Mulliez E, Padovani D, Atta M, Alcouffe C, Fontecave M
Laboratoire de Chimie et Biochimie des Centres Rédox Biologiques, DBMS-CB, CEA/CNRS/Université Joseph Fourier, 17, avenue des Martyrs, 38054 Grenoble Cedex 09, France.
Biochemistry. 2001 Mar 27;40(12):3730-6. doi: 10.1021/bi001746c.
In its active form, Escherichia coli class III ribonucleotide reductase homodimer alpha(2) relies on a protein free radical located on the Gly(681) residue of the alpha polypeptide. The formation of the glycyl radical, namely, the activation of the enzyme, involves the concerted action of four components: S-adenosylmethionine (AdoMet), dithiothreitol (DTT), an Fe-S protein called beta or "activase", and a reducing system consisting of NADPH, NADPH:flavodoxin oxidoreductase, and flavodoxin (fldx). It has been proposed that a reductant serves to generate a reduced 4Fe-4S cluster absolutely required for the reductive cleavage of AdoMet and the generation of the radical. Here, we suggest that the one-electron reduced form of flavodoxin (SQ), the only detectable product of the in vitro enzymatic reduction of flavodoxin, can support the formation of the glycyl radical. However, the redox potential of the Fe-S center of the enzyme is shown to be approximately 300 mV more negative than that of the SQ/fldx couple and not shifted to a more positive value by AdoMet binding. It is also more negative than that of the HQ/SQ couple, HQ being the fully reduced form of flavodoxin. Our interpretation is that activation of ribonucleotide reductase occurs through coupling of the reduction of the Fe-S center by flavodoxin to two thermodynamically favorable reactions, the oxidation of the cluster by AdoMet, yielding methionine and the 5'-deoxyadenosyl radical, and the oxidation of the glycine residue to the corresponding glycyl radical by the 5'-deoxyadenosyl radical. The second reaction plays the major role on the basis that a Gly-to-Ala mutation results in a greatly decreased production of methionine.
处于活性形式时，大肠杆菌III类核糖核苷酸还原酶同型二聚体α(2)依赖于位于α多肽Gly(681)残基上的蛋白质自由基。甘氨酰自由基的形成，即酶的激活，涉及四个组分的协同作用：S-腺苷甲硫氨酸（AdoMet）、二硫苏糖醇（DTT）、一种名为β或“激活酶”的铁硫蛋白，以及由NADPH、NADPH：黄素氧还蛋白氧化还原酶和黄素氧还蛋白（fldx）组成的还原系统。有人提出，一种还原剂用于生成AdoMet还原裂解及自由基生成所绝对必需的还原型4Fe-4S簇。在此，我们认为，黄素氧还蛋白体外酶促还原的唯一可检测产物——黄素氧还蛋白的单电子还原形式（SQ），能够支持甘氨酰自由基的形成。然而，该酶铁硫中心的氧化还原电位比SQ/fldx电对的氧化还原电位约低300 mV，并且不会因AdoMet结合而向更正的值移动。它也比HQ/SQ电对的氧化还原电位更负，HQ是黄素氧还蛋白的完全还原形式。我们的解释是，核糖核苷酸还原酶的激活是通过黄素氧还蛋白对铁硫中心的还原与两个热力学有利反应偶联实现的，这两个反应分别是：AdoMet氧化簇，生成甲硫氨酸和5'-脱氧腺苷自由基；5'-脱氧腺苷自由基将甘氨酸残基氧化为相应的甘氨酰自由基。基于甘氨酸到丙氨酸的突变导致甲硫氨酸产量大幅下降这一事实，第二个反应起主要作用。
