Green J, Dalton H
Department of Biological Sciences, University of Warwick, Coventry, United Kingdom.
J Biol Chem. 1988 Nov 25;263(33):17561-5.
Protein A of soluble methane monooxygenase (EC 1.14.13.25) of Methylococcus capsulatus (Bath) is the hydroxylase component of the enzyme complex, capable of inserting an atom of oxygen into methane. The protein possesses an unusual non-heme iron center consisting of two mu-hydroxo-bridged antiferromagnetically coupled iron atoms. It was possible to remove the iron center of protein A by subjecting the protein to freeze/thaw cycles or by dialysis against 8-hydroxyquinoline. Incubation of iron-depleted protein A with iron-EDTA and dithiothreitol resulted in the reassembly of the iron center of protein A as judged by restoration of enzyme activity (typically approximately 3 times the original activity) and by ESR spectroscopic methods. Reconstitution was inhibited by a number of metal ions, but none of the metals tested proved capable of replacing iron in the oxidation of methane. A number of iron-chelating agents exhibited weak inhibition of reconstitution. The KM(app) for iron in the reconstitution reaction was 133 microM. In vitro translation of total RNA isolated from M. capsulatus (Bath) produced protein A subunits equal to the native molecular weight, suggesting that there is no precursor form of this protein. Addition of the in vitro translated protein A to the reconstitution system resulted in low levels of hydroxylase activity suggesting that the subunits of protein A are self-assembling, a supposition supported by the presence of only one protein band on nondenaturing gels. Taken together, these data suggest that the subunits of protein A are synthesized in their native, mature form and self-assemble into an apoprotein A, into which the iron center is inserted.
荚膜甲基球菌（巴斯）可溶性甲烷单加氧酶（EC 1.14.13.25）的蛋白质A是该酶复合物的羟化酶组分，能够将一个氧原子插入甲烷中。该蛋白质具有一个不寻常的非血红素铁中心，由两个μ-羟基桥连的反铁磁耦合铁原子组成。通过对蛋白质进行冻融循环或用8-羟基喹啉透析，可以去除蛋白质A的铁中心。用铁-乙二胺四乙酸和二硫苏糖醇孵育缺铁的蛋白质A，根据酶活性的恢复（通常约为原始活性的3倍）和电子顺磁共振光谱法判断，导致蛋白质A的铁中心重新组装。许多金属离子抑制了重组，但所测试的金属都不能在甲烷氧化中替代铁。一些铁螯合剂对重组表现出微弱的抑制作用。重组反应中铁的表观米氏常数为133微摩尔。从荚膜甲基球菌（巴斯）分离的总RNA的体外翻译产生了与天然分子量相等的蛋白质A亚基，这表明该蛋白质没有前体形式。将体外翻译的蛋白质A添加到重组系统中导致羟化酶活性水平较低，这表明蛋白质A的亚基是自组装的，这一假设得到了非变性凝胶上仅出现一条蛋白带的支持。综上所述，这些数据表明蛋白质A的亚基以其天然、成熟的形式合成，并自组装成脱辅基蛋白A，铁中心插入其中。
