Site IQ in mitochondrial complex I generates S1QEL-sensitive superoxide/hydrogen peroxide in both the reverse and forward reactions.

Superoxide/hydrogen peroxide production by site I_Q in complex I of the electron transport chain is conventionally assayed during reverse electron transport (RET) from ubiquinol to NAD. However, S1QELs (specific suppressors of superoxide/hydrogen peroxide production by site I_Q) have potent effects in cells and in vivo during presumed forward electron transport (FET). Therefore, we tested whether site I_Q generates S1QEL-sensitive superoxide/hydrogen peroxide during FET (site I_Qf), or alternatively, whether RET and associated S1QEL-sensitive superoxide/hydrogen peroxide production (site IQr) occurs in cells under normal conditions. We introduce an assay to determine if electron flow through complex I is thermodynamically forward or reverse: on blocking electron flow through complex I, the endogenous matrix NAD pool will become more reduced if flow before the challenge was forward, but more oxidised if flow was reverse. Using this assay we show in the model system of isolated rat skeletal muscle mitochondria that superoxide/hydrogen peroxide production by site I_Q can be equally great whether RET or FET is running. We show that sites I_Qr and I_Qf are equally sensitive to S1QELs, and to rotenone and piericidin A, inhibitors that block the Q-site of complex I. We exclude the possibility that some sub-fraction of the mitochondrial population running site I_Qr during FET is responsible for S1QEL-sensitive superoxide/hydrogen peroxide production by site I_Q. Finally, we show that superoxide/hydrogen peroxide production by site I_Q in cells occurs during FET, and is S1QEL-sensitive. [ABSTRACT FROM AUTHOR]