Low-Temperature Raman Spectroscopy of Halorhodopsin from Natronomonas pharaonis: Structural Discrimination of Blue-Shifted and Red-Shifted Photoproducts

From the low-temperature absorption and Raman measurements of halorhodopsin from Natronomonas pharaonis ( p HR), we observed that the two photoproducts were generated after exciting p HR at 80 K by green light. One photoproduct was the red-shifted K intermediate ( p HR K ) as the primary photointermediate for Cl – pumping, and the other was the blue-shifted one ( p HR hypso ), 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 p HR, and we revealed the chromophore structures of the two photointermediates from their Raman spectra for the first time. p HR hypso had the distorted all- trans chromophore, while p HR K contained the distorted 13- cis form. The present results revealed that the structural analyses of p HR K carried out so far at ∼80 K potentially included a significant contribution from p HR hypso . p HR hypso was efficiently formed via the photoexcitation of p HR K, indicating that p HR hypso was likely a side product after photoexcitation of p HR K . The formation of p HR hypso suggested that the active site became tight in p HR K due to the slight movement of Cl –, and the back photoisomerization then produced the distorted all- trans chromophore in p HR hypso .