Singlet oxygen produced by photodynamic action causes inactivation of the mitochondrial permeability transition pore

We have studied the effects of singlet oxygen produced by photodynamic action on the cyclosporin A-sensitive permeability transition (PT) in isolated rat liver mitochondria. Mitochondria were incubated with 3 mu M hematoporphyrin and irradiated at 365 nm with a fluence rate of 25 watts/m(2). For short durations of irradiation (60 s) the adenine nucleotide translocase was inactivated, but mitochondria retained their ability to form a proton electrochemical gradient and accumulated Ca2+ and P-i at the same rate as non-irradiated controls, Strikingly, however, the oxidative effects of photodynamic action prevented opening of the PT pore which is normally induced by Ca2+ plus P-i or by treatment with diethyl pyrocarbonate (a histidine reagent) or diamide (a thiol oxidant), We show that the most likely targets for photodynamic action are critical histidines that undergo degradation. Irradiated, hematoporphyrin-loaded mitochondria treated with diethyl pyrocarbonate or diamide still undergo the PT when treated with phenylarsine oxide, which reacts with a critical dithiol involved in pore modulation (Petronilli, V,, Costantini, P,, Scorrano, L., Colonna, R,, Passamonti, S,, and Pernardi, P, (1994) J, Biol. Chem. 269, 16638-16642), These data suggest (i) that the dithiol cysteines are not oxidized by photodynamic action, but rather became inaccessible to oxidants; and (ii) that irradiation of hematoporphyrin-loaded mitochondria does not lead to pore denaturation, but rather to site-selective inactivation of discrete pore functional domains.