An alternative mechanism can be proposed for the excitation and relaxation of the photosensitizers during the triplet energy transfer: the stepwise procedure where the vertical relaxation of the triplet state comes first and RIHT comes later, as shown in Figure . Note that the mechanism of the concerted or stepwise pathway is still under debate because of the undetectable phosphorescence for hydroxyanthraquinones. Two types of vertical relaxation were examined through DFT and time-dependent density functional theory (TD-DFT) calculations: the AQ series that (i) undergoes ESIHT in a stepwise manner or (ii) does not transfer their hydrogen upon excitation. In both of these cases, the reorganization energy is negligible and the singlettriplet energy gap is the only factor that determines the triplet energy transfer. According to our calculations, the ESIHT-assisted models exhibit an adequate energy gap to facilitate the triplet energy process. HQ, Ali, Qui, and Pur have an energy gap ranging from 10.8 to 22.0 kcal/mol that is insufficient for converting Taken together, our computational studies highlight the importance of additional hydroxyl groups and reveal that the position of hydrogen bond donors are key to facilitate the triplet energy transfer. The peaks assigned to oxidized Aβ40 species generated by photoactivation were further probed by ESI-MS 2 to identify the oxidized amino acid residues (Figure ). Aβ40 peptides photooxygenated with Pur (Group A) and Alo (Group B) to different extents were selected for tandem MS measurements. Figure exemplifies the collision-induced dissociation (CID) experiments on the singly oxidized peak of Aβ40 (1,448 m/z) obtained by photoexcited Pur. We observed b fragments smaller than b35 and larger than b12 in their nonoxidized and oxidized forms, respectively. The b fragments between b13 and b34 were found in both nonoxidized and oxidized forms. These observations imply that Pur oxidizes either His13, His14, or Met35 separately, but it is unable to simultaneously modify more than one residue. On the contrary, as disclosed in In the inhibition experiments, metal-free or metal-Aβ was freshly prepared and treated for 24 h with the AQ series, as shown in Figure . For light-exposed samples, Aβ species added with the AQ series were photoirradiated for 1 h prior to 24 h incubation. As portrayed in Figure , under dark conditions (grey gels), no significant change in the MW distribution of both metal-free and metal-Aβ40 was observed even with the addition of the AQ series. Notably, in the presence of light, the resultant Aβ40 species with the AQ series (blue gels) showed a divergent MW distribution, relative to Aβ40 only. Treatment of metal-free Aβ40 with light-activated 9,10-AQ, HQ, Ali, Qui, and Pur gave rise to increased signal intensities between ca. 15-35 kDa. Note that the AQ series can produce superoxide anion radicals; 61 however, we could not monitor them because the excitation and emission wavelengths used for the assays overlapped with those of the compounds. On the contrary, the illuminated Aβ40 samples with Dan, Rhe, and Alo greatly affected the MW distribution exhibiting new gel bands throughout ca. 15-240 kDa. Smearing bands over ca. 240 kDa were spotted for the samples added with both Group A and Group B indicating that the light-driven oxidation of metal-free Aβ40 leads to smaller peptide ensembles that can penetrate the gel matrix.