Facile electron transfer during formation of cluster X and kinetic competence of X for tyrosyl radical production in protein R2 of ribonucleotide reductase from mouse.
Facile electron transfer during formation of cluster X and kinetic competence of X for tyrosyl radical production in protein R2 of ribonucleotide reductase from mouse.
复制标题
X簇形成过程中的轻松电子转移以及X在小鼠核糖核苷酸还原酶蛋白R2中产生酪氨酰自由基的动力学能力。
DOI：
10.1021/bi011797p
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发表时间：
2002
期刊：
影响因子：
2.9
通讯作者：
BollingerJr,JMartin
中科院分区：
文献类型：
--
作者：
Yun,Danny;Krebs,Carsten;Gupta,GovindP;Iwig,DavidF;Huynh,BoiHanh;BollingerJr,JMartin
本文用停流吸收、冷冻淬灭电子顺磁共振和穆斯堡尔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）簇的形成动力学和机理。该反应包括（1）通过R2载脂蛋白获得Fe（II）离子，（2）在所得羧酸根桥接的二铁（II）簇上活化分子氧以形成氧化的中间体二铁物质，和（3）通过这些中间体之一单价氧化Y177以形成稳定的自由基，伴随或随后形成相邻的μ-（氧代）二铁（III）簇。这些数据建立了氧化二铁中间体光谱相似的良好的特点，正式Fe（III）Fe（IV）簇X的反应theEscherichia coliR 2蛋白之前的Y177自由基的反应序列，可能是Y177氧化剂。由于X中间体的形成（1）需要在添加分子氧之后将“额外的”还原当量转移到掩埋的二铁簇，并且（2）观察到相对于反应中的其它步骤是快速的，因此本数据表明，与先前提出的相反，该还原当量的转移对于Y177自由基形成不是速率限制的（施密特，P. P.，美国罗瓦，Katterle，B.，Thelander，L.，和Gräslund，A.（1998）J.Biol.Chem.273，21463 - 21472）。事实上，X的形成（kobs= 13 ± 3 s-1，在5 °C和0.95 mM O2下）和中间体衰变产生Y177自由基（kobs= 5 ± 2 s-1）都比由载脂蛋白和Fe（II）水溶液形成反应性Fe（II）−R2复合物（kobs= 0.29 ± 0.03 s-1）快得多，后者是总体上最慢的步骤。簇X是Y177自由基形成的中间体，还原当量的转移相对容易，这一结论意味着小鼠R2和E. coliR 2反应在机理上是相似的。
The kinetics and mechanism of formation of the tyrosyl radical and μ-(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 μ-(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 theEscherichia coliR2 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 (kobs= 13 ± 3 s-1at 5 °C and 0.95 mM O2) and the decay of the intermediate to give the Y177 radical (kobs= 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(kobs= 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 andE. coliR2 reactions are mechanistically similar.