Differentiating between apparent and actual rates of H(2)O(2) metabolism by isolated rat muscle mitochondria to test a simple model of mitochondria as regulators of H(2)O(2) concentration

Mitochondria are often regarded as a major source of reactive oxygen species (ROS) in animal cells, with H(2)O(2) being the predominant ROS released from mitochondria; however, it has been recently demonstrated that energized brain mitochondria may act as stabilizers of H(2)O(2) concentration (Starkov et al. [1]) based on the balance between production and the consumption of H(2)O(2), the later of which is a function of [H(2)O(2)] and follows first order kinetics. Here we test the hypothesis that isolated skeletal muscle mitochondria, from the rat, are able to modulate [H(2)O(2)] based upon the interaction between the production of ROS, as superoxide/H(2)O(2), and the H(2)O(2) decomposition capacity. The compartmentalization of detection systems for H(2)O(2) and the intramitochondrial metabolism of H(2)O(2) leads to spacial separation between these two components of the assay system. This results in an underestimation of rates when relying solely on extramitochondrial H(2)O(2) detection. We find that differentiating between these apparent rates found when using extramitochondrial H(2)O(2) detection and the actual rates of metabolism is important to determining the rate constant for H(2)O(2) consumption by mitochondria in kinetic experiments. Using the high rate of ROS production by mitochondria respiring on succinate, we demonstrate that net H(2)O(2) metabolism by mitochondria can approach a stable steady-state of extramitochondrial [H(2)O(2)]. Importantly, the rate constant determined by extrapolation of kinetic experiments is similar to the rate constant determined as the [H(2)O(2)] approaches a steady state.