To prepare molecular model systems we utilized coordinates of heavy atoms from the structures PDB ID 6TEG and PDB ID 6SGG . The first structure refers to the RutA-FMN-O2-uracil complex, which we sometimes call below for brevity as the ROU complex. The second structure corresponds to the complex without uracil, RutA-FMN-O2 (the RO complex). Hydrogen atoms were added assuming the conventional protonation states of the polar residues at neutral pH -Arg, Lys (positively charged); Glu, Asp (negatively charged); the histidine residues were assumed in the neutral state. Water molecules resolved in the crystal structure were kept in the model system, and solvation water box was built using the visual molecular dynamics (VMD) program. The CHARMM36 force field topology and parameters were employed along with the FMN parameters in the reduced form (total charge -3) taken from Ref. and those for the molecular oxygen taken from Ref. , while water molecules were treated as TIP3P. Classical MD trajectories were simulated with the NAMD 3.0 software package. The isothermal-isobaric (NPT) ensemble at P = 1 atm and T = 300 K using the Nosé-Hoover Langevin piston pressure control and the Langevin dynamics were employed. Periodic boundary conditions and the particle mesh Ewald algorithm to account for the longrange electrostatic interactions were applied, whereas the non-bonded interaction cut-off parameter was set to 12 Å; integration step was set to 1 fs. A harmonic constraint potential of 1 kcal/Å 2 was applied to the CA atoms of the protein, except for the residues #290-310 of the region that poorly resolved in the crystal structures. In sum, over 500 ns of combined trajectories were produced. The QM/MM MD trajectories were computed with the TeraChem/NAMD via an appropriate interface. Calculations were initiated from selected frames of classical MD. The energies and forces in QM parts were computed with the density functional theory (DFT) with the range-separated ωb97X functional and the D3 dispersion correction. The 6-31G** basis set was employed for carbon and hydrogen atoms and 6-31+G** for nitrogen and oxygen atoms. The MM part was treated by the CHARMM36 force field, water molecules were described by the TIP3P model.