Femtosecond stimulated emission pumping experiments from bacteriorhodopsins fluorescent state show for the first time that it is a photocycle intermediate, with a constant nonisomerized structure throughout its 0.5 psec lifetime. Implications concerning internal conversion dynamics are discussed.

The primary events in the photosynthetic retinal protein bacteriorhodopsin (bR) are reviewed in light of photophysical and photochemical experiments with artificial bR in which the native retinal polyene is replaced by a variety of chromophores. Focus is on retinals in which the “critical” C13=C14 bond is locked with respect to isomerization by a rigid ring structure. Other systems include retinal oxime and non-isomerizable dyes noncovalently residing in the binding site. The early photophysical events are analyzed in view of recent pump–probe experiments with sub-picosecond time resolution comparing the behavior of bR pigments with those of model protonated Schiff bases in solution. An additional approach is based on the light-induced cleavage of the protonated Schiff base bond that links retinal to the protein by reacting with hydroxylamine. Also described are EPR experiments monitoring reduction and oxidation reactions of a spin label covalently attached to various protein sites. It is concluded that in bR the initial relaxation out of the Franck–Condon (FC) state does not involve sub-stantial C13=C14 torsional motion and is considerably catalyzed by the protein matrix. Prior to the decay of the relaxed fluorescent state (FS or I state), the protein is activated via a mechanism that does not require double bond isomerization. Most plausibly, it is a result of charge delocalization in the excited state of the polyene (or other) chromophores. More generally, it is concluded that proteins and other macromolecules may undergo structural changes (that may affect their chemical reactivity) following optical excitation of an appropriately (covalently or non-covalently) bound chromophore. Possible relations between the light-induced changes due to charge delocalization, and those associated with C13=C14 isomerization (that are at the basis of the bR photocycle), are discussed. It is suggested that the two effects may couple at a certain stage of the photocycle, and it is the combination of the two that drives the cross-membrane proton pump mechanism.

In this study, the ultrafast pump-probe spectroscopy of the all-trans protonated Schiff base of retinal (trans-PSB) in solution, is compared to that of two retinal analogues, trans-PSB5.12 and 13-cis-PSB5.13, in which C-13=C-14 torsional motion is inhibited by a rigid five-membered ring structure. The objective is to obtain measures of internal conversion (IC) dynamics in these polyenes. Contrasting the results with those obtained for the same pigments when attached to their opsin protein, serve to appreciate the protein role in catalyzing energy transduction in bacteriorhodopsin. Several major features appear to be common to all three PSBs: (i) A 50-100 fs process due to a primary relaxation out of the Franck-Condon (FC) region, (ii) A subsequent biexponential decay (t(1) = 1-2 ps and t(2) = 4-7 ps) of the fluorescent state (FS) assumed to be due to IC, and (iii) Spectral modulations in the FS emission. The three are only marginally effected by locking of the C-13=C-14 bond. With respect to features (i) and (iii) the PSB model compounds behave analogously to the related retinal protein bacteriorhodopsin (bR). However, this does not apply to the FS decay. While in bR, the IC takes place with a 0.5 ps decay time, locking of the C-13=C-14 bond in bR markedly increases the FS lifetime to similar to 15 ps. These observations demonstrate the crucial role played by the protein in directing the isomerization action to the active double bond and enhancing the rate of IC. They also prove that these coordinates are not exclusive pathways of IC in the isolated PSB of retinal. The mechanism of ground-state repopulation in the PSBs is discussed in light of these results.