Removal and Reconstitution of the Carotenoid Antenna of Xanthorhodopsin

Abstract

Salinixanthin, a C40-carotenoid acyl glycoside, serves as a light-harvesting antenna in the retinal-based proton pump xanthorhodopsin of Salinibacter ruber. In the crystallographic structure of this protein, the conjugated chain of salinixanthin is located at the protein–lipid boundary and interacts with residues of helices E and F. Its ring, with a 4-keto group, is rotated relative to the plane of the π-system of the carotenoid polyene chain and immobilized in a binding site near the β-ionone retinal ring. We show here that the carotenoid can be removed by oxidation with ammonium persulfate, with little effect on the other chromophore, retinal. The characteristic CD bands attributed to bound salinixanthin are now absent. The kinetics of the photocycle is only slightly perturbed, showing a 1.5-fold decrease in the overall turnover rate. The carotenoid-free protein can be reconstituted with salinixanthin extracted from the cell membrane of S. ruber. Reconstitution is accompanied by restoration of the characteristic vibronic structure of the absorption spectrum of the antenna carotenoid, its chirality, and the excited-state energy transfer to the retinal. Minor modification of salinixanthin, by reducing the carbonyl C=O double bond in the ring to a C-OH, suppresses its binding to the protein and eliminates the antenna function. This indicates that the presence of the 4-keto group is critical for carotenoid binding and efficient energy transfer.

Characterization of an Unconventional Rhodopsin from the Freshwater Actinobacterium Rhodoluna lacicola

“…Ammonium persulfate has been used to selectively oxidize carotenoid pigments in both the presence and absence of another rhodopsin, xanthorhodopsin (XR). When XR is present, the rhodopsin-bound retinal is not affected by ammonium persulfate (36). Ammonium persulfate treatment of membrane fractions prepared from E. coli expressing actinorhodopsin and retinal verified that the oxidation does not affect actinorhodopsin-bound retinal (data not shown).…”

“…Ammonium persulfate has been shown to selectively oxidize carotenoid pigments while leaving rhodopsin-bound retinal intact (36). Membranes prepared from R. lacicola in 10 mM HEPES (pH 7.1) and 3% ␤-OG were diluted with the same buffer until the absorbance peaks of the carotenoids were well resolved.…”

Salinibacter: an extremely halophilic bacterium with archaeal properties

“…The carotenoid can be removed from the xanthorhodopsin complex by ammonium persulfate oxidation, leaving a functional xanthorhodopsin with a slightly slower photocycle. Salinixanthin addition enables reconstitution of the native complex (Imasheva et al, 2011). Interactions between the retinal of xanthorhodopsin and salinixanthin were characterized using circular dichroism spectroscopy of artificial pigments derived from synthetic retinal analogues with different absorption maxima (Smolensky & Sheves, 2009).…”

Ultrafast Carotenoid to Retinal Energy Transfer in Xanthorhodopsin Revealed by the Combination of Transient Absorption and Two‐Dimensional Electronic Spectroscopy

“…Note also that the negligible shift of the CAR transitionsi nX Ra nd RXR indicates that CAR and RET are not forming ad elocalized excitons tate, as they maintain their individualityi nb oths amples. This is furtherc orroborated by the observed retention of carotene chirality in circulard ichroism spectra [21,22] of both XR and RXR. [15] This observation points toward aF çrster EET mechanism between localized CAR and RET states,w hich was previously postulated for the XR system.…”