Cytochrome c-mediated oxidation of hydroethidine and mito-hydroethidine in mitochondria: Identification of homo- and heterodimers

Here we report that ferricytochrome c (cyt c3+) induces oxidation of hydroethidine (HE) and mitochondria-targeted hydroethidine (Mito-HE or MitoSOX Red) forming highly characteristic homo- and heterodimeric products. Using an HPLC-electrochemical (EC) method, several products were detected from cyt c3+-catalyzed oxidation of HE and Mito-HE and characterized by mass spectrometry and NMR techniques as follows: homodimers (HE-HE, E+-E+, Mito-HE-Mito-HE, and Mito-E+-Mito-E+) and heterodimers (HE-E+ and Mito-HE-Mito-E+), as well as the monomeric ethidium (E+) and mito-ethidium (Mito-E+). Similar products were detected when HE and Mito-HE were incubated with mitochondria. In contrast, mitochondria depleted of cyt c3+ were much less effective in oxidizing HE or Mito-HE to corresponding dimeric products. Unlike E+ or Mito-E+, the dimeric analogs (E+-E+ and Mito-E+-Mito-E+) were not fluorescent. Superoxide (O2
• −) or Fremy's salt reacts with Mito-HE to form a product, 2-hydroxy-mito-ethidium (2-OH-Mito-E+) that was detected by HPLC. We conclude that HPLC-EC but not the confocal and fluorescence microscopy is a viable technique for measuring superoxide and cyt c3+-dependent oxidation products of HE and Mito-HE in cells. Superoxide detection using HE and Mito-HE could be severely compromised due to their propensity to undergo oxidation.