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

Abstract

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.