Chromophore Dynamics in the PYP Photocycle from Femtosecond Stimulated Raman Spectroscopy

Femtosecond stimulated Raman spectroscopy (FSRS) is used to examine the structural dynamics of the para-hydroxycinnamic acid (HCA) chromophore during the first 300 Ps of the photoactive yellow protein (PYP) photocycle, as the system transitions from its vertically excited state to the early ground state cis intermediate, I-0. A downshift in both the C-7=C-8 and C-1=O stretches upon photoexcitation reveals that the chromophore has shifted to an increasingly quinonic form in the excited state, indicating a charge shift from the phenolate moiety toward the C-9=O carbonyl, which continues to increase for 170 Is. In addition, there is a downshift in the C-9=O carbonyl out-of-plane vibration on an 800 fs time scale as PYP transitions from its excited state to I-0, indicating that weakening of the hydrogen bond with Cys69 and out-of-plane rotation of the C-9=O carbonyl are key steps leading to photoproduct formation. HOOP intensity increases on a 3 ps time scale during the formation of I-0, signifying distortion about the C-7=C-8 bond. Once on the I-0 surface, the C-7=C-8 and C-1=O stretches blue shift, indicating recovery of charge to the phenolate, while persistent intensity in the HOOP and carbonyl out-of-plane modes reveal HCA to be a cissoid structure with significant distortion about the C-7=C-8 molecular plane.