Photoisomerization Quantum Yield and Apparent Energy Content of the K Intermediate in the Photocycles of Bacteriorhodopsin, Its Mutants D85N, R82Q, and D212N, and Deionized Blue Bacteriorhodopsin

A visible-pump͞UV-probe transient absorption is used to characterize the ultrafast dynamics of bacteriorhodopsin with 80-fs time resolution. We identify three spectral components in the 265-to 310-nm region, related to the all-trans retinal, tryptophan (Trp)-86 and the isomerized photoproduct, allowing us to map the dynamics from reactants to products, along with the response of Trp amino acids. The signal of the photoproduct appears with a time delay of Ϸ250 fs and is characterized by a steep rise (Ϸ150 fs), followed by additional rise and decay components, with time scales characteristic of the J intermediate. The delayed onset and the steep rise point to an impulsive formation of a transition state on the way to isomerization. We argue that this impulsive formation results from a splitting of a wave packet of torsional modes on the potential surface at the branching between the all-trans and the cis forms. Parallel to these dynamics, the signal caused by Trp response rises in Ϸ200 fs, because of the translocation of charge along the conjugate chain, and possible mechanisms are presented, which trigger isomerization.ultrafast spectroscopy ͉ retinal proteins ͉ translocation of charge ͉ structural dynamics T he different biological functions of retinal proteins (vision, ion pumping, light signaling, etc.) rely on the ultrafast isomerization of the retinal chromophore (1). This process has been intensively investigated by fs pump-probe and f luorescence up-conversion spectroscopy in the visible (VIS) and͞or near-IR (2-13), revealing the retinal excited-state dynamics before isomerization and the build-up of the isomerized form. Time-resolved VIS-pump͞mid-IR probe experiments on bacteriorhodopsin (bR) established that the isomerization time is Ն400 fs (14). Indeed, the excited-state lifetime of 300 -500 fs (12, 15) goes hand-in-hand with the rise times observed for the 13-cis photoproduct (3,4,14), whereas a longer time scale of 3-4 ps has been attributed to vibrational relaxation of the photoproduct (14).The mechanism leading to the isomer is still subject to debate. Using Ͻ5-fs pulses, Kobayashi et al. (11) were able to monitor changes in the high-frequency vibrational spectrum of the excited state, suggesting that retinal is in a twisted, nonplanar configuration during the initial 200 fs. They also showed that the high-frequency modes were modulated with a period of Ϸ200 fs, corresponding to the low-frequency torsional modes observed in transient absorption experiments at 800-nm probe wavelength (16). That the system is all-trans-like during the first 200 fs is in line with Zhong et al. (17), who carried out transient absorption studies on wild-type bR and bR reconstituted with retinal analogs that cannot undergo isomerization and found identical dynamics during the initial 200 fs. In addition, McCamant et al. (18) recently showed by fs-stimulated Raman spectroscopy that the high-frequency modes (1,000-1,500 cm Ϫ1 ) decay on a time scale of Ϸ250 fs. They argued that this observation points to the excited-sta...

mentioning

confidence: 99%

Insights into excited-state and isomerization dynamics of bacteriorhodopsin from ultrafast transient UV absorption

Schenkl

Mourik

Friedman

et al. 2006

“…The chromophore undergoes light-induced isomerization and masters proton transfer across the membrane (7)(8)(9)(10)(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.…”

mentioning

confidence: 99%

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

Logunov

Volkov

Braun

et al. 2001

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...

“…According to the generally accepted scheme (29) the majority of the excited molecules decays to the J intermediate. The next step of the photocycle is the formation of the K intermediate, which can be described by exponential kinetics with a time constant ( 2 ) of 3 and 6 ps for the native and the acid blue form, respectively (31,32). If these kinetics are associated with a jump of protons from one metastable position to another, as indicated by previous direct electric measurements (6)(7)(8), on a macroscopic scale this corresponds to an accelerated proton current, leading to terahertz emission.…”

Section: Model Ii: Intramolecular Electron Transfer (Resonant Opticalmentioning

confidence: 91%

“…As a result of the prolonged kinetics, the slow negative phase in the terahertz signal emitted by the acid blue sample is shifted toward longer times and somewhat reduced in amplitude. In contrast to the native bR, the acid blue form decays by a biexponential process with characteristic time constants of 11 ϭ 1.7 ps and 12 ϭ 18 ps with an amplitude ratio of a 11 :a 12 ϭ 1:0.3 (31,32). Calculating with these kinetic parameters (listed also in Table 1) results in an almost completely positive simulated curve (Fig.…”

Section: Model Ii: Intramolecular Electron Transfer (Resonant Opticalmentioning

confidence: 97%

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

Groma

Hebling

Kozma

et al. 2008

The kinetics of electrogenic events associated with the different steps of the light-induced proton pump of bacteriorhodopsin is well studied in a wide range of time scales by direct electric methods. However, the investigation of the fundamental primary charge translocation phenomena taking place in the functional energy conversion process of this protein, and in other biomolecular assemblies using light energy, has remained experimentally unfeasible because of the lack of proper detection technique operating in the 0.1-to 20-THz region. Here, we show that extending the concept of the familiar Hertzian dipole emission into the extreme spatial and temporal range of intramolecular polarization processes provides an alternative way to study ultrafast electrogenic events on naturally ordered biological systems. Applying a relatively simple experimental arrangement based on this idea, we were able to observe light-induced coherent terahertz radiation from bacteriorhodopsin with femtosecond time resolution. The detected terahertz signal was analyzed by numerical simulation in the framework of different models for the elementary polarization processes. It was found that the principal component of the terahertz emission can be well described by excited-state intramolecular electron transfer within the retinal chromophore. An additional slower process is attributed to the earliest phase of the proton pump, probably occurring by the redistribution of a H bond near the retinal. The correlated electron and proton translocation supports the concept, assigning a functional role to the light-induced sudden polarization in retinal proteins. coherent terahertz emission ͉ excited-state kinetics ͉ nonlinear spectroscopy ͉ sudden polarization ͉ ultrafast charge separation