Excited-State Mixing in the LOV Domain Proteins: Possible Physics behind the Difference in the Transient Absorption and Transient Stimulated Raman Spectroscopy

It remains uncertain whether excited electronic state mixing occurs in the flavin cofactor of the light-oxygen-voltage-sensing (LOV) domain. In this study, we present transient absorption and femtosecond stimulated Raman spectra of both free and EL222 binding flavin mononucleotide (FMN). We observed a change in the shape of the excited-state absorption around 800 nm in the S₁ state transient absorption after binding to EL222, alongside a relative intensity increase of the N₁-C₂ and C₂═O₂ stretching modes in the S₁ state Raman spectra. Based on the previous calculated geometric differences between the ππ* and nπ* states, we propose a probable electronic state mixing in EL222 binding FMN. This mixing is favored by the nonsymmetric hydrogen bonding interaction between the flavin O₄ atom and the asparagine residue and fewer hydrogen bonds with the O₂ atom in EL222.