Subpicosecond resonance Raman spectra of the early intermediates in the photocycle of bacteriorhodopsin

We present the results of two-pump and probe femtosecond experiments designed to follow the relaxation dynamics of the lowest excited state (S 1) populated by different modes. In the first mode, a direct (S0 3 S1) radiative excitation of the ground state is used. In the second mode, an indirect excitation is used where the S 1 state is populated by the use of two femtosecond laser pulses with different colors and delay times between them. The first pulse excites the S 0 3 S1 transition whereas the second pulse excites the S 1 3 Sn transition. The nonradiative relaxation from the Sn state populates the lowest excited state. Our results suggest that the S 1 state relaxes faster when populated nonradiatively from the Sn state than when pumped directly by the S0 3 S1 excitation. Additionally, the S n 3 S1 nonradiative relaxation time is found to change by varying the delay time between the two pump pulses. The observed dependence of the lowest excited state population as well as its dependence on the delay between the two pump pulses are found to fit a kinetic model in which the S n state populates a different surface (called S 1) than the one being directly excited (S 1). The possible involvement of the Ag type states, the J intermediate, and the conical intersection leading to the S 0 or to the isomerization product (K intermediate) are discussed in the framework of the proposed model. Halobacterium salinarum (Halobium salinarium) is a member of Halobacteria, which is part of the domain Archaea. In an anaerobic condition under light illumination, H. salinarum demonstrates a pH decrease in cell suspension and ATP synthesis (1, 2), the main features of photosynthesis. The photosynthetic activity in H. salinarum was attributed to bacteriorhodopsin (bR), a 26-kDa protein that is hexagonally packed within the purple membrane (1-4). Since the discovery of the purple membrane, its enigmatically efficient photosynthesis is in the center of the most active research. To our surprise, the archaic organism effectively utilizes more than 50% of the absorbed light energy (5, 6).Retinal, a light-absorbing polyene, is the bR chromophore and is linked to its Lys-216 by a protonated Schiff base. The chromophore undergoes light-induced isomerization and masters proton transfer across the membrane (7-11). The photoisomerization of retinal initiates a series of retinal protein structural reconformations of the bacteriorhodopsin that lead to the formation of different intermediates and finally return it to its initial state with all-trans-retinal. Overall, this photocycle consists of at least seven intermediates of bR with different visible absorption spectra and lifetimes bR 568 3 S 1 3 J 3 K 630 3 L 550 3 M 412 3 N 520 3 O 640 3 bR 568 (4).Retinal in solution shows relatively slow and nonselective photoisomerization around several double bonds (12-14) with low yield. The specific ultrafast, all-trans, 13-cis photoisomerization of the retinal in bR is the purple membrane photosynthesis key event which has a quantum yield of 55%. The p...

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confidence: 75%

Section: Discussionmentioning

confidence: 95%

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confidence: 99%

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The relaxation dynamics of the excited electronic states of retinal in bacteriorhodopsin by two-pump-probe femtosecond studies

Logunov

Volkov

Braun

et al. 2001

“…These observations are best explained by a small conformational relaxation of the chromophore and its immediate protein environment that occurs as the photoproduct evolves from a distorted form to a relaxed all-trans geometry (6,7,21). Conformational changes in both rhodopsin and bacteriorhodopsin on time scales >700 fs have been observed by time-resolved vibrational techniques (5,20,22).…”

Section: Methodsmentioning

confidence: 99%

The first step in vision occurs in femtoseconds: complete blue and red spectral studies.

Peteanu

Schoenlein

Wang

et al. 1993

185

168

Femtosecond transient absorption measurements of the ci-trans isomerization of the visual pigment rhodopsin clarify the interpretation of the dynamics of the first step in vision. We present femtosecond time-resolved spectra as well as kinetic measurements at specific wavelengths between 490 and 670 nm using 10-fs probe pulses centered at 500 and 620 nm following a 35-fs pump pulse at 500 nm. The expanded spectral window beyond that available (500-570 nm) in our previous study [ Vision is initiated when a photon is absorbed by the seven a-helical, membrane-bound protein called rhodopsin (1). After optical excitation, the 11-cis-retinal prosthetic group of rhodopsin is converted to an all-trans primary photoproduct in an efficient and apparently barrierless isomerization reaction ( Fig. 1). This product, which was first identified as bathorhodopsin in low-temperature absorption experiments, has an absorption maximum of =z560 nm (2). (13) with 35-fs pump and 10-fs probe pulses centered at 500 nm. Subsequently, Callender and co-workers (14) presented 300-fs time resolution data at 620 nm, which they interpreted in terms of a 200-fs twisting process on the excited-state surface, followed by a 3-ps excited-state to ground-state photoproduct transition. To resolve this disagreement, we present here a more complete series of femtosecond experiments on rhodopsin in which the probe wavelength range is extended to the red by using 10-fs pulses centered at 620 nm. Narrow-bandwidth kinetic traces at a selection of probe wavelengths as well as differential spectral measurements extending from 490 to 670 nm have been obtained. These results establish, in agreement with our earlier report, that the formation time for the rhodopsin photoproduct is 200 fs, and they provide key evidence that clarifies the interpretation of the primary photochemistry of vision. EXPERIMENTAL PROCEDURESThe method for generating 35-fs pump pulses at 500 nm as well as 10-fs probe pulses centered at 500 and 620 nm have been described elsewhere (12,13,15). The experimental configuration used to obtain differential transmittance spectra and time-resolved measurements is identical to that used in ref. 13. Briefly, the average pump power is ==3 ,W at a repetition rate of400 Hz. The pump (==0.5 mJ/cm2) and probe (O0.04 mJ/cm2) beams are crossed in a 0.3-mmjet offlowing bovine rhodopsin (15 OD/cm in ammonyx-LO). The flow rate is sufficient to replace the photolyzed sample between successive pump pulses. Two methods are used to measure transient changes in absorption (AT/T) as a function ofprobe delay time after photolysis of the rhodopsin. Differential spectra at particular pump-probe delay times are obtained by dispersing the probe pulse in a spectrometer after it passes through the sample and imaging it onto a dual diode array. Kinetic information at specific wavelengths is obtained by filtering the probe pulse after the sample (-"7-nm bandpass) and combining differential detection with lock-in amplification as the pump-probe delay time is continu...

“…Time-resolved resonance Raman (RR) spectroscopy has been used to determine changes in the vibrational degrees of freedom associated with these early intermediates (2,(15)(16)(17)(18)(19)(20). Although these studies have yet to firmly characterize the structural nature of J-625 (16,18,20), it appears that J-625 contains a highly twisted retinal [deduced from the strong HOOP modes observed in the 900-to 1000-cm-1 region (16,18)].…”

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confidence: 99%

Primary picosecond molecular events in the photoreaction of the BR5.12 artificial bacteriorhodopsin pigment.

Delaney

Brack

Atkinson

et al. 1995

The picosecond dynamics of the photoreaction of an artificial bacteriorhodopsin (BR) pigment containing a retinal in which a five-membered ring spans the C-12 to C-14 positions of the polyene chain (BR5.12) is examined by using time-resolved absorption and fluorescence and resonance Raman spectroscopy. The ring within the retinal chromophore of BR5.12 blocks the C-13=C-14 isomerization proposed to be a primary step in the energy storage /transduction mechanism in the BR photocycle. Relative to the native BR pigment (BR-570), the absorption spectrum of BR5.12 is red-shifted by 8 nm. The fluorescence spectrum of BR5.12 closely resembles that of BR-570 although the relative fluorescence yield is higher ("10-fold