Primary reaction intermediates of Type-I photosensitized lipid oxidation as revealed by time-resolved optical spectroscopies

Chlorophyll a (Chl a) as a lipophilic photosensitizer can induce biomembrane destruction via Type-II reaction involving singlet oxygen (O-1(2)) as a primary initiator. Type-I oxidation by an excited-state photosensitizer reacting directly with lipid substrate also contributes but the primary intermediate remains to be verified experimentally. We have investigated the reaction dynamics initiated by Chl a-photosensitization involving oxygen and the -C=C-moieties of lipids in the membranes of small unilamellar vesicles (SUVs) prepared from phospholipids of different degree of unsaturation (0, 1 or 2). Under anaerobic condition, femtosecond time resolved absorption (fs-TA) combined with spectroelectrochemical spectroscopies validated the formation of Chl a(center dot-) in subpicoseconds, and time resolved fluorescence spectroscopy revealed the rapid quenching of (1)Chl a* with a rate constant of (28 ns)(-1). These ultrafast processes, independent of oxygen, are ascribed to the electron transfer reaction from a -C=C-moiety to (1)Chl a* as an initiation step of Type-I reaction. On longer timescales, ns- TA spectroscopy unraveled the drastic quenching of 3Chl a* by either -C=C-or O-2, and the quenching by O-2 was found to be 20 times more efficient. This together with the O-1(2)-luminesence analysis prove the involvement of both types of photosensitization. In addition, HPLC-MS spectroscopy confirmed the ketonic, the alcoholic and the core-aldehyde products of lipid oxidation. Moreover, the oxygen dependent partition between Type-I and Type-II reactions is discussed on a detailed kinetics basis, showing that the two mechanisms of lipid oxidation are equally important under an oxygen concentration of 1.3x10(-5) M at room temperature.