尿素对钼铁蛋白的活性和金属原子簇稳定性的影响

THE EFFECT OF UREA ON THE ACTIVITY AND STABILITY OF METALLOCLUSTER OF THE MoFe PROTEIN OF NITROGENASE FROM AZOTOBACTER VINELANDII

当尿素浓度高于０．５ ｍ ｏＬ／Ｌ时，棕色固氮菌（Ａｚｏｔｏｂａｃｔｅｒｖｉｎｅｌａｎｄｉｉ）固氮酶钼铁蛋白的乙炔还原活性呈指数下降；而经厌氧缓冲系统稀释并保温后，又可得到明显恢复。尿素对邻菲口罗啉从还原的或部分缺失Ｐ－ｃｌｕｓｔｅｒ 的ＭｏＦｅ 蛋白中螯合金属原子簇的Ｆｅ 原子均有较大的促进作用。还原ＭｏＦｅ 蛋白在尿素梯度凝胶电泳中的迁移率：在０～１．５ ｍ ｏｌ／Ｌ内，无明显变化；在１．５～５．０ ｍ ｏｌ／Ｌ内，线性变小；在５．０～８．０ ｍ ｏｌ／Ｌ内，则呈平缓状态。结果表明：（１）尿素对不同状态的ＭｏＦｅ 蛋白金属原子簇的影响程度不尽相同，而对蛋白质的变构作用是尿素影响ＭｏＦｅ 蛋白的活性和金属原子簇稳定性的主要原因；（２）ＭｏＦｅ 蛋白的构象与金属原子簇密切相关；（３）ＭｏＦｅ 蛋白在变性过程中。

When the MoFe protein from Azotobacter vinelandii was treated with more than 0.5 mol/L of urea anaerobically,the C 2H 2 reduction activity of the treated protein was exponentially decreased.However,the activity could be significantly restored after the treated protein was diluted with the buffer system and followed by incubation at 15 ℃.The urea had remarkable enhancement on chelation of Fe atoms from the reducted MoFe protein and the P cluster deficient MoFe protein by O phenanthroline(O phen),respectively.The migration of the reducted MoFe protein on the urea gradient gel electrophoresis did not significantly change from 0 to 1.5 mol/L urea ,linearly became smaller from 1.5 to 5.0 mol/L urea,and reached a stable state from 5.0 to 8.0 mol/L urea.The results indicated that:(1) The effect of urea on the activity and the stability of metallocluster of the MoFe protoin was mainly attributed to the conformational change of the protein in urea,moveover,the effect of urea on the metallocluster might not be all the same if the states of MoFe protein were different;(2) There was a close relationship between the conformation and the metalloclusters of MoFe protein;(3) In the course of the denaturation,the decrease in the activity of MoFe protein probably happened prior to the conformational change of the whole molecule.