Scavenging of H₂O₂ by mouse brain mitochondria

Abstract

Mitochondrial reactive oxygen species (ROS) metabolism is unique in that mitochondria both generate and scavenge ROS. Recent estimates of ROS scavenging capacity of brain mitochondria are surprisingly high, ca. 9-12 nmol H₂O₂/min/mg, which is ~100 times higher than the rate of ROS generation. This raises a question whether brain mitochondria are a source or a sink of ROS. We studied the interaction between ROS generation and scavenging in mouse brain mitochondria by measuring the rate of removal of H₂O₂ added at a concentration of 0.4 μM, which is close to the reported physiological H₂O₂ concentrations in tissues, under conditions of low and high levels of mitochondrial H₂O₂ generation. With NAD-linked substrates, the rate of H₂O₂ generation by mitochondria was ~50–70 pmol/min/mg. The H₂O₂ scavenging dynamics was best approximated by the first order reaction equation. H₂O₂ scavenging was not affected by the uncoupling of mitochondria, phosphorylation of added ADP, or the genetic ablation of glutathione peroxidase 1, but decreased in the absence of respiratory substrates, in the presence of thioredoxin reductase inhibitor auranofin, or in partially disrupted mitochondria. With succinate, the rate of H₂O₂ generation was ~2,200–2,900 pmol/min/mg; the scavenging of added H₂O₂ was masked by a significant accumulation of generated H₂O₂ in the assay medium. The obtained data were fitted into a simple model that reasonably well described the interaction between H₂O₂ scavenging and production. It showed that mitochondria are neither a sink nor a source of H₂O₂, but can function as both at the same time, efficiently stabilizing exogenous H₂O₂ concentration at a level directly proportional to the ratio of the H₂O₂ generation rate to the rate constant of the first order scavenging reaction.