Trolox, a Water-Soluble Analogue of α-Tocopherol, Photoprotects the Surface-Exposed Regions of the Photosystem II Reaction Center in Vitro. Is This Physiologically Relevant?

Can Trolox, a water-soluble analogue of alpha-tocopherol and a scavenger of singlet oxygen (O-1(2)), provide photoprotection, under high irradiance, to the isolated photosystem II (PSII) reaction center (RC)? To answer the question, we studied the endogenous production of O-1(2) in preparations of the five-chlorophyll PSII RC (RCS) containing only one beta-carotene molecule. The temporal profile of O-1(2) emission at 1270 nm photogenerated by RCS in D2O followed the expected biexponential behavior, with a rise time, unaffected by Trolox, of 13 +/- 1 mu s and decay times of 54 +/- 2 mu s (without Trolox) and 38 +/- 2 mu s (in the presence of 25 mu M Trolox). The ratio between the total (k(t)) and chemical (k(r)) bimolecular rate constants for the scavenging of O-1(2) by Trolox in aqueous buffer was calculated to be similar to 1.3, with a k(t) of (2.4 +/- 0.2) x 10(8) M-1 s(-1) and a k(r) of (1.8 +/- 0.2) x 10(8) M-1 s(-1), indicating that most of the O-1(2) photosensitized by methylene blue chemically reacts with Trolox in the assay buffer. The photoinduced oxygen consumption in the oxygen electrode, when RCS and Trolox were mixed, revealed that Trolox was a better O-1(2) scavenger than histidine and furfuryl alcohol at low concentrations (i.e., <1 mM). After its incorporation into detergent micelles in unbuffered solutions, Trolox was able to photoprotect the surface-exposed regions of the D1-D2 heterodimer, but not the RCS pigments, which were oxidized, together with the membrane region of the protein matrix of the PSII RC, by O-1(2). These results are discussed and compared with those of studies dealing with the physiological role of tocopherol molecules as a O-1(2) scavenger in thylakoid membranes of photosynthetic organisms.