Electronic State Mixing and Its Stimulated Raman Signature: A Case Study Using Free FMN and LOV Domain Proteins

In our previous investigation of the excited-state Raman spectra of free flavin mononucleotide (FMN) and the light-oxygen-voltage (LOV) sensitive protein EL222, we observed intensity variations and a red shift in the N 1 –C 2 stretching modes within the S 1 state Raman spectroscopy of EL222. This phenomenon was attributed to potential S 1 /S 2 state mixing. However, the existence of T 1 /T 2 mixing in EL222 remained undetermined. To address this, we employed an avoided crossing model to simulate the influence of electronic state mixing on Raman spectroscopy. Our results indicate that mixing induces redshift or blueshift in the 0–1 Raman transition frequency and alters the equilibrium position, consequently affecting the corresponding Raman intensity. The analysis further suggests that S 1 /S 2 state mixing in EL222 occurs within the weak coupling regime. Additionally, experimental data revealed potential Raman signatures indicative of T 1 /T 2 mixing in EL222.