A melatonin‐based fluorescence method for the measurement of mitochondrial complex III function in intact cells

Mitochondrial complex III ( MC ‐3) plays a pivotal role in electron transfer and oxidative phosphorylation. Impaired MC ‐3 functions may contribute to a variety of diseases by interrupting normal bioenergetics and increasing reactive oxygen production and oxidative stress. Currently, MC ‐3 function is assessed by measuring the cytochrome c reductase activity spectrophotometrically in isolated mitochondria or MC ‐3. The cytoplasmic microenvironment critical for mitochondrial complex functions may be depleted during these isolation processes. The development of a reliable method to measure MC ‐3 activities in intact cells or tissues is highly desirable. This report describes a novel fluorescence‐based method to assess MC ‐3 functions, i.e., Q i site electron transfer, in the intact cells. Human mesangial and teratocarcinoma NT 2 cells were used to demonstrate that melatonin‐induced oxidation of 2′,7′‐dichlorodihydrofluorescein ( H 2 DCF ) was inhibited by antimycin A , the MC ‐3 Q i site‐specific inhibitor, but not by myxothiazol, the MC ‐3 Q o site‐specific inhibitor, nor rotenone, the mitochondrial complex I inhibitor. These results indicate that melatonin‐induced oxidation of H 2 DCF is reflecting MC ‐3 Q i site electron transfer activities. Modifying structures of the side groups at the R 3 and R 5 positions of the indole ring of melatonin diminished its efficacy for inducing H 2 DCF oxidation, suggesting a specific interaction of melatonin with the MC ‐3 Q i site. These results suggest that the fluorogenic property of melatonin‐induced H 2 DCF oxidation provides a MC ‐3 Q i site electron transfer‐specific measurement in intact cells. Interestingly, using this method, the Q i site electron transfer activity in transformed or immortalized cells was found to be significantly higher than the nontransformed cells.