TIME‐RESOLVED RESONANCE RAMAN STUDIES ON THE PHOTOCHEMICAL CYCLE OF BACTERIORHODOPSIN

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Abstract
Abstract— Kinetic resonance Raman (RR) experiments were designed to study the time‐behaviour of the retinal‐binding protein bacteriorhodopsin (BR) in its photochemical cycle. The unphotolyzed chro‐mophore B‐570 and the two intermediates L‐550 and M‐412 were probed by the characteristic C=C stretching vibrations of the retinal moiety. Time resolution was achieved with a spinning cell as flow system in combination with two CW lasers in a pump‐probe configuration. RR spectra were probed at 475 nm at various delay times between pump and probe event. The deconvolution of the spectra into the various components B‐570, L‐550 and M‐412 was carried out by curve fitting procedures. It was found that at pH7.4 L‐550 decays — with a time‐constant of 62 μs — not completely but to a residual level of 35% of its initial value. This intermediate L‐amplitude finally disappears in the ms‐range (4.5 ms) synchroneously with the intermediate M‐412. An analogeous time‐behaviour was found at pH 4.6. In the basic range also an “L” ‐intermediate could be identified which is coupled to the long‐lived M‐component. To explain the peculiar time‐dependence it is proposed that during the fast decay of L a dynamic equilibrium between L and M is established. Then during the reconstitution of B‐570 the two intermediates disappear synchroneously. A molecular model is presented in which the dynamic equilibrium between L and M is explained by an oscillatory motion of a proton from the Schiff base group of the chromophore to its counterion.