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 (~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.
Keywords: femtosecond-stimulated Raman spectroscopy (FSRS); flavins; kinetic isotope effect (KIE); lifetime distribution analysis (LDA); light-oxygen-voltage (LOV) photosensors; maximum entropy method; photobiology; photochemistry; protein structural dynamics; time-resolved vibrational spectroscopy; transient visible absorption (visTA) spectroscopy.