Protomer-Dependent Electronic Spectroscopy and Photochemistry of the Model Flavin Chromophore Alloxazine

Flavin chromophores play key roles in a wide range of photoactive proteins, but key questions exist in relation to their fundamental spectroscopic and photochemical properties. In this work, we report the first gas-phase spectroscopy study of protonated alloxazine (AL∙H⁺), a model flavin chromophore. Laser photodissociation is employed across a wide range (2.34⁻5.64 eV) to obtain the electronic spectrum and characterize the photofragmentation pathways. By comparison to TDDFT quantum chemical calculations, the spectrum is assigned to two AL∙H⁺ protomers; an N5 (dominant) and O4 (minor) form. The protomers have distinctly different spectral profiles in the region above 4.8 eV due to the presence of a strong electronic transition for the O4 protomer corresponding to an electron-density shift from the benzene to uracil moiety. AL∙H⁺ photoexcitation leads to fragmentation via loss of HCN and HNCO (along with small molecules such as CO₂ and H₂O), but the photofragmentation patterns differ dramatically from those observed upon collision excitation of the ground electronic state. This reveals that fragmentation is occurring during the excited state lifetime. Finally, our results show that the N5 protomer is associated primarily with HNCO loss while the O4 protomer is associated with HCN loss, indicating that the ring-opening dynamics are dependent on the location of protonation in the ground-state molecule.