SINGLET OXYGEN PRODUCED BY PHOTODYNAMIC-ACTION CAUSES INACTIVATION OFTHE 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 m u 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 nuc leotide translocase was inactivated, but mitochondria retained their a bility to form a proton electrochemical gradient and accumulated Ca2and P-i at the same rate as non-irradiated controls, Strikingly, howev er, 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, hematop orphyrin-loaded mitochondria treated with diethyl pyrocarbonate or dia mide 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 P ernardi, P, (1994) J, Biol. Chem. 269, 16638-16642), These data sugges t (i) that the dithiol cysteines are not oxidized by photodynamic acti on, but rather became inaccessible to oxidants; and (ii) that irradiat ion of hematoporphyrin-loaded mitochondria does not lead to pore denat uration, but rather to site-selective inactivation of discrete pore fu nctional domains.