Department of Chemistry and Biochemistry and Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, Georgia 30332, USA.
J Am Chem Soc. 2013 May 1;135(17):6380-3. doi: 10.1021/ja3032949. Epub 2013 Apr 17.
Ribonucleotide reductase (RNR) catalyzes conversion of nucleoside diphosphates (NDPs) to 2'-deoxynucleotides, a critical step in DNA replication and repair in all organisms. Class-Ia RNRs, found in aerobic bacteria and all eukaryotes, are a complex of two subunits: α2 and β2. The β2 subunit contains an essential diferric-tyrosyl radical (Y122O(•)) cofactor that is needed to initiate reduction of NDPs in the α2 subunit. In this work, we investigated the Y122O(•) reduction mechanism in Escherichia coli β2 by hydroxyurea (HU), a radical scavenger and cancer therapeutic agent. We tested the hypothesis that Y122OH redox reactions cause structural changes in the diferric cluster. Reduction of Y122O(•) was studied using reaction-induced FT-IR spectroscopy and [(13)C]aspartate-labeled β2. These Y122O(•) minus Y122OH difference spectra provide evidence that the Y122OH redox reaction is associated with a frequency change to the asymmetric vibration of D84, a unidentate ligand to the diferric cluster. The results are consistent with a redox-induced shift in H-bonding between Y122OH and D84 that may regulate proton-transfer reactions on the HU-mediated inactivation pathway in isolated β2.
核酶(RNR)催化核苷二磷酸(NDP)转化为 2'-脱氧核苷酸,这是所有生物中 DNA 复制和修复的关键步骤。在需氧细菌和所有真核生物中发现的 I 类 RNR 是由两个亚基组成的复合物:α2 和β2。β2 亚基含有必需的二铁酪氨酸自由基(Y122O(•))辅助因子,该辅助因子用于启动 NDP 在 α2 亚基中的还原。在这项工作中,我们通过羟脲(HU)研究了大肠杆菌β2 中的 Y122O(•)还原机制,HU 是一种自由基清除剂和癌症治疗剂。我们检验了以下假设:Y122OH 氧化还原反应引起二铁簇的结构变化。使用反应诱导的傅里叶变换红外光谱和 [(13)C]天冬氨酸标记的β2 研究了 Y122O(•)的还原。这些 Y122O(•)减去 Y122OH 差谱提供了证据,表明 Y122OH 氧化还原反应与 D84 的不对称振动的频率变化有关,D84 是二铁簇的一个单齿配体。结果与 Y122OH 和 D84 之间的氢键的氧化还原诱导位移一致,该位移可能调节 HU 介导的失活途径中质子转移反应在分离的β2 中。