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.

ELI Beamlines Facility Extreme Light Infrastructure ERIC 25241 Dolni Brezany Czech Republic

Faculty of Science Charles University 12800 Prague Czech Republic

Institute of Biotechnology of the Czech Academy of Sciences 25250 Vestec Czech Republic

Institute of Molecular Science University of Valencia 46980 Paterna Spain

The Hamburg Centre for Ultrafast Imaging Hamburg University 22761 Hamburg Germany

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Light-dependent flavin redox and adduct states control the conformation and DNA-binding activity of the transcription factor EL222

On the feasibility of time-resolved X-ray powder diffraction of macromolecules using laser-driven ultrafast X-ray sources

Genetically encoded non-canonical amino acids reveal asynchronous dark reversion of chromophore, backbone, and side-chains in EL222