present, can an oxygen‐tolerant complex be isolated. Quantitative SDS/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 [2 Fe‐2 S] clusters present in the protein and not by the amount of protein. Measurements of the amount of iron and sulfide of Fe/S protein II showed that the iron and sulfide content of the protein was variable. The maximum values found indicate that Fe/S protein II contains two [2Fe‐2S] clusters per dimer of 26 kDa. 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 two [2Fe‐2S] clusters per dimer of 26 kDa. When Fe/S protein II contains less [2 Fe‐2S] clusters, more protein is necessary to obtain full protection. The three‐component nitrogenase complex is also oxygen stable in the presence of MgATP or MgADP.Analysis in the ultracentrifuge showed that the major fraction of the reconstituted complex has a sedimentation coefficient centered around 34S. A small fraction (1, 8–12 molecules of Av2 and 4–6 molecules of Fe/S protein II containing two [2Fe‐2S] clusters per dimer of 26 kDa.