On the Nature of the Primary Light-Induced Events in Bacteriorhodopsin:  Ultrafast Spectroscopy of Native and C13=C14 Locked Pigments

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 C13C14 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 C13C14 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 C13C14. 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 ⇔ 13-cis isomerization dynamics.