5122
J. Phys. Chem. B 1999, 103, 5122-5130
On the Nature of the Primary Light-Induced Events in Bacteriorhodopsin: Ultrafast Spectroscopy of Native and C13)C14 Locked Pigments T. Ye,† N. Friedman,‡ Y. Gat,‡ G. H. Atkinson,§ M. Sheves,*,‡ M. Ottolenghi,*,† and S. Ruhman*,† Department of Physical Chemistry, The Hebrew UniVersity, Jerusalem 91904, Israel, Department of Organic Chemistry, The Weizmann Institute of Science, RehoVot 76100, Israel, and Department of Chemistry and Optical Science Center, UniVersity of Arizona, Tucson, Arizona 85721 ReceiVed: December 4, 1998
The primary light-induced events in the photosynthetic retinal protein bacteriorhodopsin (bR) are investigated by ultrafast optical spectroscopy over the 440-1000 nm spectral range. The study compares the early dynamics of the native all-trans pigment bR570 with those of two synthetic analogues, bR5.12 and bR5.13, in which isomerization around the critical C13dC14 bond is blocked by a five-membered ring into all-trans and 13-cis configurations, respectively. Nearly identical spectral evolution is observed in both native and artificial systems over the first 100-200 fs of probe delay. During this period stimulated near-IR (∼900 nm) emission, and intense ∼460 nm absorption bands, due to analogous fluorescent I states (denoted as I460, I5.12 and I5.13, respectively), appear concurrently within 30 fs. In all systems continuous spectral shifting over tens of femtoseconds is observed in the 500-700 nm range. Native bR goes on to produce the J625 absorption band within ∼1 ps, which is accompanied by disappearance of the I460 emission and absorption features. In bR5.12 and bR5.13, aside from minor spectral modifications, the analogous dramatic changes associated with I5.12 and I5.13 are arrested beyond the first ∼100 fs, reverting uniformly to the initial ground state with exponential time constants of 19 ps and 11 ps, respectively. Analysis of the data calls for a major revision of models previously put forward for the primary events in bacteriorhodopsin. The close likeness of initial transient spectral evolution in both native and artificial pigments, despite the locking of the active isomerization coordinate in the synthetic chromophores, demonstrates that in bR570 the ultrafast changes in transmission leading to I460, previously believed to involve C13dC14 torsion, must be associated with other modes. The detailed comparison conducted here also identifies which of the later spectral changes in the native system requires torsional flexibility in C13dC14. Similarity of 660 nm probing data in both synthetic and native chromophores demonstrates that the sub-picosecond dynamic features uncovered at this probing wavelength commonly attributed to the evolution of J625, are not, as previously thought, reliable measures of all-trans S 13-cis isomerization dynamics.
I. Introduction Bacteriorhodopsin (bR) is the photosynthetic representative of a series of retinal proteins, also including visual pigments, the chloride ion pump halorhodopsin (hR), and sensory rhodopsins. (For a series of comprehensive reviews on retinal proteins, see ref 1.) bR acts as a proton pump, driven by the absorption of visible light by the all-trans retinal chromophore (Structure A) which is attached to the protein via a protonated Schiff base linkage with Lys-216. Light absorption by all-trans bR, denoted as bR570, is associated with the schematic photocycle shown in Scheme 1:
SCHEME 1 hν
2 µs
3 ps
bR570 98 [S1FC(H), I460, J625] 98 K610 98 50 µs
2 ms
8 ms
L550 {\} M412 {\} (N540, O640) 98 bR570 Subscripts refer to the corresponding absorption maxima, S1FC represents the primarily generated Franck-Condon excited state, also denoted as H, and time values are approximate transition times between the various photointermediates. * Authors to whom correspondence should be addressed. † The Hebrew University. ‡ The Weizmann Institute of Science. § University of Arizona.
