Hematoporphyrin (HP)-mediated photooxidative stress can either prevent or activate the mitochondrial permeability transition (PT) depending on the site of porphyrin/target localization and on the light dose. Under irradiation with moderate light doses, HP situated in matrix-exposed sites of the PT pore (PTP) promotes photosensitization of key His residues leading to PT inhibition. Under irradiation with high light doses, PT is re-activated through photomodification of external Cys residues by vicinal HP. Here we checked whether the peculiar photosensitizing properties of HP on the PTP were maintained in mitoplasts (inner membrane preparations) obtained by treatment with proper digitonin concentrations. Mitoplast purity was verified by enzymatic analysis of the outer membrane marker monoamino oxidase and by electron microscopy. In analogy with the results obtained in intact mitochondria, irradiation of HP-treated mitoplasts at low light doses caused PT inhibition that was counteracted by diethyl pyrocarbonate, indicating that it resulted from photomodification of PTP-regulating His residues. At variance from mitochondria, however, in mitoplasts the PT could not be reactivated after exposure to prolonged irradiation periods, yet opening of a CsA-sensitive PTP could be still observed upon addition of the membrane-impermeant, thiol-oxidant copper-o-phenanthroline [Cu(OP)2], indicating that mitoplasts retain the external PTP-regulating sulfhydryls. Ablation of PT re-activation in mitoplasts was specific for dicarboxylic porphyrins endowed with protoporphyrin IX (PP) configuration, such as deuteroporphyrin (DP) and PP itself, which exhibit nanomolar affinity for the outer membrane-associated translocator protein of 18 kDa (TSPO, formerly called peripheral benzodiazepine receptor); whereas PP-unrelated porphyrins did not affect mitochondria or mitoplasts under irradiation. We suggest that in intact mitochondria thiol-sensitizing HP interferes with the PTP through interaction with specific regions of the TSPO
Characterization of the permeability transition pore in mitoplasts exposed to photooxidative stress
Petronilli Valeria;Bernardi Paolo;Ricchelli Fernanda
2010
Abstract
Hematoporphyrin (HP)-mediated photooxidative stress can either prevent or activate the mitochondrial permeability transition (PT) depending on the site of porphyrin/target localization and on the light dose. Under irradiation with moderate light doses, HP situated in matrix-exposed sites of the PT pore (PTP) promotes photosensitization of key His residues leading to PT inhibition. Under irradiation with high light doses, PT is re-activated through photomodification of external Cys residues by vicinal HP. Here we checked whether the peculiar photosensitizing properties of HP on the PTP were maintained in mitoplasts (inner membrane preparations) obtained by treatment with proper digitonin concentrations. Mitoplast purity was verified by enzymatic analysis of the outer membrane marker monoamino oxidase and by electron microscopy. In analogy with the results obtained in intact mitochondria, irradiation of HP-treated mitoplasts at low light doses caused PT inhibition that was counteracted by diethyl pyrocarbonate, indicating that it resulted from photomodification of PTP-regulating His residues. At variance from mitochondria, however, in mitoplasts the PT could not be reactivated after exposure to prolonged irradiation periods, yet opening of a CsA-sensitive PTP could be still observed upon addition of the membrane-impermeant, thiol-oxidant copper-o-phenanthroline [Cu(OP)2], indicating that mitoplasts retain the external PTP-regulating sulfhydryls. Ablation of PT re-activation in mitoplasts was specific for dicarboxylic porphyrins endowed with protoporphyrin IX (PP) configuration, such as deuteroporphyrin (DP) and PP itself, which exhibit nanomolar affinity for the outer membrane-associated translocator protein of 18 kDa (TSPO, formerly called peripheral benzodiazepine receptor); whereas PP-unrelated porphyrins did not affect mitochondria or mitoplasts under irradiation. We suggest that in intact mitochondria thiol-sensitizing HP interferes with the PTP through interaction with specific regions of the TSPOI documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
