Abstract
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 νC
8
-C
9
marker modes for solution phase PYP in the P and I
0
states, from which we derive that the first intermediate state I
0
, 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 I
0
correlates with the conversion from the trans to the cis state. For the P and I
0
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 to have a strong influence. The C
7
=C
8
-(C
9
=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 a X-ray diffraction study of PYP crystals. In the case of I
0
, experiment and theory are only in agreement when the C
7
=C
8
-(C
9
=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 in going from the electronic ground P state, via the electronic excited P* state to the intermediate I
0
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.