Zhang J H, Kurtz D M, Xia Y M, Debrunner P G
Department of Chemistry, University of Georgia, Athens 30602.
Biochim Biophys Acta. 1992 Aug 21;1122(3):293-8. doi: 10.1016/0167-4838(92)90407-5.
A recent report from this laboratory (Zhang, J.-H., Kurtz, D.M., Jr., Xia, Y.-M. and Debrunner, P.G. (1991) Biochemistry 30, 583-589) described a procedure for reconstitution of a functional di-iron site in the octameric, non-heme iron O2-carrying protein, hemerythrin by addition of ferrous salts to apoprotein, followed by slow dilution of the denaturant. Although the resulting protein contained its full complement of iron, i.e., 2 Fe per subunit, about 30% of the iron was found to remain ferrous under ambient O2, i.e., this iron was incapable of forming an O2 adduct. In this report a method is described for obtaining essentially fully functional hemerythrin by passage of the freshly reconstituted protein through an [oxy/30% non-functional----met----deoxy----oxy redox cycle. UV/vis absorption and 57Fe Mössbauer spectroscopies show that little or no non-functional iron remains in the reconstituted oxyhemerythrin after the redox cycle. Quantitations of protein and diiron sites show that, during the first step of the redox cycle, the non-functional iron is converted to a form that is spectroscopically indistinguishable from that of native methemerythrin. Far-UV circular dichroism shows that the secondary structure of this reconstituted methemerythrin is essentially identical to that of native protein. Non-denaturing polyacrylamide gel electrophoresis shows that the size and charge of the native and reconstituted proteins before and after redox cycling are essentially identical. These results indicate that the non-functional iron is converted to a functional form by the redox cycling, and that the key step in this conversion is the [oxy/30% non-functional]----met transformation.
本实验室最近的一份报告（Zhang, J.-H., Kurtz, D.M., Jr., Xia, Y.-M. 和 Debrunner, P.G. (1991) Biochemistry 30, 583 - 589）描述了一种在八聚体、非血红素铁的氧携带蛋白蚯蚓血红蛋白中重建功能性双铁位点的方法，即向脱辅基蛋白中添加亚铁盐，随后缓慢稀释变性剂。尽管所得蛋白质含有其全部的铁，即每个亚基含2个铁原子，但发现在环境氧气下约30%的铁仍为亚铁状态，也就是说这种铁无法形成氧加合物。在本报告中，描述了一种通过使新重建的蛋白质经历[氧/30%非功能性----高铁----脱氧----氧氧化还原循环]来获得基本完全功能性蚯蚓血红蛋白的方法。紫外/可见吸收光谱和57Fe穆斯堡尔光谱表明，在氧化还原循环后，重建的氧合蚯蚓血红蛋白中几乎没有非功能性铁残留。蛋白质和双铁位点的定量分析表明，在氧化还原循环的第一步中，非功能性铁转化为一种在光谱上与天然高铁蚯蚓血红蛋白无法区分的形式。远紫外圆二色性表明，这种重建的高铁蚯蚓血红蛋白的二级结构与天然蛋白质基本相同。非变性聚丙烯酰胺凝胶电泳表明，氧化还原循环前后天然蛋白质和重建蛋白质的大小和电荷基本相同。这些结果表明，非功能性铁通过氧化还原循环转化为功能性形式，并且这种转化的关键步骤是[氧/30%非功能性]----高铁转变。
