Time‐resolved resonance Raman studies of bacteriorhodopsin and its intermediates K₅₉₀ and L₅₅₀: Biological implications

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 590 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 590 is formed photochemically in ca. 5 ps from the parent chromophore BR 570 and in H 2 O decays into the product L 550 with a time constant of ca. 1.2 μs (21°C). Using a flow velocity of ca. 20 m s −1 and a beam waist of 40 μm, the integrated relative concentrations of BR 570 , K 590 and L 550 in the beam were 58:33:10, respectively. By this method the ‘late’ K intermediate which is accumulated on a time scale of ca. 1 μs was probed. The spectra of the mixture were recorded with a spectral step width of 0.2 cm −1 and a band width of 4 cm −1 . Up to 100 scans were accumulated to obtain the high quality which is required for subtraction and band‐fitting procedures. The contributions of BR 570 and L 550 , whose spectra were obtained in separate experiments, were subtracted leaving the pure spectra of K 590 . Experiments were performed in H 2 O and D 2 O suspensions. Structural changes of the chromophore during the transition from K 590 to L 550 were inferred from the RP 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 β‐ionone ring of retinal. It is proposed that this electrostatic interaction is important for the biological function of bR.