Photostationary states of halorhodopsin (HR, a retinal protein in the halobacterial membrane) and their further thermal conversions were investigated at 140–230 K by absorption spectroscopy in the visible. The difference spectra confirm several steps of the all-trans-HR photocycle, in the presence of chloride, proposed earlier on the basis of room temperature flash spectroscopy. Thus, at 140 K, the spectra reveal the HR600 → HR520 reaction, and at 170–230 K the HR640→HR578 and the HR520 → HR578 reactions can be seen. No evidence for the expected HR520 ⇄ HR640 process was found, however. From the difference spectra at various temperatures, exact absorption spectra of HR600 and HR520 were calculated, and an estimate of the HR640 spectrum in a mixture also containing HR520 was obtained. The low-temperature absorption maxima of HR578 and its photointermediates relate to the room temperature maxima differently from what is expected from the spectra of the corresponding intermediates in the bacteriorhodopsin photocycle.
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