Mitochondrial Complex I superoxide production is attenuated by uncoupling

Complex I, i.e. proton-pumping NADH:quinone oxidoreductase, is an essential component of the mitochondrial respiratory chain but produces superoxide as a side-reaction. However, conditions for maximum superoxide production or its attenuation are not well understood. Unlike for Complex III, it has not been clear whether a Complex I-derived superoxide generation at forward electron transport is sensitive to membrane potential or protonmotive force. In order to investigate this, we used Amplex Red for H2O2 monitoring, assessing the total mitochondrial superoxide production in isolated rat liver mitochondria respiring at state 4 as well as at state 3, namely with exclusive Complex I substrates or with Complex I substrates plus succinate. We have shown for the first time, that uncoupling diminishes rotenone-induced H2O2 production also in state 3, while similar attenuation was observed in state 4. Moreover, we have found that 5-(N-ethyl-N-isopropyl) amiloride is a real inhibitor of Complex I H+ pumping (IC50 of 27 μM) without affecting respiration. It also partially prevented suppression by FCCP of rotenone-induced H2O2 production with Complex I substrates alone (glutamate and malate), but nearly completely with Complexes I and II substrates. Sole 5-(N-ethyl-N-isopropyl) amiloride alone suppressed 20% and 30% of total H2O2 production, respectively, under these conditions. Our data suggest that Complex I mitochondrial superoxide production can be attenuated by uncoupling, which means by acceleration of Complex I H+ pumping due to the respiratory control. However, when this acceleration is prevented by 5-(N-ethyl-N-isopropyl) amiloride inhibition, no attenuation of superoxide production takes place.