Ca2+ Enhances ROS Generation from Inhibited Complex I but not from Inhibited Complex III with Nadh-Linked Substrate

Abstract

Mitochondrial electron transport chain complexes can be major sources of ROS. Several mechanisms are responsible for modulating ROS production, possibly including mitochondrial Ca2+ uptake. Here we tested effects of added buffer CaCl2 on ROS generation from complex I in the presence of rotenone, and from complex III in the presence of antimycin A. Guinea pig heart mitochondria (n=6) were isolated by differential centrifugation and suspended in respiration media containing amplex red and horseradish peroxidase to measure the rate of H2O2 generation. Increasing concentrations of buffered CaCl2 were added to the mitochondrial suspension. Complex I substrate pyruvate (10 mM) or complex II substrate succinate (10 mM) was added followed by either rotenone (10 μM) or antimycin A (5 μM) to block complex I or III, respectively. Compared to no added CaCl2 in the respiratory buffer, the slope of the H2O2 signal in the presence of pyruvate + rotenone increased respectively by 1.3±0.1, 2.1±0.2, 3.4±0.4, 4.5±0.3 times with 10, 25, 50, and 100 μM added external CaCl2. In contrast, H2O2 generation from complex III in the presence of antimycin A did not change with increasing CaCl2, whereas H2O2 generation from complex I in the presence of succinate (due to reversed electron flow) decreased with increasing buffer CaCl2. Moreover, H2O2 generation from complex III in the presence of antimycin A and rotenone in mitochondria supported with succinate did not change with increased buffer CaCl2. We conclude that adding CaCl2 to the buffer enhances H2O2 generation from complex I only during blocked downstream electron transport. This emphasizes the impact of matrix Ca2+ loading on electron leak leading to free radical formation only under conditions of inhibited electron flow at complex I.