Immobility of the chromophore in bacteriorhodopsin

The purple membrane of Halobacterium halobium contains a single protein, bacteriorhodopsin, which has one molecule of retinal covalently bound to a lysine residue. Light absorption by the chromophore initiates a photochemical cycle which is accompanied by proton translocation from the cytoplasm to the medium and thereby produces an electrochemical gradient across the cell membrane [ 11. The way in which the chromophore interacts with the protein is not yet understood well. In such a stage, it is of importance to answer the question whether the chromophore has any rotational freedom in the binding site. Earlier studies of transient absorption dichroism of the purple membrane have shown that rotational motions of the protein in the membrane are absent in the time range from 104-lo3 s [2-41. However, the reported values of absorption anisotropy are considerably lower than the theoretical maximum. This suggests a possibility that the chromophore and/or the protein has a freedom of rotation whose relaxation time is in the ps or shorter time region [S]. To clarify this point, we have carried out two independent experiments in which the rotational motions with 10-9-10-2 s relaxation times can be monitored. One was the ns time-resolved fluorescence depolarization [6] and the other the transient absorption dichroism [7]. In the former experiment, we investigated NaBH,-reduced purple membrane in which the chromophores were converted to fluorescent derivatives but the hexagonal crystalline structure was maintained [ 11. The results show that the rotational motion of the chromophore as well as the protein in