Resonant optical rectification in bacteriorhodopsin

Abstract

The relative role of retinal isomerization and microscopic polarization in the phototransduction process of bacteriorhodopsin is still an open question. It is known that both processes occur on an ultrafast time scale. The retinal trans3cis photoisomerization takes place on the time scale of a few hundred femtoseconds. On the other hand, it has been proposed that the primary lightinduced event is a sudden polarization of the retinal environment, although there is no direct experimental evidence for femtosecond charge displacements, because photovoltaic techniques cannot be used to detect charge movements faster than picoseconds. Making use of the known high second-order susceptibility (2) of retinal in proteins, we have used a nonlinear technique, interferometric detection of coherent infrared emission, to study macroscopically oriented bacteriorhodopsin-containing purple membranes. We report and characterize impulsive macroscopic polarization of these films by optical rectification of an 11-fs visible light pulse in resonance with the optical transition. This finding provides direct evidence for charge separation as a precursor event for subsequent functional processes. A simple two-level model incorporating the resonant second-order optical properties of retinal, which are known to be a requirement for functioning of bacteriorhodopsin, is used to describe the observations. In addition to the electronic response, long-lived infrared emission at specific frequencies was observed, reflecting charge movements associated with vibrational motions. The simultaneous and phase-sensitive observation of both the electronic and vibrational signals opens the way to study the transduction of the initial polarization into structural dynamics.R etinal proteins play an essential role in a broad range of light-driven biological processes, including vision (1), energy transduction (2), and circadian control (3). All these functions involve both the conversion of light energy into charge separation and retinal isomerization, but the interplay of these processes is the subject of intense debate. The retinal protein of which the initial photochemistry is most extensively studied is the photosynthetic protein bacteriorhodopsin (bR). This protein colors the purple membrane of halobacteria and acts as a light-driven proton pump by means of a multistep process termed the photocycle (2). In the traditional model for the initial transduction step in this cycle is directly light-driven trans3cis isomerization of retinal (4-6); this model has been recently extended by including excited-state skeletal stretching (5, 7). However, experiments with modified bR containing nonisomerizable retinal analogs challenged this model by showing that the initial photo-induced events are not associated with retinal isomerization (8-10). In fact, in an early alternative hypothesis (11), the essential process was proposed to be dielectric relaxation of the protein as a response to sudden polarization upon retinal excitation (11-13). Recent molecular dynamic...

Terahertz radiation from bacteriorhodopsin reveals correlated primary electron and proton transfer processes

“…2 Inset. This range, the upper limit of which was determined by the time resolution of our experimental setup, fills an important part of the gap between the frequency regions accessible by electronic measuring techniques (5-7) and coherent infrared emission methods (16). In a control experiment the sample was pumped at 775 nm where the absorption spectrum of bR vanishes.…”

“…This technical limit excluded the direct observation of the ultrafast charge translocation events taking place in the retinal chromophore and its proximity during the excited state and right after its relaxation. However, in both early (10) and recent literature (11)(12)(13)(14)(15)(16), more and more evidence indicates that these initial polarization processes possess a functional role in triggering the isomerization of retinal and initiating the proton pump.…”

“…The time evolution of the retinal polarization was followed indirectly by observing the transient absorption of the neighboring tryptophan residues (14,15), resulting in an unresolved component assigned to the Franck-Condon polarization and a second phase developed in Ϸ200 fs and attributed to the charge translocation along the conjugate chain. A more direct observation of the Franck-Condon polarization was carried out by the detection of the corresponding resonant optical rectification signal from bR in coherent infrared emission experiments (16,19). The sensitive heterodyne detection technique applied in those studies covered the extremely high 20-to 50-THz range, but still did not provide any information on the detailed kinetics of electron polarization in the retinal chromophore or on the primary events of the proton translocation.…”

Dephasing-Induced Vibronic Resonances in Difference Frequency Generation Spectroscopy

“…18 DFG has further found applications in semiconductors. 10,11 In this paper we focus on an ultrafast DFG technique known as coherence emission spectroscopy/optical rectification, 19,20 whereby two femtosecond visible pulses resonant with an electronic transition create vibrational coherences in both the ground and the excited electronic states. The generated heterodyne detected infrared field (both amplitude and phase) reveals vibrational modes strongly coupled to the photoexcitation.…”

“…The generated heterodyne detected infrared field (both amplitude and phase) reveals vibrational modes strongly coupled to the photoexcitation. This technique has been applied using 11 fs pulses to study protein vibrational motions coupled to an electronically excited cofactor in photoactivable single crystals (The photodissociation of the heme cofactor in ordered crystals of myoglobin 19 and the retinal trans f cis photoisomerization in oriented films for bacteriorhodopsin 20 ). These experiments have opened up new possibilities for probing protein structure and for following concerted motions induced by an external femtosecond trigger.…”