856
J. Phys. Chem. B 2001, 105, 856-859
Femtosecond Dynamics of Rhodopsin Photochemistry Probed by a Double Pump Spectroscopic Approach Ming Yan,† Louis Rothberg,‡ and Robert Callender*,§,| Lawrence LiVermore National Laboratory, LiVermore, California 94550, Department of Chemistry, UniVersity of Rochester, Rochester, New York 14627, and Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461 ReceiVed: June 5, 2000; In Final Form: October 24, 2000
Femtosecond pump-probe measurements of the excitation pathway of bovine rhodopsin to bathorhodopsin show varying transient events in the photoinduced absorption spectrum. In the spectral region where bathorhodopsin absorption dominates in the rhodopsin-bathorhodopsin difference spectrum, subpicosecond (200-600 fs) and 3 ps signals are observed. In the region where rhodopsin absorption dominates, a 3-5 ps transient is observed, which follows rhodopsin repopulation. To unravel the different models of the rhodopsinbathorhodopsin dynamics that have been proposed to explain these transient observations, we have employed a three-beam experiment. The first pump beam launches the photochemistry, a second pump pulse interrupts the spontaneous evolution of the trajectory of excited rhodopsin at a delayed time from the first pump pulse, and a weak third beam probes changes in the amount of bathorhodopsin that is formed in response to the second pump beam. We first find that bathorhodopsin can be photoexcited, presumably back to rhodopsin, by reversing the initial photochemistry. We find that bathorhodopsin is formed very promptly (within the 300 fs resolution of our experiments). Surprisingly, the kinetics in the absorbance signal near 605 nm is found to be the superposition of two independent signals, one being characterized by events on the subpicosecond time scale and the other at 3 ps. The results suggest that the trajectory of excited rhodopsin splits into two pathways very early: one of which results in bathorhodopsin formation on the ∼200 fs time scale and another which lives in the excited state for ca. 3 ps before undergoing internal conversion to rhodopsin.
Rhodopsin is a membrane-bound protein, embedded in the outer segments of the rods and cones that make up the retina. It is responsible for the absorption of light and transmitting this information in order to give rise to neural excitation (cf. refs 1 and 2). Excellent progress has been made in understanding the initial rhodopsin photochemistry relevant to vision.3-7 It has been known for some time that absorption of a photon by the retinal chromophore (structure 1) of rhodopsin, which is cis
about the C11dC12 double bond, results in a cis-trans isomerization about this bond forming a trans-like, but twisted conformation.3-5 The equilibrated product, fully formed on the 3 ps time scale,3-5 is called bathorhodopsin because its absorption maximum is red-shifted to 543 nm from the 500 nm * Corresponding author. Phone: 718-430-3024. Fax: 718-430-8565. E-mail
[email protected]. † Lawrence Livermore National Laboratory. ‡ University of Rochester. § Albert Einstein College of Medicine. | On leave from the City College.
absorption maximum of rhodopsin in the bovine pigment, the subject of this paper. The energy surfaces from rhodopsin to bathorhodopsin about the C11dC12 double bond reaction coordinate in the ground state and the first excited state are well understood qualitatively as well as quantitatively. A number of observations argue that the excited-state surface is barrrierless.8-10 In the ground state, conversely, there is an unusually large potential barrier between the cis and trans states, which must exist because rhodopsin is necessarily very stable thermally,11 and the energy separation between the cis and trans positions in the ground state is about 60% of the absorbed photon’s energy.11-13 In agreement with these experimental observations, Birge and co-workers calculated the excited and ground-state potential surfaces and proposed a picture where dynamical evolution of a wave packet on these surfaces determines the initial event.14 Their calculations predict curve crossing from the excited state to the isomerized (bathorhodopsin) ground state using the MillerGeorge theory such that coherence is retained in photoproduct. Transient spectroscopic studies6 have directly observed the coherent torsional oscillations in the bathorhodopsin product state and showed that the isomerization is surprisingly fast (∼200 fs), a factor of 3 faster than predicted. Further femtosecond pump-probe studies employing a range of probes in the red and near-IR suggest a multidimensional potential energy surface for isomerization probably involving carbon-carbon bond stretching in addition to bond rotational motion quite early in the dynamics (
