Moderate dependence of ROS formation on Δψm in isolated brain mitochondria supported by NADH-linked substrates

The membrane potential (Delta Psi m) dependence of the generation of reactive oxygen species (ROS) in isolated guinea-pig brain mitochondria respiring on NADH-linked substrates (glutamate plus malate) was addressed. Depolarization by FCCP was without effect on H2O2 formation in the absence of bovine serum albumin (BSA). Addition of BSA (0.025%) to the assay medium hyperpolarized mitochondria by 6.1 +/- 0.9 mV (from 169 +/- 3 to 175.1 +/- 2.1 mV) and increased the rate of H2O2 formation from 207 +/- 4.5 to 312 +/- 12 pmol/min/mg protein. Depolarization by FCCP (5-250 nM) in the presence of BSA decreased H2O2 formation but only to the level observed in the absence of BSA. Rotenone stimulated the formation of H2O2 both in the absence and presence of BSA. It is suggested that H2O2 formation in mitochondria supported by NADH-linked substrates is sensitive to changes in Delta psi m only when mitochondria are highly polarized and even then, 60% of ROS generation is independent of Delta Psi m. This is in contrast to earlier reports on the highly Delta Psi m sensitive ROS formation related to reverse electron flow observed in well-coupled succinate-supported mitochondria.