The method allows localizing the sites responsible for ROS production inside the electron transport chain. The sites of ROS production are identified by measuring the rate of H2O2 production with different combinations of substrates and inhibitors specific for different segments of the respiratory chain. For example, pyruvate/malate and glutamate/malate are complex I-linked substrates, and succinate is a complex II-linked substrate. Rotenone inhibits specifically at complex I, TTFA at complex II and antimycin A at complex III.
It is well known that the rate of oxygen radical production increases as a function of the degree of reduction of the autoxidazable electron carrier responsible for ROS generation (Boveris and Chance, 1973). Blocking the respiratory chain with an inhibitor increases the reduction state of electron carriers on the substrate side of the inhibitor, whereas those in the oxygen (opposite) side change to a more oxidized state. Thus, an increase in oxygen radical production following the addition of an inhibitor means that the oxygen radical generator is located on the substrate side of the block. Conversely, if oxygen radical production decreases after addition of the inhibitor, the generator must be situated on the oxygen side. Thus, the higher H2O2 production observed in the presence than in the absence of rotenone in pyruvate/ malate-supplemented rat mitochondria (Figure 16.1A) indicates that they produce ROS at complex I since this is the only complex situated on the substrate side of the inhibitor in this experiment.
Similarly, the classically described increase in H2O2 production after addition of antimycin A to succi-nate(+rotenone)-supplemented rat heart mitochondria (Figure 16.1B) is due to ROS production at complex III. On the other hand, in contrast with the experiment with pyruvate/malate, addition of rotenone to mitochondria respiring with succinate decreases H2O2 production, indicating that part of the ROS generated with this substrate comes from complex I (Herrero and Barja, 1997; Lambert and Brand, 2004). In this experiment rotenone blocks the reverse flow of electrons from succinate to complex I and thus its capacity for ROS production. Similar experiments can be used to localize the sites involved in changes in ROS generation induced by experimental manipulations in animals.
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