Pötsch S, Lendzian F, Ingemarson R, Hörnberg A, Thelander L, Lubitz W, Lassmann G, Gräslund A
Department of Biophysics, Stockholm University, Arrhenius Laboratories, S-106 91 Stockholm, Sweden.
J Biol Chem. 1999 Jun 18;274(25):17696-704. doi: 10.1074/jbc.274.25.17696.
The ferrous iron/oxygen reconstitution reaction in protein R2 of mouse and Escherichia coli ribonucleotide reductase (RNR) leads to the formation of a stable protein-linked tyrosyl radical and a mu-oxo-bridged diferric iron center, both necessary for enzyme activity. We have studied the reconstitution reaction in three protein R2 mutants Y177W, Y177F, and Y177C of mouse RNR to investigate if other residues at the site of the radical forming Tyr-177 can harbor free radicals. In Y177W we observed for the first time the formation of a tryptophan radical in protein R2 of mouse RNR with a lifetime of several minutes at room temperature. We assign it to an oxidized neutral tryptophan radical on Trp-177, based on selective deuteration and EPR and electron nuclear double resonance spectroscopy in H2O and D2O solution. The reconstitution reaction at 22 degrees C in both Y177F and Y177C leads to the formation of a so-called intermediate X which has previously been assigned to an oxo (hydroxo)-bridged Fe(III)/Fe(IV) cluster. Surprisingly, in both mutants that do not have successor radicals as Trp. in Y177W, this cluster exists on a much longer time scale (several seconds) at room temperature than has been reported for X in E. coli Y122F or native mouse protein R2. All three mouse R2 mutants were enzymatically inactive, indicating that only a tyrosyl radical at position 177 has the capability to take part in the reduction of substrates.
小鼠和大肠杆菌核糖核苷酸还原酶（RNR）的蛋白质R2中的亚铁/氧重构反应会导致形成稳定的蛋白质连接的酪氨酸自由基和一个μ-氧桥联二价铁中心，这两者都是酶活性所必需的。我们研究了小鼠RNR的三种蛋白质R2突变体Y177W、Y177F和Y177C中的重构反应，以研究在形成自由基的Tyr-177位点的其他残基是否能够容纳自由基。在Y177W中，我们首次在小鼠RNR的蛋白质R2中观察到色氨酸自由基的形成，其在室温下的寿命为几分钟。基于在H2O和D2O溶液中的选择性氘代以及EPR和电子核双共振光谱，我们将其归为Trp-177上的氧化中性色氨酸自由基。在Y177F和Y177C中于22℃进行的重构反应导致形成所谓的中间体X，该中间体先前已被归为氧（羟基）桥联的Fe（III）/Fe（IV）簇。令人惊讶的是，在这两个不像Y177W中的Trp那样具有后续自由基的突变体中，该簇在室温下存在的时间尺度要长得多（几秒），比大肠杆菌Y122F或天然小鼠蛋白质R2中的X的报道时间要长。所有三种小鼠R2突变体均无酶活性，这表明只有177位的酪氨酸自由基有能力参与底物的还原。
