Sub-Millisecond Photoinduced Dynamics of Free and EL222-Bound FMN by Stimulated Raman and Visible Absorption Spectroscopies

Time-resolved femtosecond-stimulated Raman spectroscopy (FSRS) provides valuable information on the structural dynamics of biomolecules. However, FSRS has been applied mainly up to the nanoseconds regime and above 700 cm(-1), which covers only part of the spectrum of biologically relevant time scales and Raman shifts. Here we report on a broadband (similar to 200-2200 cm(-1)) dual transient visible absorption (visTA)/FSRS set-up that can accommodate time delays from a few femtoseconds to several hundreds of microseconds after illumination with an actinic pump. The extended time scale and wavenumber range allowed us to monitor the complete excited-state dynamics of the biological chromophore flavin mononucleotide (FMN), both free in solution and embedded in two variants of the bacterial light-oxygen-voltage (LOV) photoreceptor EL222. The observed lifetimes and intermediate states (singlet, triplet, and adduct) are in agreement with previous time-resolved infrared spectroscopy experiments. Importantly, we found evidence for additional dynamical events, particularly upon analysis of the low-frequency Raman region below 1000 cm(-1). We show that fs-to-sub-ms visTA/FSRS with a broad wavenumber range is a useful tool to characterize short-lived conformationally excited states in flavoproteins and potentially other light-responsive proteins.

Automated summary

A broadband dual transient visible absorption (visTA)/femtosecond stimulated Raman spectroscopy (FSRS) set-up was developed to monitor the complete excited-state dynamics of flavin mononucleotide (FMN) in solution and embedded in bacterial light-oxygen-voltage (LOV) photoreceptor EL222. The set-up covers a wide range of time delays and Raman shifts, providing valuable information on short-lived conformationally excited states. Additional dynamical events were found in the low-frequency Raman region below 1000 cm(-1).