Nitroxide destruction and flavin-photosensitized damage in inner mitochondrial membranes

Abstract

Nitroxide free radicals lost their paramagnetic absorption spectrum when they were illuminated with visible light in aged but not freshly isolated inner mitochondrial membranes (SMP's). The action spectrum of spin loss rates coincided with a flavin absorption spectrum. Spin loss consisted of one-electron reduction and "destruction", the latter being defined as spin loss that cannot be reversed by ferricyanide oxidation. By placing SMP's in gas permeable tubing and illuminating alternately under nitrogen and air, it was possible to discriminate qualitatively between spin reduction and destruction. Aerobic spin loss consisted entirely of destruction. When aged SMP's were centrifuged, spin loss was observed in supernatants but not pellets. Flavin fluorescence was observed in the supernatants, suggesting that free flavins catalyzed spin loss. However, addition of exogenous flavins to fresh SMP's did not cause spin loss; hence some other factor was required to cause nitroxide destruction. This factor accumulates during either aerobic or anaerobic aging of SMP's and may be present in mitochondria. Supernatants of aged SMP's, when added to freshly isolated SMP's, induced rapid nitroxide destruction, slightly accelerated photodamage to succinate oxidase, and considerably increased photo-induced lipid peroxidation.