Department of Biophysics, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
Departments of Neurology and Pediatrics, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
Int J Mol Sci. 2019 Mar 6;20(5):1143. doi: 10.3390/ijms20051143.
In a previous study on chromate toxicity, an increase in the 2Fe2S electron paramagnetic resonance (EPR) signal from mitochondria was found upon addition of chromate to human bronchial epithelial cells and bovine airway tissue ex vivo. This study was undertaken to show that a chromate-induced increase in the 2Fe2S EPR signal is a general phenomenon that can be used as a low-temperature EPR method to determine the maximum concentration of 2Fe2S centers in mitochondria. First, the low-temperature EPR method to determine the concentration of 2Fe2S clusters in cells and tissues is fully developed for other cells and tissues. The EPR signal for the 2Fe2S clusters N1b in Complex I and/or S1 in Complex II and the 2Fe2S cluster in xanthine oxidoreductase in rat liver tissue do not change in intensity because these clusters are already reduced; however, the EPR signals for N2, the terminal cluster in Complex I, and N4, the cluster preceding the terminal cluster, decrease upon adding chromate. More surprising to us, the EPR signals for N3, the cluster preceding the 2Fe2S cluster in Complex I, also decrease upon adding chromate. Moreover, this method is used to obtain the concentration of the 2Fe2S clusters in white blood cells where the 2Fe2S signal is mostly oxidized before treatment with chromate and becomes reduced and EPR detectable after treatment with chromate. The increase of the g = 1.94 2Fe2S EPR signal upon the addition of chromate can thus be used to obtain the relative steady-state concentration of the 2Fe2S clusters and steady-state concentration of Complex I and/or Complex II in mitochondria.
在先前关于铬酸盐毒性的研究中，发现向人支气管上皮细胞和牛气道组织中添加铬酸盐后，线粒体中的 2Fe2S 电子顺磁共振（EPR）信号增加。本研究旨在表明，铬酸盐诱导的 2Fe2S EPR 信号增加是一种普遍现象，可以用作低温 EPR 方法来确定线粒体中 2Fe2S 中心的最大浓度。首先，充分开发了用于其他细胞和组织的低温 EPR 方法来确定细胞和组织中 2Fe2S 簇的浓度。由于这些簇已经被还原，所以在 Complex I 中的 N1b 和/或 S1 中的 2Fe2S 簇以及在大鼠肝组织中的黄嘌呤氧化还原酶中的 2Fe2S 簇的 EPR 信号的强度不会发生变化；然而，添加铬酸盐后，Complex I 中的末端簇 N2 和位于末端簇之前的 N4 的 EPR 信号减少。令我们感到惊讶的是，添加铬酸盐后，位于 Complex I 中的 2Fe2S 簇之前的 N3 簇的 EPR 信号也减少了。此外，该方法用于获得白细胞中 2Fe2S 簇的浓度，其中在用铬酸盐处理之前，2Fe2S 信号主要被氧化，并且在用铬酸盐处理后变得还原并且可通过 EPR 检测到。因此，添加铬酸盐后 g = 1.94 的 2Fe2S EPR 信号的增加可用于获得 2Fe2S 簇的相对稳态浓度和线粒体中 Complex I 和/或 Complex II 的稳态浓度。
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