Retinal β‑Ionone Ring–Salinixanthin Interactions in Xanthorhodopsin: A Study Using Artificial Pigments

Xanthorhodopsin (xR) is a retinal protein that contains, in addition to the retinal chromophore, a carotenoid (salinixanthin) that functions as a light-harvesting antenna [Balashov, S. P., et al. (2005) Science 309, 2061–2064]. The center–center distance between the two polyene chains is 12–13 Å, but the distance between the two rings of retinal and salinixanthin is surprisingly small (∼5 Å) with an angle of ∼45° [Luecke, H., et al. (2008) Proc. Natl. Acad. Sci. U.S.A. 105, 16561–16565]. We aimed to clarify the role of the β-ionone ring in the binding of retinal to apo-xR, as well as a possible role that the β-ionone ring plays in fixation of the salinixanthin 4-keto ring. The binding of native retinal and series of synthetic retinal analogues modified in the β-ionone ring to apo-xR was monitored by absorption and circular dichroism (CD) spectroscopies. The results indicate that the β-ionone ring modification significantly affected formation of the retinal–protein covalent bond as well as the pigment absorption and CD spectra. It was observed that several retinal analogues, modified in the retinal β-ionone ring, did not bind to apo-xR and did not form the pigment. Also, none of these analogues induced the fixation of the salinixanthin 4-keto ring. In addition, we show that the native retinal within its binding site adopts exclusively the 6-s-trans ring–chain conformation.