Topology of superoxide and hydrogen peroxide production in mitochondria.

摘要

The mitochondrial electron transport chain is the major cellular source of superoxide anion (O₂·−) largely originating from autoxidation of ubisemiquinone. O₂· −, generated in this manner, can be vectorially released towards the mitochondrial matrix or the intermembrane space, for ubisemiquinone formation occurs on the inner membrane both near the matrix (Q_I site) and the intermembrane space (Q_O site). The vectorial release of O ₂·− into the matrix space is a well-established notion, while the release of O₂·− towards the intermembrane space has not been explored. To address this issue, liver mitoplasts, mitochondria with portions of the outer membrane removed, and heart mitochondria were generated as a model system to study O₂ ·− production into the intermembrane space. EPR analysis in conjunction with spin traps of antimycin-supplemented mitoplasts revealed the formation of a DMPO-hydroxyl adduct originating from the spontaneous decay of a DMPO-superoxide adduct. The EPR signal was (a) abrogated by superoxide dismutase, (b) competitively decreased by exogenous ferricytochrome c and (c) broadened by the membrane impermeable spin broadening agent chromium trioxalate. These results confirm the production and release of O₂·− towards the cytosolic side of the inner mitochondrial membrane. In isolated heart mitochondria O ₂·− was also measured diffusing across the porous outer membrane. In the presence of glutamate/malate, heart mitochondria are estimated to release 0.025 nmol/min/mg protein of O₂ ·− into the cytoplasm, a value increased to 0.50 nmol/min/mg in the presence of antimycin. These EPR estimates were confirmed by two other approaches: (1) hydroethidine, a dye that is specifically oxidized by O₂·−; (2) p-hydroxyphenylacetate and horseradish peroxidase, which measures H₂O₂ originating from O₂·− disproportionation.; The mitochondrial respiratory chain is therefore responsible for O ₂·− in the matrix, intermembrane space, and cytoplasm, H₂O₂, originating from dismutation of O₂·− generated by the respiratory chain, can also be formed in the matrix, intermembrane space, and cytoplasm. In addition, catalase in heart mitochondria is shown to play only a minor role in modulating H₂O₂ steady-state levels in the matrix. These results provide a new topology of O₂·− and H₂O₂ production in mitochondria.