Energy transfer from antenna pigments to the reaction center is common in chlorophyll-based photosynthesis but never been observed in retinal-based ion pumps and photoreceptors. Here we describe xanthorhodopsin, a retinal protein/carotenoid complex in the eubacterium Salinibacter ruber, a proton pump. Difference absorption spectra measured under a variety of conditions and action spectra for pumping indicate that this protein contains two chromophores: retinal and the carotenoid, salinixanthin, in a molar ratio of about 1:1. The two chromophores strongly interact, and light energy absorbed by the carotenoid is efficiently transferred to the retinal and used for transmembrane proton transport.The extreme halophile Salinibacter ruber isolated from salt crystallizer ponds (1,2) can be grown in aerobic heterotrophic culture in 4 M NaCl. This eubacterium accumulates high concentrations of KCl to adapt to the high ionic strength (3), as the haloarchaea in the same environment. After several days of growth, Salinibacter ruber acquires a deep red color, from salinixanthin which constitutes nearly 100% of its carotenoid content and whose chemical structure was recently established (4). It was proposed to provide protection from photodamage and to stabilize the cell membrane because both the polyene and the fatty acid part of this carotenoid acyl glycoside will be immersed in the lipid bilayer (4). We report here that salinixanthin is not the only pigment in the Salinibacter ruber cell membrane, and heterotrophy is not the only source of energy for this organism. These cells contain an unusual retinal protein, which uses salinixanthin to assist harvesting light energy in a wider spectral range and utilizes it for transmembrane proton transport. Thus, it is a light-driven proton pump similar to bacteriorhodopsin (5) and the archaerhodopsins (6) of the archaea, the proteorhodopsins of planktobacteria (7), and leptosphaeria rhodopsin of an eukaryote (8), but with two chromophores. We term it here xanthorhodopsin. Its novel carotenoid antenna is a feature shared with chlorophyll-based light-harvesting complexes and reaction centers (9,10).Illumination of cell membrane vesicles prepared from Salinibacter produces acidification of the medium, which is abolished by the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) (Fig. 1A). When assayed in 1 M Na 2 SO 4 , these light-dependent pH changes are unaffected by the presence of chloride ions (not shown). Thus, the vesicles contain an outward-directed light-driven proton pump like bacteriorhodopsin, and lack a

Homologous to bacteriorhodopsin and even more to proteorhodopsin, xanthorhodopsin is a light-driven proton pump that, in addition to retinal, contains a noncovalently bound carotenoid with a function of a light-harvesting antenna. We determined the structure of this eubacterial membrane protein-carotenoid complex by X-ray diffraction, to 1.9-Å resolution. Although it contains 7 transmembrane helices like bacteriorhodopsin and archaerhodopsin, the structure of xanthorhodopsin is considerably different from the 2 archaeal proteins. The crystallographic model for this rhodopsin introduces structural motifs for proton transfer during the reaction cycle, particularly for proton release, that are dramatically different from those in other retinal-based transmembrane pumps. Further, it contains a histidine-aspartate complex for regulating the pK a of the primary proton acceptor not present in archaeal pumps but apparently conserved in eubacterial pumps. In addition to aiding elucidation of a more general proton transfer mechanism for light-driven energy transducers, the structure defines also the geometry of the carotenoid and the retinal. The close approach of the 2 polyenes at their ring ends explains why the efficiency of the excited-state energy transfer is as high as Ϸ45%, and the 46°angle between them suggests that the chromophore location is a compromise between optimal capture of light of all polarization angles and excited-state energy transfer.carotenoid antenna ͉ energy transfer ͉ retinal protein ͉ salinixanthin ͉ X-ray structure

At pH >7, proteorhodopsin functions as an outward-directed proton pump in cell membranes, and Asp-97 and Glu-108, the homologues of the Asp-85 and Asp-96 in bacteriorhodopsin, are the proton acceptor and donor to the retinal Schiff base, respectively. It was reported, however [Friedrich, T. et al. (2002) J. Mol. Biol., 321, 821-838], that proteorhodopsin transports protons also at pH <7 where Asp-97 is protonated and in the direction reverse from that at higher pH. To explore the roles of Asp-97 and Glu-108 in the proposed pumping with variable vectoriality, we compared the photocycles of D97N and E108Q mutants, and the effects of azide on the photocycle of the E108Q mutant, at low and high pH. Unlike at high pH, at a pH low enough to protonate Asp-97 neither the mutations nor the effects of azide revealed evidence for the participation of the acidic residues in proton transfer, and as in the photocycle of the wild-type protein, no intermediate with unprotonated Schiff base accumulated. In view of these findings, and the doubts raised by absence of charge transfer after flash excitation at low pH, we revisited the question whether transport occurs at all under these conditions. In both oriented membrane fragments and liposomes reconstituted with proteorhodopsin, we found transport at high pH but not at low pH. Instead, proton transport activity followed the titration curve for Asp-97, with an apparent pK(a) of 7.1, and became zero at the pH where Asp-97 is fully protonated.