Absorption spectra of halorhodopsin (HR), a retinal protein in the halobacterial membrane, and its photostationary states were determined at 80 K. The absorption lines appear to narrow upon cooling, thereby revealing complex spectral fine structure of the main absorption band in the visible region, characteristic of conformational substates of HR. Illumination causes (1) the redistribution of these substates and consequent changing of the fine structure (“hole-burning”) and (2) the appearance of a hypsoproduct of undefined nature, in addition to the previously described bathoproduct HR600. Bacteriorhodopsin, a related retinal pigment, gives rise only to the bathointermediate (i.e., K590) under these conditions. After warming of illuminated HR to 110 K, and recooling to 80 K, relaxation of the illumination-induced change in spectral fine structure, and decay of the hypsoproduct but not the bathoproduct, was observed. The results are explained with a model in which one ensemble of HR conformational substates at 80 K is converted to another in a photoequilibrium via the excited state, which also produces the batho- and hypsoproducts. The original ensemble can be regained through thermal pathways at a somewhat higher temperature, and only the bathoproduct will decay thermally into the next intermediate of the HR photocycle.
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