**Acknowledgements**

fluorescent probe that is specifically targeted to mitochondria. The resulting confocal images revealed that the green fluorescence of MitoTracker Green colocalize with the red fluorescence of MitoSOX Red indicating that MitoSOX Red is oxidized within mitochondria (**Figure 8**). To determine whether MitoSOX oxidation is a superoxide-dependent process, the cells were pre-

PEG-SOD, MitoSOX Red fluorescence was negligible (**Figure 8**, panels B and E). This result indicates that the majority of the atpenin A5-induced MitoSOX fluorescence is superoxide

is a succinate-dependent process by preincubating the cells with NPA before adding atpenin A5. As shown in **Figure 8** panels C and F, the MitoSOX Red fluorescence intensity in the NPAtreated cells was markedly reduced indicating that the atpenin A5-induced MitoSOX Red oxidation is a succinate-dependent event. Collectively, these data indicate that atpenin A5 induces

and HT-29 cells. In agreement with the results from the isolated mitochondria, addition of atpenin A5 to the MitoSOX Red-loaded dermal fibroblasts did not display any red fluorescence

of dermal fibroblasts. But dermal fibroblasts showed an increment in red fluorescence with antimycin A (**Figure 8** panel H). This observation shows that the mitochondria of the dermal

ROS are produced from the mitochondrial complex II of the cancer cells (DLD-1 and HT-29) but not from the normal cells (dermal fibroblasts) when the entry of electrons to its quinonebinding site is blocked with 10 μM atpenin A5. However, previous studies in our laboratory have revealed that quinone reduction in the mitochondrial complex II can be completely inhibited at the tested concentration of atpenin A5 [8, 10]. Therefore, the difference in ROS production between cancer cells and normal cells may be attributed to a difference in the complex IIs that is upstream of the atpenin A5-binding site. Since the dicarboxylate-binding site inhibitor, NPA, could inhibit the ROS production induced by atpenin A5 in cancer cells, it is concluded that the ROS-producing site must be within the complex II, upstream of the atpenin A5 inhibition site(Q site) and downstream of NPA-binding site (dicarboxylate-binding site). According to previous evidence, the most likely ROS-producing sites of mitochondrial complex II are FAD [35–37] site and the Q site [37–39]. Observations of this study show that the ROS-generating site of complex II in atpenin A5-treated cancer cells is FAD but not Q site.

In order to explain this observation, we analyzed the published data on the crystal structures of flavoproteins, which are available in both the substrate bound and unbound forms [40–42]. They have shown that the capping domain in Fp subunit rotates during the enzyme catalysis. When the active site is not occupied by the substrate, the capping domain is rotated away from the active site creating a solvent channel linking the active site and the surrounding aqueous environment. In the substrate bound state, the capping domain is rotated inward concealing the active site, preventing the access of solvent to the active site [37]. Thus, closed conformation acquired during enzyme catalysis appears to be vital for

dependent. Next, we attempted to determine whether the atpenin A5-induced O<sup>2</sup>

−

production from the mitochondrial complex II within intact DLD-1

when the Q site of complex III is inhibited by antimycin A.

−

generated within cells. In the presence of

−

production in mitochondria

production

incubated with PEG-SOD, which can scavenge O<sup>2</sup>

−

−

(**Figure 8** panel G) indicating that atpenin A5 does not induce O2

succinate-dependent O2

102 Mitochondrial Diseases

fibroblasts can produce O<sup>2</sup>

**4. Discussion**

Ministry of Education, Culture and Sports in Japan is gratefully acknowledged for their financial support.
