Mitoflash generated at the Qo site of mitochondrial Complex III

Abstract

The previous research has shown that mitochondrial flash (mitoflash) genesis are functionally and mechanistically integrated with mitochondrial electron transport chain (ETC) energy metabolism. However, the response of mitoflash to superoxide is not entirely consistent with the response of MitoSOX Red. The generation mechanism of mitoflash is still unclear. Here, we investigated mitoflash activities, using the different combinations of ETC substrates and inhibitors, in permeabilized cardiomyocytes or hearts. We found that blocking the complete electron flow, from Complex I to IV, with any one of ETC inhibitors including rotenone (Rot), antimycin A (AntA), myxothiazol (Myxo), stigmatellin, and sodium cyanide, will lead to the abolishment of mitoflashes triggered by substrates in adult permeabilized cardiomyocytes. However, Myxo boosted mitoflashes triggered by the reverse electron of N,N,N′,N′‐tetramethyl‐p‐phenylenediamine/ascorbate. Moreover, Rot and AntA furtherly enhanced mitoflash activity rather than depressed it, suggesting that mitoflashes generated at the Complex III Qo site. Meanwhile, the inhibition of Complex III protein expression resulted in the activity of Complex III decrease, which decreased mitoflash frequency. The function defect (no change of protein level) of the Qo site of Complex III in aging hearts augmented mitoflash generation confirmed the Qo site function was critical to mitoflash genesis. Thus, our results indicate that mitoflash detected by circularly permuted yellow fluorescent protein is generated at the Qo site of Complex III.

The Role of Oxidative Stress in the Aging Heart

“…Aging modifies the binding locus of mixothiazole in the QO site of cytochrome b [ 141 ]. The functional deficiency in the complex III QO site triggers electron leakage increasing ROS production in interfibrillar mitochondria [ 142 ].…”

Letter to the editor: Compromised hepatic mitochondrial fatty acid oxidation and reduced markers of mitochondrial turnover in human NAFLD

“…It is well known that mitochondrial superoxide generated from the electron leak from Complexes I and III of electron transport chain during ATP production, and dysfunctional complex I and III trigger more superoxide generation. [ 3 ] We think that it is necessary to provide evidence of complex I and III activity not only for the ROS accumulation but also for the less energetic metabolism.…”