Heyne, K; Mohammed, OF; Usman, A; Dreyer, J; Nibbering, ETJ; Cusanovich, MA

JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 2005, 127(51), p. 18100-18106

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 v(C₈- 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 I₀, experiment and theory are only in agreement when the C₇=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.

DOI 10.1021/ja051210k