Succinate-driven oxidation via complex II (CII) may have a significant contribution towards the high rates of production of
reactive oxygen species (ROS) by mitochondria. Here, we show that the CII Q site inhibitor
thenoyltrifluoroacetone (TTFA) blocks
succinate +
rotenone-driven ROS production, whereas the
complex III (CIII) Qo inhibitor
stigmatellin has no effect, indicating that CII, not CIII, is the ROS-producing site. The complex I (CI) inhibitor
rotenone partially reduces the ROS production driven by high
succinate levels (5 mm), which is commonly interpreted as being due to inhibition of a reverse electron flow from CII to CI. However, experimental evidence presented here contradicts the model of reverse electron flow. First, ROS levels produced using
succinate +
rotenone were significantly higher than those produced using
glutamate +
malate +
rotenone. Second, in
tumor mitochondria,
succinate-driven ROS production was significantly increased (not decreased) by
rotenone. Third, in liver mitochondria,
rotenone had no effects on
succinate-driven ROS production. Fourth, using isolated heart or
hepatoma (AS-30D) mitochondria, the CII Qp anti-
cancer drug mitochondrially targeted
vitamin E succinate (MitoVES) induced elevated ROS production in the presence of low levels of
succinate(0.5 mm), but
rotenone had no effect. Using sub-mitochondrial particles, the Cu-based anti-
cancer drug Casiopeina II-gly enhanced
succinate-driven ROS production. Thus, the present results are inconsistent with and question the interpretation of reverse electron flow from CII to CI and the
rotenone effect on ROS production supported by
succinate oxidation. Instead, a thermodynamically more favorable explanation is that, in the absence of CIII or complex IV (CIV) inhibitors (which, when added, facilitate reverse electron flow by inducing accumulation of
ubiquinol, the CI product), the CII redox centers are the major source of
succinate-driven ROS production.