Abstract
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.
Highlights
Most of the iron sulfur (Fe-S) site signals in mitochondrial Complex I, including the four-iron four-sulfur clusters (4Fe4S) and two-iron two-sulfur clusters (2Fe2S), can be detected using electron paramagnetic resonance (EPR) when these clusters are in the reduced state (Figure 1)
EPR spectra were obtained from white blood cells with and without the addition of 400 μM chromate (Figure 2)
Of interest for this study is the very substantial increase of the g = 1.94 2Fe2S signal for cells treated with chromate, which reached a signal intensity as large as that detected for chromate-treated human bronchial epithelial cells and bovine airways [1]
Summary
Most of the iron sulfur (Fe-S) site signals in mitochondrial Complex I, including the four-iron four-sulfur clusters (4Fe4S) and two-iron two-sulfur clusters (2Fe2S), can be detected using electron paramagnetic resonance (EPR) when these clusters are in the reduced state (Figure 1). Other Fe-S signals, primarily from the three-iron four-sulfur cluster (3Fe4S) in aconitase, are detected by EPR when they are in the oxidized state. An increase in the g = 1.94 EPR signal attributed to 2Fe2S sites in Complex I and/or Complex II in mitochondria upon addition of chromate to human bronchial epithelial cells and bovine airways treated ex vivo is thought to be an important biomarker for Cr(VI) exposure [1]. It occurred to us that the intensity of this g = 1.94 2Fe2S signal could be more widely used to determine the relative concentration of Complex I and/or Complex II in mitochondria.
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