Scherings G, Haaker H, Wassink H, Veeger C
Eur J Biochem. 1983 Oct 3;135(3):591-9. doi: 10.1111/j.1432-1033.1983.tb07693.x.
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-2S] 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 [2Fe-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 (less than 30%) sediments with values centered around 111 S. This suggests an average mass for the oxygen-stable nitrogenase complex of 1.5 MDa. Taking into account the determined stoichiometry of the individual proteins, the molecular composition of the oxygen-stable nitrogenase complex is presumably 4 molecules of AV1,8--12 molecules of aAV2 and 4--6 molecules of Fe/S protein II containing two [2Fe-2S] clusters per dimer of 26 kDa.
定义了一些条件，在这些条件下，来自棕色固氮菌的对氧敏感的固氮酶组分可以受到所谓的铁硫蛋白II的保护，防止被氧灭活。结果表明，通过三种蛋白质形成复合物可以实现氧保护。通过凝胶色谱法研究了复合物的形成。只有当三种蛋白质处于氧化态且存在MgCl2时，才能分离出耐氧复合物。对这种复合物进行定量SDS/聚丙烯酰胺凝胶电泳，得到固氮酶组分2/固氮酶组分1（Av2/Av1）的平均比值为2.4±0.5。铁硫蛋白II的保护作用与该蛋白质中存在的[2Fe-2S]簇的数量相关，而不是与蛋白质的数量相关。对铁硫蛋白II中铁和硫化物含量的测量表明，该蛋白质的铁和硫化物含量是可变的。所发现的最大值表明，铁硫蛋白II每26 kDa二聚体含有两个[2Fe-2S]簇。当铁硫蛋白II与Av1的比例为1.1±0.2时，可获得铁硫蛋白II的完全保护；该铁硫蛋白II每26 kDa二聚体含有两个[2Fe-2S]簇。当铁硫蛋白II含有较少的[2Fe-2S]簇时，需要更多的蛋白质才能获得完全保护。在MgATP或MgADP存在下，三组分固氮酶复合物对氧也稳定。超速离心分析表明，重组复合物的主要部分沉降系数集中在34S左右。一小部分（小于30%）沉降系数集中在111S左右。这表明耐氧固氮酶复合物的平均质量为1.5 MDa。考虑到所确定的各个蛋白质的化学计量，耐氧固氮酶复合物的分子组成可能是4个AV1分子、8 - 12个aAV2分子和4 - 6个铁硫蛋白II分子，每26 kDa二聚体含有两个[2Fe-2S]簇。
