Effect of a LOV Protein Matrix on Flavin Photocycle Probed by Transient Resonance Raman Spectroscopy and Theoretical Calculations

Y. Liu^{1, 2},^* P. C. Andrikopoulos¹, A.S. Chaudhari¹, A. Picchiotti^{1, 3}, N. Lenngren², M. Rebarz², M. Kloz², B. Schneider², J. Hajdu^{2, 4}, J. Andreasson^{2, 5}, G. Fuertes¹

¹ Institute of Biotechnology of the Czech Academy of Sciences, BIOCEV, Průmyslová 595, CZ-252 50 Vestec, Czechia

² Institute of Physics of the Czech Academy of Sciences, ELI Beamlines, Za Radnicí 835, CZ-252 41 Dolní Břežany, Czechia

³ Deutsches Elektronen-Synchrotron DESY, Notkestrasse 85 DE-22607 Hamburg, Germany

⁴ Department of Cell and Molecular Biology, Uppsala University, Husargatan 3 (Box 596), SE-751 24 Uppsala, Sweden

⁵ Condensed Matter Physics, Department of Physics, Chalmers University of Technology, SE-412 96 Gothenburg, Sweden

Yingliang.liu2@eli-beams.eu

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.[1] 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 O_4’ 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.