Extended Data Fig. 7: Computation of IR spectra.

Computation of IR spectra. Selected QM/MM calculated (*) and experimental vibrational frequencies of vibrational modes diagnostic of the chromophore geometry for the Pfr models as well as Lumi-F model in H₂O and D₂O. Frequencies are given in cm⁻¹. (*) The crystal structure of Agp2 in the Pfr state (PDB:6G1Y) was used as template for generating the initial structure for the Pfr and Lumi-F models. Unresolved regions in the crystal were restored by three-dimensional homology modelling using SWISS MODEL and hydrogens were added to the crystallographic structure according to predictions based on Karlsberg2 + . His248 and His278 were modeled as charge neutral each with a proton at Ne position. The Lumi-F model was generated based on the Pfr model by simply rotating the pyrrole ring D around 180 degrees. The chromophore binding site of the energy minimized- and thermally equilibrated solvated protein models were geometry optimized at QM/MM level. Accordingly, the biliverdin chromophore, the side chains of Cys13 and the pyrrole water were treated quantum mechanically at the B3LYP/6-31 G* level of theory while the protein matrix, solvent water and ions were described molecular mechanically using CHARMM36 force field. Relaxation during the minimization was allowed only for atoms located within a 20 Å-radius sphere centered at N22 of the BV cofactor. The charge-shifted scheme in combination with the electrostatic embedding approach was used to couple the QM and the MM region. The QM/MM optimized geometries were further used as input for subsequent frequency calculation of exclusively the QM fragment. These computations were performed at B3LYP/6-31 G* level of theory using GAUSSIAN09 following the same protocol as described previously in ref. ⁷⁹. Scaling of force constants, normal mode analysis as well as correction of the QM Hessian matrix were performed using the programs developed in our group.