pB(2) intermediate of the photoactive yellow protein: structure and excitation energies

pB(2) is the last electronically excited intermediate of the photoactive yellow protein (PYP) before it thermally reverts to the dark state. We investigate the structure of pB(2) by quantum refinement and QM/MM methods and compare our results with a previously published crystal structure ( 1TS6 ). We find differences in the chromophore geometries, mostly with regard to torsion angles, that lead to a somewhat higher degree of planarity than in 1TS6 . Quantum chemical calculations at the DFT/MRCI level show that these geometry changes affect the excitation energies and oscillator strengths for transitions into the two lowest singlet excited states. The DFT/MRCI results for the presently determined structures agree well with the experimental spectrum. A classical molecular dynamics simulation of free water molecules surrounding the fixed-in-space PYP indicates hydrogen bonding between water and the solvent-exposed pB(2) chromophore, since one water molecule is constantly found around the phenolic OH group. Including this additional water molecule in the quantum refinement yields improved electron density maps with a good fit of previously unresolved densities. On the other hand, this extra water molecule has little influence on the chromophore structure and the calculated excitation energies. The overall best pB(2) structure from the present work comes from quantum refinement with electrostatics and with an additional water molecule near the phenolic OH group of the chromophore (model 4).