Site I(Q) 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(Q)f), or alternatively, whether RET and associated S1QEL-sensitive superoxide/hydrogen peroxide production (site I(Q)r) 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(Q)r and I(Q)f 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(Q)r 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.