Structural evolution of the chromophore in the primary stages of trans/cis isomerization in photoactive yellow protein

We have studied the structural changes induced by optical excitation of the chromophore in wild-type photoactive yellow protein (PYP) in liquid solution with a combined approach of polarization-sensitive ultrafast infrared spectroscopy and density functional theory calculations. We identify the νC₈-C₉ marker modes for solution phase PYP in the P and I₀ states, from which we derive that the first intermediate state I₀ that appears with a 3 ps time constant can be characterized to have a cis geometry. This is the first unequivocal demonstration that the formation of I₀ correlates with the conversion from the trans to the cis state. For the P and I₀ states we compare the experimentally measured vibrational band patterns and anisotropies with calculations and find that for both trans and cis configurations the planarity of the chromophore has a strong influence. The C₇=C₈-(C₉=O)-S moiety of the chromophore in the dark P state has a trans geometry with the C=O group slightly tilted out-of-plane, in accordance with the earlier reported structure obtained in an X-ray diffraction study of PYP crystals. In the case of I ₀, experiment and theory are only in agreement when the C ₇=C₈-(C₉=O)-S moiety has a planar configuration. We find that the carboxylic side group of Glu46 that is hydrogen-bonded to the chromophore phenolate oxygen does not alter its orientation on going from the electronic ground P state, via the electronic excited P* state to the intermediate I0 state, providing conclusive experimental evidence that the primary stages of PYP photoisomerization involve flipping of the enone thioester linkage without significant relocation of the phenolate moiety.