Extended Data Fig. 1: QM/MM approach for electronic transitions.

QM/MM approach for electronic transitions. Excitation energies for the S₀ → S₁ transitions were computed using RI-CC2/cc-pVDZ for different models with protonated and deprotonated pyrrole rings by transferring the proton to the bulk or to adjacent amino acid side chains (see (f)). The model with a fully protonated BV chromophore is consistent with the Pfr ground state and has an S₀ → S₁ excitation energy of 15645 cm⁻¹, 2347 cm⁻¹ higher than the experimentally measured absorption maximum. Steady state fluorescence spectra of Agp2-WT are not available due to its ultrafast excited state deactivation. Calculated transition energies differ slightly from the experimental ones, but relative transition energies differences are more precise. Thus, we compare spectral shifts. We transferred the proton from ring B, C, and D to bulk water, to H248 or D196, or changed the protonation state of H248 from ε to δ (see (f)). (a) and (b): Difference in electrostatic potential between first excited state and ground state of BV in Pfr. Upon excitation the electron density increases at ring B (blue color). Negative values (blue) indicate a decrease when going from the ground to the first excited state, while positive values (red) indicate an increase. The electrostatic potential is mapped onto the electron density at an isovalue of 0.02. (a) Difference in electrostatic potential with only BV (excluding propionates) inside the QM region. (b) Same as in (a) but with Y251. For both QM regions the potential increases near the D ring (bottom right) and decreases near the B ring (upper left). c) and d): Density differences between first excited state and ground state of BV in Pfr. Negative values (blue), positive values (red, the density difference is visualised for an isovalue of ±0.001). (c) Transition density with transition dipole moment (TDM) vector for the S₀ → S₁ transition. The TDM(x,y,z)=(4.72, 1.60, 0.43) a.u. (d) Electron density difference (EDD) between first excited state and ground state of BV in Pfr. The EDD was computed with CC2/cc-pVDZ and shows how the electron density shifts upon excitation from the ground (blue) to the first excited state (red). Changes in the EDD are mostly located on the B and C ring of BV. Contribution of Y251 to the excitation is negligible. There are also other tyrosines, for example Y165, but the electronic TDM of these tyrosines are located perpendicular to the TDM of BV, excluding an efficient coupling. (e) Table: Excitation from the ground state (S₀) to the first excited state (S₁) for S₀- and S₁-optimised structures (absorption and emission, respectively), as well as S₀ and S₁ transition dipole moments (TDM).