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 α-tocopherol and a scavenger of singlet oxygen (1O2), provide photoprotection, under high irradiance, to the isolated photosystem II (PSII) reaction center (RC)? To answer the question, we studied the endogenous production of 1O2 in preparations of the five-chlorophyll PSII RC (RC5) containing only one β-carotene molecule. The temporal profile of 1O2 emission at 1270 nm photogenerated by RC5 in D2O followed the expected biexponential behavior, with a rise time, unaffected by Trolox, of 13 ± 1 μs and decay times of 54 ± 2 μs (without Trolox) and 38 ± 2 μs (in the presence of 25 μM Trolox). The ratio between the total (kt) and chemical (kr) bimolecular rate constants for the scavenging of 1O2 by Trolox in aqueous buffer was calculated to be ∼1.3, with a kt of (2.4 ± 0.2) × 108 M–1 s–1 and a kr of (1.8 ± 0.2) × 108 M–1 s–1, indicating that most of the 1O2 photosensitized by methylene blue chemically reacts with Trolox in the assay buffer. The photoinduced oxygen consumption in the oxygen electrode, when RC5 and Trolox were mixed, revealed that Trolox was a better 1O2 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 RC5 pigments, which were oxidized, together with the membrane region of the protein matrix of the PSII RC, by 1O2. These results are discussed and compared with those of studies dealing with the physiological role of tocopherol molecules as a 1O2 scavenger in thylakoid membranes of photosynthetic organisms.