Femtosecond dynamics of rhodopsin photochemistry probed by a double pump spectroscopic approach

Femtosecond pump-probe measurements of the excitation pathway of bovine rho dopsin to bathorhodopsin show varying transient events in the photoinduced absorption spectrum. In the spectral region where bathorhodopsin absorption dominates in the rhodopsin-batharhodopsin difference spectrum, subpicoseco nd (200-600 fs) and 3 ps signals are observed. In the region where rhodopsi n absorption dominates, a 3-5 ps transient is observed, which follows rhodo psin repopulation. To unravel the different models of the rhodopsin-bathorh odopsin dynamics that have been proposed to explain these transient observa tions, we have employed a three-beam experiment. The first pump beam launch es the photochemistry, a second pump pulse interrupts the spontaneous evolu tion of the trajectory of excited rhodopsin at a delayed time from the firs t pump pulse, and a weak third beam probes changes in the amount of bathorh odopsin that is formed in response to the second pump beam. We first find t hat bathorhodopsin can be photoexcited, presumably back to rhodopsin, by re versing the initial photochemistry. We find that bathorhodopsin is formed v ery promptly (within the 300 fs resolution of our experiments). Surprisingl y, the kinetics in the absorbance signal near 605 nm is found to be the sup erposition of two independent signals, one being characterized by events on the subpicosecond time scale and the other at 3 ps. The results suggest th at the trajectory of excited rhodopsin splits into two pathways very early: one of which results in bathorhodopsin formation on the similar to 200 fs time scale and another which lives in the excited state for ca. 3 ps before undergoing internal conversion to rhodopsin.