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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Photo-ribonucleotide reductase β2 by selective cysteine labeling with a radical phototrigger
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Photo-ribonucleotide reductase β2 by selective cysteine labeling with a radical phototrigger

机译:通过自由基光触发选择性半胱氨酸标记的光-核糖核苷酸还原酶β2

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摘要

Photochemical radical initiation is a powerful tool for studying radical initiation and transport in biology. Ribonucleotide reduc-tases (RNRs), which catalyze the conversion of nucleotides to deoxynucleotides in all organisms, are an exemplar of radical mediated transformations in biology. Class la RNRs are composed of two subunits: α2 and p2. As a method to initiate radical formation photochemically within p2, a single surface-exposed cysteine of the (52 subunit of Escherichia coli Class la RNR has been labeled (98%) with a photooxidant ([Re] = tricarbonyl (1,10-phenanthroline)(methylpyridyl)rhenium(l)). The labeling was achieved by incubation of S355C-β2 with the 4-(bromomethyl) pyridyl derivative of [Re] to yield the labeled species, [Re]-S355C-p2. Steady-state and time-resolved emission experiments reveal that the metal-to-ligand charge transfer (MLCT) excited-state ~3[Re]~* is not significantly perturbed after bioconjugation and is available as a phototrigger of tyrosine radical at position 356 in the β2 subunit; transient absorption spectroscopy reveals that the radical lives for microseconds. The work described herein provides a platform for photochemical radical initiation and study of proton-coupled electron transfer (PCET) in the β2 subunit of RNR, from which radical initiation and transport for this enzyme originates.
机译:光化学自由基引发是研究生物学中自由基引发和转运的强大工具。核糖核苷酸还原酶(RNRs)是所有生物中催化核苷酸转化为脱氧核苷酸的催化剂,是生物学中自由基介导的转化的典范。 1a类RNR由两个亚基组成:α2和p2。作为一种在p2内以光化学方式引发自由基形成的方法,已用光氧化剂([Re] =三羰基(1,10-菲咯啉)标记(98%)的单表面暴露半胱氨酸(大肠杆菌Ia类RRN类的52个亚基) )(甲基吡啶基)r(1))的标记是通过将S355C-β2与[Re]的4-(溴甲基)吡啶基衍生物孵育以产生标记的物质[Re] -S355C-p2而实现的。以及时间分辨发射实验表明,生物缀合后金属-配体电荷转移(MLCT)激发态〜3 [Re]〜*不会受到明显干扰,可以作为β2356位酪氨酸自由基的光触发剂亚基;瞬态吸收光谱法揭示了该基团的寿命为微秒,本文所述工作为光化学基团引发和研究RNRβ2亚基中的质子偶联电子转移(PCET)提供了一个平台,据此该基团可进行基团引发和转运酶起源特斯

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