Bobrowski Krzysztof, Hug Gordon L, Pogocki Dariusz, Marciniak Bronislaw, Schöneich Christian
Institute of Nuclear Chemistry and Technology, 03-195 Warsaw, Poland.
J Phys Chem B. 2007 Aug 16;111(32):9608-20. doi: 10.1021/jp071191w. Epub 2007 Jul 21.
The recent study on the *OH-induced oxidation of calmodulin, a regulatory "calcium sensor" protein containing nine methionine (Met) residues, has supported the first experimental evidence in a protein for the formation of S therefore N three-electron bonded radical complexes involving the sulfur atom of a methionine residue and the amide groups in adjacent peptide bonds. To characterize reactions of oxidized methionine residues in proteins containing multiple methionine residues in more detail, in the current study, a small model cyclic dipeptide, c-(L-Met-L-Met), was oxidized by *OH radicals generated via pulse radiolysis and the ensuing reactive intermediates were monitored by time-resolved UV-vis spectroscopic and conductometric techniques. The picture that emerges from this investigation shows there is an efficient formation of the Met (S therefore N) radicals, in spite of the close proximity of two sulfur atoms, located in the side chains of methionine residues, and in spite of the close proximity of sulfur atoms and oxygen atoms, located in the peptide bonds. Moreover, it is shown, for the first time, that the formation of Met(S therefore N) radicals can proceed directly, via H+-transfer, with the involvement of hydrogen from the peptide bond to an intermediary hydroxysulfuranyl radical. Ultimately, the Met(S therefore N) radicals decayed via two different pH-dependent reaction pathways, (i) conversion into sulfur-sulfur, intramolecular, three-electron-bonded radical cations and (ii) a proposed hydrolytic cleavage of the protonated form of the intramolecular, three-electron-bonded radicals [Met(S therefore N)/Met(S therefore NH)+] followed by electron transfer and decarboxylation. Surprisingly, also alpha-(alkylthio)alkyl radicals enter the latter mechanism in a pH-dependent manner. Density functional theory computations were performed on the model c-(L-Met-Gly) and its radicals in order to obtain optimizations and energies to aid in the interpretation of the experiments on c-(L-Met-L-Met).
最近对钙调蛋白（一种含有九个甲硫氨酸（Met）残基的调节性“钙传感器”蛋白）的羟基诱导氧化研究，为蛋白质中涉及甲硫氨酸残基硫原子和相邻肽键中酰胺基团形成S=N三电子键自由基复合物提供了首个实验证据。为了更详细地表征含有多个甲硫氨酸残基的蛋白质中氧化甲硫氨酸残基的反应，在本研究中，一个小的模型环二肽c-（L-甲硫氨酸-L-甲硫氨酸）被脉冲辐射产生的羟基自由基氧化，随后通过时间分辨紫外可见光谱和电导技术监测产生的反应中间体。尽管位于甲硫氨酸残基侧链的两个硫原子靠得很近，并且位于肽键中的硫原子和氧原子也靠得很近，但这项研究得出的结果表明，Met（S=N）自由基能有效形成。此外，首次表明，Met（S=N）自由基的形成可以通过质子转移直接进行，肽键中的氢参与形成中间的羟基硫烷基自由基。最终，Met（S=N）自由基通过两种不同的pH依赖性反应途径衰减，（i）转化为分子内硫-硫三电子键自由基阳离子，（ii）分子内三电子键自由基[Met（S=N）/Met（S=NH）+]的质子化形式发生水解裂解，随后进行电子转移和脱羧。令人惊讶的是，α-（烷硫基）烷基自由基也以pH依赖性方式参与后一种机制。对模型c-（L-甲硫氨酸-甘氨酸）及其自由基进行了密度泛函理论计算，以获得优化结构和能量，有助于解释c-（L-甲硫氨酸-L-甲硫氨酸）的实验结果。