Of special interest are the early light-induced ultrafast dynamic events, most commonly investigated by time-resolved pump-probe spectroscopies (for recent reviews, see refs 2 and 3). These events terminate with the formation of the K610 intermediate, which is the first phototransient which may be trapped at cryogenic temperatures. A major problem is the characterization of the early protein and chromophore dynamics, with special emphasis on determining the exact time scale of the C13dC14 (all-trans) f 13-cis isomerization, which is a basic feature of the bR570 photocycle. These ultrafast dynamic features are closely related to key questions such as the causes for the high quantum yield and selectivity of the photoreaction, as well as the molecular mechanism by which light energy is initially stored and subsequently used by the protein in the pump process. Early absorption experiments with picosecond time resolution4 indicated that K610 is preceded by a further red-shifted precursor denoted5 as J625. An analysis5 of the first subpicosecond experiments of Ippen et al.6 indicated that J625 is not the primary photointermediate, suggesting that it grows-in over a 100
19
2-4
19
Rh(Oct)
700 nm absorption range of J625 (features 2 and 3) and those of the decay of I460 at ∼460 and ∼900 nm. Moreover, both features 2 and 3 are assumed to reflect a substantial trans f 13-cis tortional motion. Both assumptions are invalidated by our present data (vide supra). (b) As outlined above, our data do not exclude a mechanism involving two initial molecular pathways, initiating from the S1FC state. This will not be consistent with the Class 1 model of Figure 9. (c) Most Class 1 and Class 2 (but not Class 3) models contradict our major conclusion, namely, that I460 cannot be substantially twisted around C13dC14. (d) The conclusion that the molecular changes mirrored in the ∼500 fs decay of I460 must require substantial torsional motion around C13dC14 contradicts the Class 3 model of Figure 9, in which I460 is on the nonreactive pathway. Our present data do not lead to an unequivocal mechanistic alternative to the above models, but they do provide feasible options. Thus, the suggestion of two distinct pathways (see above), would imply a Class 2 mechanism in which two I states (II and III) are generated from S1FC. However, in variance with the Class 2 models of Figure 9, neither II nor III can represent a C13dC14 isomerized chromophore. This conclusion, which is valid independently of any specific single or double pathway mechanism, implies that other coordinates associated with the chromphore and/or with the surrounding protein matrix should play a role in the early events in bR, hR, and possibly visual pigments. This is in keeping with the Fourier transform analysis of Akyama et al. of the optical absorption spectra of bR570 and bR5.12.28 However, neither the latter study nor our present experimental data can account on a molecular level for the nature of these early processes. A study of the primary reorganization of the excited state, also accounting for the excitation wavelength dependence of the bR spontaneous fluorescence, was recently carried out by Wexler et al.34 A multi-mode analysis identified vibrational degrees of freedom of the retinal chromophore, as well as protein (dielectric) relaxation coordinates, which might be associated with the primary relaxation out of the FranckCondon state. Specific information in this respect was provided by the general ab initio model for protonated Schiff bases of Garavelli et al.,29 which leads to the conclusion that, in retinal proteins, the initial excited-state motion out of the S1FC state is dominated by CdC stretching, rather than by twisting modes. This relaxation results in elongation of a central double bond, associated with change in bond order of the excited state. A recent time-resolved resonance Raman study in bR57030 is indeed indicative of changes in the CdC stretching frequency in the fluorescent state. Such a process may well be associated with the formation of the I460 states. Evidently, if branching into reactive and nonreactive pathways occurs from S1FC, it implies that additional relaxation of the polyene and/or the surrounding protein, should play a role in these early stages of the photoreaction. In other words, changes along one or more additional coordinates are required for differentiating between different fluorescent states such as II and III. It is important to emphasize that while our data are compatible with a Class 2 mechanism in which the (nonisomerized) I460 t II state is on
0.2
τ2
2-4
τ3
τ4
ref
the reactive pathway, we cannot exclude the alternative approach19,20 in which I460 is on the nonreactive pathway. In such a case I460 t III, and II will be identified with the 40-80 fs decay in the 520-560 nm range. Discrimination between these two alternatives will be the subject of future studies. We finally consider the “phase 3” features which in the native system has been interpreted in some models as representing the growing-in of J625 in several hundreds femtoseconds. In our previous report it was suggested22 that an analogous “J-like” species, denoted as T5.12, was formed in the photolysis of bR5.12. In fact, in view of the close similarity in the dynamic features at 660 nm between the native and locked molecules (Figures 6 and 8c), it is tempting to consider that even in the locked systems a species akin to J625 is formed. This would imply that J625 still retains the original all-trans configuration. However, in the context of the broad spectral coverage of the current results, the similarities of appearance in the data collected at 660 nm may be coincidental. In the locked species, excess absorption at 660 nm decays concurrently with I5.12, while in the native system, J625 persists after the decay of I460. Moreover, in the locked system, we do not observe an absorbance decay that would match the 300-500 fs evolution at 660 nm. Thus, an alternative plausible assignment of T5.12 is that it reflects a red band of the I5.12 state, as suggested by the analyses of refs 14-16. This raises the question of why the slower rise in absorption near 660 nm in bR5.12 has no observable counterpart in the near-IR and blue bands assigned to I5.12. A plausible explanation is that this very slight rise in OD is magnified in appearance due to the proposed cancellation of emission and absorption in this spectral range. A conclusive answer will have to await further investigation. V. Conclusions In the present study we have addressed the early light-induced events in bacteriorhodopsin. We have shown that the primary dynamic process following light absorption does not involve the C13dC14 torsional coordinate and that other chromophore and/or protein coordinates must be involved in the very early branching from the Franck-Condon excited state into reactive and nonreactive pathways. Establishing the nature of such pathways which is required from an identification of early species such as I460, and J625 is still an open question, and should be the subject of future investigations. These conclusions most probably also apply to the closely related chloride pump halorhodopsin and possibly to visual pigments. Acknowledgment. This research was supported by grants from the Israel National Science Foundation, founded by the Israel Academy of Sciences and Humanities, Centers of Excellence Program, and from the James-Franck and L. Farkas (HUMinerva) Research Centers. References and Notes (1) The Photophysics and Photochemistry of Retinal Proteins; Ottolenghi, M., Sheves, M., Eds.; Isr. J. Chem. 1995, 35 (3/4), pp 193-515.
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