Forward and reverse electron transfer with the Y356DOPA-β2 heterodimer of E-coli ribonucleotide reductase
Forward and reverse electron transfer with the Y356DOPA-β2 heterodimer of E-coli ribonucleotide reductase
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DOI：
10.1021/ja0685607
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发表时间：
2007-02-28
影响因子：
15
通讯作者：
Stubbe, JoAnne
中科院分区：
文献类型：
--
作者：
Seyedsayamdost, Mohammad R.;Stubbe, JoAnne
E.大肠杆菌核糖核苷酸还原酶催化核苷酸转化为脱氧核苷酸，由α 2和β 2两个亚基组成。β 2含有一个稳定的二铁酪氨酰基自由基（Y-122中心点），这是催化所必需的。α 2含有发生核苷酸还原的活性位点，以及控制底物特异性和转换速率的效应和活性位点。在这项研究中，我们已经使用内含肽方法来产生β 2的异源二聚体，其在残基356处含有非天然氨基酸3，4-二羟基苯丙氨酸（DOPA）（DOPA-beta β '）。在该异二聚体中，β-单体是全长的（残基1-375），而β '-单体是截短的并且仅含有残基1-353。加入α 2、CDP和ATP效应物后，DOPA-β β ′产生DOPA中心点，伴随着Y-122中心点的损失。通过EPR对DOPA中心点稳定性的分析表明，DOPA中心点-β β ′可以再氧化Y-122，从而再生Y-122中心点。这些结果首次直接证明了从残基356到Y-122的反向电子转移。
E. coli ribonucleotide reductase catalyzes the conversion of nucleotides to deoxynucleotides, and consists of two subunits, alpha 2 and beta 2. beta 2 contains a stable diiron tyrosyl radical (Y-122 center dot) that is essential for catalysis. alpha 2 harbors the active site, where nucleotide reduction occurs, as well as effector and activity sites which control substrate specificity and turnover rates. In this study, we have used intein methodology to generate a heterodimer of beta 2 containing the unnatural amino acid 3,4-dihydroxyphenylalanine (DOPA) at residue 356 (DOPA-beta beta'). In this heterodimer, the beta-monomer is full-length (residues 1-375), whereas the beta'-monomer is truncated and only contains residues 1-353. DOPA-beta beta', upon addition of alpha 2, CDP, and ATP effector, generates a DOPA center dot concomitant with loss of the Y-122 center dot. Analysis of DOPA center dot stability by EPR reveal that DOPA center dot-beta beta' can reoxidize Y-122 thereby regenerating the Y-122 center dot. These results, for the first time, directly demonstrate back electron transfer from residue 356 to Y-122.