Chromophore Interaction in Xanthorhodopsin—Retinal Dependence of Salinixanthin Binding^†

Abstract

Xanthorhodopsin is a light-driven proton pump in the extremely halophilic bacterium Salinibacter ruber. Its unique feature is that besides retinal it has a carotenoid, salinixanthin, with a light harvesting function. Tight and specific binding of the carotenoid antenna is controlled by binding of the retinal. Addition of all-trans retinal to xanthorhodopsin bleached with hydroxylamine restores not only the retinal chromophore absorption band, but causes sharpening of the salinixanthin bands reflecting its rigid binding by the protein. In this report we examine the correlation of the changes in the two chromophores during bleaching and reconstitution with native all-trans retinal, artificial retinal analogues, and retinol. Bleaching and reconstitution both appear to be multi-stage processes. The carotenoid absorption changes during bleaching occurred not only uponhydrolysis of the Schiff base but continued while the retinal was leaving its binding site. In the case of reconstitution, the 13-desmethyl analogue formed the protonated Schiff base slower than retinal, and provided the opportunity to observe changes in carotenoid binding at various stages. The characteristic sharpening of the carotenoid bands, indicative of its reduced conformational heterogeneity in the binding site, occurs already when the retinal occupies the binding site but the covalent bond to Lys-227 via a Schiff base is not yet formed. This is confirmed by the results for retinol reconstitution, where the Schiff base does not form but the carotenoid exhibits its characteristic spectral change from the binding.

Reconstitution of Gloeobacter violaceus Rhodopsin with a Light-Harvesting Carotenoid Antenna

“…In the difference spectrum, “spectrum 4 minus spectrum 2”, the sharp positive bands at 521 and 486 nm are from increase of extinction at the maximum of the carotenoid, whereas the negative band at 532 nm is from narrowing of the band (Figure 3B, spectrum 1). These features are similar to those observed for binding of salinixanthin to xantho-opsin upon addition of retinol (23) (Figure 3B, spectrum 2), and upon reconstitution of carotenoid-free xanthorhodopsin with salinixanthin (our unpublished result). Incorporation of the carotenoid occurs with a time-constant of ca.…”

“…The carotenoid loses the resolution of the vibronic bands upon hydrolysis, as in xanthorhodopsin, but to a somewhat lesser extent. The results indicate that conformation of the carotenoid is controlled by the retinal, apparently from direct interaction of their rings as in xanthorhodopsin (20, 23). Unlike in xanthorhodopsin, however, the carotenoid retains a somewhat structured spectrum even after complete hydrolysis of the retinal Schiff base.…”

Carotenoid response to retinal excitation and photoisomerization dynamics in xanthorhodopsin

“…Cell membranes were then washed in 100 mMNaCl and in distilled water. Finally, membrane fractions containing XR were collected by centrifugation [19]. Purple membranes containing BR were obtained as described previously [20].…”

Crystallographic structure of xanthorhodopsin, the light-driven proton pump with a dual chromophore

“…Because energy transfer is from the shortlived S 2 carotenoid level (4), there must be a short distance and favorable geometry between the 2 chromophores to account for its high (40-50%) efficiency. Close interaction of the 2 chromophores is indicated by dependence of the carotenoid conformation on the presence of the retinal in the protein (1,5,6) and spectral changes of the carotenoid during the photochemical transformations of the retinal (1), but, as for the proteorhodopsin family of proteins, no direct structural information has been available (4,7). Unexpectedly, the crystallographic structure of xanthorhodopsin we report here reveals not only the location of the antenna but also striking differences from the archaeal retinal proteins, bacteriorhodopsin and archaerhodopsin.…”