Vibrational motions associated with primary processes in bacteriorhodopsin studied by coherent infrared emission spectroscopy

Abstract

The primary energetic processes driving the functional proton pump of bacteriorhodopsin take place in the form of complex molecular dynamic events after excitation of the retinal chromophore into the Franck-Condon state. These early events include a strong electronic polarization, skeletal stretching, and all-trans-to-13-cis isomerization upon formation of the J intermediate. The effectiveness of the photoreaction is ensured by a conical intersection between the electronic excited and ground states, providing highly nonadiabatic coupling to nuclear motions. Here, we study real-time vibrational coherences associated with these motions by analyzing light-induced infrared emission from oriented purple membranes in the 750-1400 cm(-)(1) region. The experimental technique applied is based on second-order femtosecond difference frequency generation on macroscopically ordered samples that also yield information on phase and direction of the underlying motions. Concerted use of several analysis methods resulted in the isolation and characterization of seven different vibrational modes, assigned as C-C stretches, out-of-plane methyl rocks, and hydrogen out-of-plane wags, whereas no in-plane H rock was found. Based on their lifetimes and several other criteria, we deduce that the majority of the observed modes take place on the potential energy surface of the excited electronic state. In particular, the direction sensitivity provides experimental evidence for large intermediate distortions of the retinal plane during the excited-state isomerization process.

Figure 1

Interferogram between the IR emissions from native bR and GaAs reference. The interferogram shows two parts, the instantaneous electronic response around zero delay time and a signal at positive time that reflects vibrational motions of the retinal/protein system. (Inset) Amplitude enlargement (20×) of the interferogram.

Figure 2

Spectrogram of the interferogram of native and the acid blue bR sample with GaAs reference, based on sliding-window Fourier analysis with a Hann window of 400-fs length (200 fs FWHM). Arrows indicate regions of time-dependent frequencies (A) and recurrences (B–D).

Figure 3

Vibrational part of the interferogram of the native sample fitted with seven vibrational modes, and the acid blue sample fitted with nine modes.

Figure 4

Power-spectrum estimates calculated from the experimental and fitted data for the vibrations from the native and acid blue bR samples in Fig. 3. Also indicated is the spectral response of the measuring system.

Figure 5

Spectrogram of the modeled interferogram fitted to the experimental data from the native and the acid blue sample with coloring identical to that on Fig. 2. The arrows mark regions where the recurrences are reproduced by the fit.