Raman spectroscopic studies on photoreactive retinal proteins are comprehensively described, including the basic physics of Raman scattering and illustrative examples of the types of information on the structure and function of the retinal chromophoreand its environment which can be obtained from the vibrational Raman spectra. In addition, practical advice and recipes are given which should enable the reader to plan and eventually perform a Raman experiment in a photolabile retinal protein. A dominant role is played by the resonance Raman (RR) experiment with visible laser excitation which selelctively probes the retinal chromophore. Much discussion is devoted to bacteriorhodopsin (bR) and its photocycle as a paradigm for a light-inducedreaction of a retinal protein. Various time-resolved techniques are described to study the temporal evolution of the bR chromophore by probing RR spectra of intermediatestates. VibrationalRaman spectra are interpreted in terms of structure and structural changes of the chromophore. RR spectroscopic studies on halorhodopsin, sensory rhodopsin, and visual pigments are reported, as well as on modifiedproteins in which retinal analoguesare incorporated,and on sitespecific mutants. Results of ultraviolet RR experiments which selectively probe the aromatic side chains in the protein backbone are reported. In addition, a promising new technique of near-infrared Raman excitation is discussed. Finally, application of coherent anti-Stokes Raman spectroscopy (CARS) to retinal proteins is reported.

The photocycle of bacteriorhodopsin (bR) was studied at ambient temperature in aqueous suspensions of purple membranes using time-resolved resonance Raman (RR) and optical transient spectroscopy (OTS). The samples were photolyzed, and the fractional concentrations of the retinylidene chromophore in its parent state, BR570, and in the intermediate states L550, M412, N560, and O640 were determined in the time domain 20 microseconds-1 s and in the pH range 4-10.5. Two kinetically different L components could be identified. At pH 7 one fraction of L (approximately 65%) decays in 80 microseconds to M (deprotonation of the Schiff base), whereas the residual part is converted in approximately 0.5 ms to N. The RR spectra reveal only minor structural changes of the chromophore in the L-->N transition. These were attributed to a conformational change of the protein backbone [Ormos, P., Chu, K., & Mourant, J. (1992) Biochemistry 31, 6933]. With decreasing pH the L-->N transition is delayed to > 2 ms following a titration-like function with pKa approximately 6.2. The decay of M412 monitored by OTS can be fitted for each pH value by two different amplitudes and time constants (Mf, tau f; Ms, tau s; f = fast, s = slow). Both Mf and Ms consist of subcomponents which can be distinguished by their different reaction pathways (but not by OTS). Mf occurs in the reaction sequences L-->Mf-->N-->BR and L-->Mf-->O-->BR. The population of the first sequence, in which N is formed with the time constant tau f (approximately 2-4 ms, pH 6-10.5), increases with pH. Ms is also found in two different reaction sequences of the form L-->Ms-->BR. The quantitative analysis reveals that each "titration effect" can be related to a certain fraction of bR. It is proposed that each fraction can be identified with a "subspecies" of bR which undergoes an independent and individual cyclic reaction. A complete reaction scheme is set up which represents the manifold of observed phenomena. It is concluded from the pH dependence of the lifetimes of Ms and N that the reconstitution of BR570 in the reaction steps Ms-->BR and N-->BR requires the uptake of a proton from the external phase. It is argued that this proton catalyzes the reisomerization of retinal, whereas the Schiff base is internally reprotonated from Asp-85. A model for proton pumping is proposed in which the proton taken up from the external phase to catalyze the reisomerization of retinal is the one which is pumped through the membrane during the photocycle of bR.

Experiments with a spinning cell and a single continuous-wave laser beam (514 nm) in reflection geometry were performed to obtain the resonance Raman (RR) spectra of the intermediate K,,, of the retinal chromophore of bacteriorhodopsin (bR). Samples of diluted aqueous suspensions of 'purple membranes' from Halobacterium halobium were used, which entail bR as an integral protein. K,,, is formed photochemically in CQ. 5 ps from the parent chromophore BR,,, and in H,O decays into the product L , , , with a time constant of ca. 1.2 ps (21 "C). Using a flow velocity of ca. 20 m s-' and a beam waist of 40 pm, the integrated relative concentrations of BR,,, , K , , , and L,,, in the beam were 58: 33:10, respectively. By this method the 'late' K intermediate which is accumulated on a time scale of ea. 1 ps was probed. The spectra of the mixture were recorded with a spectral step width of 0.2 cm-' and a band width of 4 cm-'. Up to 100 scans were accumulated to obtain the high quality which is required for subtraction and band-fitting procedures. The contributions of BR,,, and L,,, , whose spectra were obtained in separate experiments, were subtracted leaving the pure spectra of K,,, . Experiments were performed in H,O and D,O suspensions. Structural changes of the chromophore during the transition from K,,, to L,,, were inferred from the RR spectra in the hydrogen out-of-plane, the fingerprint and the C=C/C=N stretching regions. It was concluded that in this transition the chromophore relaxes from a distorted to a planar conformation and at the same time a positive charge of the protein envelope approaches the fi-ionone ring of retinal. It is proposed that this electrostatic interaction is important for the biological function of bR.

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