Macedo Sofia, Pechlaner Maria, Schmid Walther, Weik Martin, Sato Katsuko, Dennison Christopher, Djinović-Carugo Kristina
Department for Structural and Computational Biology, Max F. Perutz Laboratories, University of Vienna, Campus Vienna Biocenter 5, 1030 Vienna, Austria.
J Synchrotron Radiat. 2009 Mar;16(Pt 2):191-204. doi: 10.1107/S0909049509003331. Epub 2009 Feb 25.
One of the first events taking place when a crystal of a metalloprotein is exposed to X-ray radiation is photoreduction of the metal centres. The oxidation state of a metal cannot always be determined from routine X-ray diffraction experiments alone, but it may have a crucial impact on the metal's environment and on the analysis of the structural data when considering the functional mechanism of a metalloenzyme. Here, UV-Vis microspectrophotometry is used to test the efficacy of selected scavengers in reducing the undesirable photoreduction of the iron and copper centres in myoglobin and azurin, respectively, and X-ray crystallography to assess their capacity of mitigating global and specific radiation damage effects. UV-Vis absorption spectra of native crystals, as well as those soaked in 18 different radioprotectants, show dramatic metal reduction occurring in the first 60 s of irradiation with an X-ray beam from a third-generation synchrotron source. Among the tested radioprotectants only potassium hexacyanoferrate(III) seems to be capable of partially mitigating the rate of metal photoreduction at the concentrations used, but not to a sufficient extent that would allow a complete data set to be recorded from a fully oxidized crystal. On the other hand, analysis of the X-ray crystallographic data confirms ascorbate as an efficient protecting agent against radiation damage, other than metal centre reduction, and suggests further testing of HEPES and 2,3-dichloro-1,4-naphtoquinone as potential scavengers.
当金属蛋白晶体暴露于X射线辐射时，首先发生的事件之一是金属中心的光还原。金属的氧化态不能总是仅从常规X射线衍射实验中确定，但在考虑金属酶的功能机制时，它可能对金属的环境以及结构数据的分析产生关键影响。在这里，紫外可见显微分光光度法用于测试选定的清除剂分别减少肌红蛋白和天青蛋白中铁和铜中心不希望的光还原的效果，而X射线晶体学用于评估它们减轻整体和特定辐射损伤效应的能力。天然晶体以及浸泡在18种不同辐射防护剂中的晶体的紫外可见吸收光谱显示，在用第三代同步加速器源的X射线束照射的前60秒内发生了显著的金属还原。在所测试的辐射防护剂中，只有铁氰化钾（III）在所用浓度下似乎能够部分降低金属光还原的速率，但程度不足以从完全氧化的晶体中记录完整的数据集。另一方面，对X射线晶体学数据的分析证实，除了金属中心还原外，抗坏血酸是一种有效的抗辐射损伤保护剂，并建议进一步测试HEPES和2,3-二氯-1,4-萘醌作为潜在的清除剂。
