Probing the Triplet State by Cryogenic Ion Trap IR Spectroscopy─Application to Protonated Lumiflavin

Flavins are essential biological cofactors and their protonation isomers (protomers) generated upon photoexcitation play a crucial role in their photochemical reactions. Here, we identify the triplet-state (T₁) protomer of protonated lumiflavin (LFH⁺) in a cryogenic ion trap by visible-pump infrared-probe photodissociation spectroscopy using H₂-tagging (H₂-tagging Vis-IRPD). This approach yields high-resolution IR spectra of cold triplet ions which, along with quantum chemical calculations, enables structural assignments and population analysis of the identified O2-<i>cis</i>@T₁ protomer of LFH⁺. Furthermore, the implementation of a tickle radio frequency technique (Vis-tRF-IRPD) effectively removes nonexcited species from the ion trap, allowing vibrational signatures of the T₁ state to be isolated. This work establishes a fundamental platform for monitoring protonation-site switching in photoexcited flavins (S_<i>n</i> or T_<i>n</i>) and demonstrates the broader utility of H₂-tagging Vis-IRPD spectroscopy for electronic-state- and conformation-resolved studies of cold molecular ions.