Yun Danny, Krebs Carsten, Gupta Govind P, Iwig David F, Huynh Boi Hanh, Bollinger J Martin
Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, Pennsylvania 16802, USA.
Biochemistry. 2002 Jan 22;41(3):981-90. doi: 10.1021/bi011797p.
The kinetics and mechanism of formation of the tyrosyl radical and mu-(oxo)diiron(III) cluster in the R2 subunit of ribonucleotide reductase from mouse have been examined by stopped-flow absorption and freeze-quench electron paramagnetic resonance and Mössbauer spectroscopies. The reaction comprises (1) acquisition of Fe(II) ions by the R2 apo protein, (2) activation of dioxygen at the resulting carboxylate-bridged diiron(II) cluster to form oxidized intermediate diiron species, and (3) univalent oxidation of Y177 by one of these intermediates to form the stable radical, with concomitant or subsequent formation of the adjacent mu-(oxo)diiron(III) cluster. The data establish that an oxidized diiron intermediate spectroscopically similar to the well-characterized, formally Fe(III)Fe(IV) cluster X from the reaction of the Escherichia coli R2 protein precedes the Y177 radical in the reaction sequence and is probably the Y177 oxidant. As formation of the X intermediate (1) requires transfer of an "extra" reducing equivalent to the buried diiron cluster following the addition of dioxygen and (2) is observed to be rapid relative to other steps in the reaction, the present data indicate that the transfer of this reducing equivalent is not rate-limiting for Y177 radical formation, in contrast to what was previously proposed (Schmidt, P. P., Rova, U., Katterle, B., Thelander, L., and Gräslund, A. (1998) J. Biol. Chem. 273, 21463-21472). Indeed, the formation of X (k(obs) = 13 +/- 3 s(-1) at 5 degrees C and 0.95 mM O(2)) and the decay of the intermediate to give the Y177 radical (k(obs) = 5 +/- 2 s(-1)) are both considerably faster than the formation of the reactive Fe(II)-R2 complex from the apo protein and Fe(II)(aq) (k(obs) = 0.29 +/- 0.03 s(-1)), which is the slowest step overall. The conclusions that cluster X is an intermediate in Y177 radical formation and that transfer of the reducing equivalent is relatively facile imply that the mouse R2 and E. coli R2 reactions are mechanistically similar.
通过停流吸收光谱、冷冻淬灭电子顺磁共振光谱和穆斯堡尔光谱，研究了小鼠核糖核苷酸还原酶R2亚基中酪氨酰自由基和μ-(氧代)二铁(III)簇的形成动力学及机制。该反应包括：(1) R2脱辅基蛋白获取Fe(II)离子；(2) 在生成的羧酸盐桥连二铁(II)簇处激活双氧，形成氧化态中间二铁物种；(3) 这些中间体之一将Y177单电子氧化形成稳定自由基，同时或随后形成相邻的μ-(氧代)二铁(III)簇。数据表明，在反应序列中，一种氧化态二铁中间体在光谱上类似于来自大肠杆菌R2蛋白反应中特征明确的形式上为Fe(III)Fe(IV)的簇X，且先于Y177自由基形成，它可能是Y177的氧化剂。由于X中间体的形成：(1) 在添加双氧后需要将一个“额外”的还原当量转移至埋藏的二铁簇；(2) 相对于反应中的其他步骤观察到其形成迅速，因此目前的数据表明，与之前所提出的情况相反（施密特，P.P.，罗瓦，U.，卡特勒，B.，特兰德，L.，和格拉斯隆德，A.（1998年）《生物化学杂志》273，21463 - 21472），这种还原当量的转移并非Y177自由基形成的限速步骤。实际上，X的形成（在5℃和0.95 mM O₂条件下，k(obs)=13±3 s⁻¹）以及中间体衰减生成Y177自由基（k(obs)=5±2 s⁻¹）都比脱辅基蛋白和Fe(II)(aq)形成活性Fe(II)-R2复合物（k(obs)=0.29±0.03 s⁻¹）快得多，而这是整个反应中最慢的步骤。簇X是Y177自由基形成过程中的中间体以及还原当量转移相对容易这两个结论意味着小鼠R2和大肠杆菌R2的反应在机制上相似。
