RESUMEN
From the low-temperature absorption and Raman measurements of halorhodopsin from Natronomonas pharaonis (pHR), we observed that the two photoproducts were generated after exciting pHR at 80 K by green light. One photoproduct was the red-shifted K intermediate (pHRK) as the primary photointermediate for Cl- pumping, and the other was the blue-shifted one (pHRhypso), which was not involved in the Cl- pumping and thermally relaxed to the original unphotolyzed state by increasing temperature. The formation of these two kinds of photoproducts was previously reported for halorhodopsin from Halobacterium sarinarum [ Zimanyi et al. Biochemistry 1989 , 28 , 1656 ]. We found that the same took place in pHR, and we revealed the chromophore structures of the two photointermediates from their Raman spectra for the first time. pHRhypso had the distorted all-trans chromophore, while pHRK contained the distorted 13-cis form. The present results revealed that the structural analyses of pHRK carried out so far at â¼80 K potentially included a significant contribution from pHRhypso. pHRhypso was efficiently formed via the photoexcitation of pHRK, indicating that pHRhypso was likely a side product after photoexcitation of pHRK. The formation of pHRhypso suggested that the active site became tight in pHRK due to the slight movement of Cl-, and the back photoisomerization then produced the distorted all-trans chromophore in pHRhypso.
