Fifth-order time-domain Raman spectroscopy of photoactive yellow protein for visualizing vibrational coupling in its excited state.

Abstract

We report fifth-order time-domain Raman spectroscopy of photoactive yellow protein (PYP), with the aim to visualize vibrational coupling in its excited state. After the ultrashort actinic pump pulse prepared the vibrational coherence and population in the excited state, the evolving vibrational structure was tracked by time-resolved impulsive stimulated Raman spectroscopy using sub-7-fs pulses. The obtained fifth-order time-domain Raman data were translated to a two-dimensional (2D) frequency-frequency correlation map, which visualizes the correlation between low- and high-frequency vibrational modes of the excited state. The 2D map of PYP reveals a cross peak, indicating the coupling between the phenolic C─O stretch mode of the chromophore and the low-frequency modes (~160 cm-1), assignable to the intermolecular motions involving the surrounding hydrogen-bonded amino acids. The unveiled coupling suggests the importance of the low-frequency vibrational motion in the primary photoreaction of PYP, highlighting the unique capability of this spectroscopic approach for studying ultrafast reaction dynamics.