In our previous work, we applied transient absorption and femtosecond stimulated Raman spectroscopy (FSRS) to free flavin mononucleotide (FMN), a biomolecule that functions as prosthetic group and cofactor in a myriad of biochemical processes. Time-resolved spectra were taken with time delays up to a few nanosecond after FMN photoexcitation and the observed Raman bands in the excited singlet S1 and triplet T1 states were assigned with the help of quantum chemistry calculations. Here in order to understand the influence of the protein environment on the photophysics of FMN, we measured FSRS of FMN bound to EL222, a photosensory receptor containing a light-oxygen-voltage (LOV) domain, with time delays from 100 femtosecond to 0.5 milliseconds. The displacement amplitude between the excited state potential energy surface (PES) minima was estimated using the displaced harmonic oscillator model. We propose a change in the topography of the potential energy surface in the S1 state due to protein-mediated mixing of the bright ππ* state and the dark nπ* state of FMN. The mixing is supported by the decrease of the corresponding transition dipole moment with a higher electronic state as a result of protein-chromophore interactions. We speculate that such a mixing may arise from non-symmetric hydrogen bonds between the O4’ atom of FMN and two surrounding asparagine residues present in the binding pocket. The mixing might also be favoured by a smaller energy gap between these two states due to favourable interactions between the FMN moiety and the LOV cage. In principle, the nπ* feature of the S1 state will decrease the electron density of FMN N5 atom and reduce its proton affinity. This provides a perspective to understand the primary photochemical reaction in the LOV domain that occurs on the T1 state rather than the S1 state.
1. P.C. Andrikopoulos, Y. Liu, A. Picchiotti, N. Lenngren, M. Kloz, A.S. Chaudhari, M. Precek, M. Rebarz, J. Andreasson, J. Hajdu, B. Schneider, G. Fuertes, Phys. Chem. Chem. Phys. 22 (2020) 6538–6552.
This work was supported by the projects ADONIS (CZ.02.1.01/0.0/0.0/16_019/0000789) and ELIBIO (CZ.02.1.01/0.0/0.0/15_003/ 0000447), both from the European Regional Development Fund and MEYS. The Institute of Biotechnology of the Czech Academy of Sciences acknowledges the institutional grant RVO 86652036.