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Visual pigments are a class of proteins found in the membranes of photoreceptor cells (for reviews, see refs. 1 and 2). Their chromophoric unit is 11-cis-retinal covalently bound in the form of a Schiff base to the c-amino group of a lysine. The absorption of a photon by a visual pigment initiates a sequence of biochemical events that eventually lead to the generation of a neural signal by a photoreceptor cell. The identity of the primary photochemical event has been a subject of considerable interest and controversy. It was originally suggested that the primary event was an isomerization of the chromophore from its 1 1-cis to an all-trans conformation (3, 4). The strongest evidence favoring this mechanism was the observation (based on spectral data at low temperature) that an artificial pigment containing a 9-cis chromophore had the same photoproduct as rhodopsin itself. It was quite reasonably concluded that the most plausible common photoproduct formed from the two cis isomers is a trans isomer. A number of picosecond absorption studies of the primary event have raised widespread doubts as to the validity of the original model. It was found (5) that the primary event is complete in less than a few picoseconds at room temperature, and it was argued that this is too short a time for isomerization to occur [although other picosecond studies have reached the opposite conclusion (6)]. More recent evidence has come from the observation that at low temperature the rate of the process is significantly inhibited by deuterium replacement of the exchangeable protons on the pigment (7). Since only one proton on the chromophore is exchangeable, it is unlikely that this would have a measurable effect on the rate of isomerization. The picosecond measurements have generated numerous models (7-11) whose major feature is a photochemical proton transfer followed by a thermal cis-trans isomerization that occurs at a later stage. However, the original evidence upon which the suggestion of a cis-trans isomerization was originally based has never been discredited and, thus, it appears necessary to find a molecular model that is consistent with the entire body of available evidence. The purpose of this paper is to present such a model.There are, in fact, a fairly large number of observations that can be used in the construction and evaluation of alternative models. For example, we have shown, by using simple thermodynamic arguments, that a significant fraction of the photon's energy is "stored" in the primary photoproduct (12). Clearly a mechanism for energy storage must be an important component of any model that is proposed. Another energetic constraint may be derived from psychophysical and electrophysiological measurements of the level of thermal noise in photoreceptor cells. We show below that these observations may be interpreted directly in molecular terms and that they require an unusually high thermal activation energy for the primary event that appears to be inconsistent with many of the models that have been proposed.The ...

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New information concerning the photochemical dynamics of bacteriorhodopsin (BR) is obtained by impulsively stimulating emission from the reactive fluorescent state. Depletion of the excited-state fluorescence leads to an equal reduction in production of later photoproducts. Accordingly, chromophores which are forced back to the ground state via emission do not continue on in the photocycle, conclusively demonstrating that the fluorescent state is a photocycle intermediate. The insensitivity of depletion dynamics to the "dump" pulse timing, throughout the fluorescent states lifetime, and the biological inactivity of the dumped population suggest that the fluorescent-state structure is constant, well-defined, and significantly different than that where crossing to the ground state takes place naturally. In conjunction with conclusions from comparing the photophysics of BR with those of synthetic analogues containing "locked" retinals, present results show that large-amplitude torsion around C13=C14 is required to go between the above structures.

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 bR 570 with those of two synthetic analogues, bR5.12 and bR5.13, in which isomerization around the critical C 13 dC 14 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 I 460 , 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 J 625 absorption band within ∼1 ps, which is accompanied by disappearance of the I 460 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 bR 570 the ultrafast changes in transmission leading to I 460 , previously believed to involve C 13 dC 14 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 C 13 dC 14. 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 J 625 , are not, as previously thought, reliable measures of all-trans S 13-cis isomerization dynamics.

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Photochemical studies of the effects of temperature, pH, and dehydration on the formation and back photoreaction of the M412 intermediate in the photocycle of light-adapted bacteriorhodopsin (bR570) are carried out. Continuous illumination experiments in the range between -40 and -90 degrees C indicate that at low temperatures branching occurs at the stage of the L550 intermediate in which a back reaction to the parent pigment competes with the formation of M412. At low temperatures the yield of M412 is markedly increased at high pH. The effect is attributed to the catalytic action of a protein group of pK congruent to 10 on the rate of the L550 leads to M412 process. Our results, taken together with previous evidence for deprotonation of a tyrosine during the L550 leads to M412 transition, suggest that the formation of a tyrosinate ion is a prerequisite for deprotonation of the Schiff base. A model is proposed in which both the Schiff base and the tyrosine translocate their protons to two acceptor groups, A1 and A2, accessible to the outside of the cell through a segment of a proton wire. The model accounts for the observation that up to two photons may be pumped per cycle. The proton-pump mechanism is analyzed in terms of a generalized kinetic scheme for pumping. In contrast to current models for proton pumping which are based on a (primary) light-induced accessibility change of the chromophore (class I models), we introduce a new class (II) of models based exclusively on pK changes. We suggest that in bR570 the Schiff base and the tyrosine are accessible to protons on the outside surface of the membrane. An analysis of the back photoreaction from M412 tends to favor class II models over previous class I models.