Xanthorhodopsin: A Proton Pump with a Light-Harvesting Carotenoid Antenna

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Science  23 Sep 2005:
Vol. 309, Issue 5743, pp. 2061-2064
DOI: 10.1126/science.1118046


Energy transfer from light-harvesting carotenoids to chlorophyll is common in photosynthesis, but such antenna pigments have not been observed in retinal-based ion pumps and photoreceptors. Here we describe xanthorhodopsin, a proton-pumping retinal protein/carotenoid complex in the eubacterium Salinibacter ruber. The wavelength dependence of the rate of pumping and difference absorption spectra measured under a variety of conditions indicate that this protein contains two chromophores, retinal and the carotenoid salinixanthin, in a molar ratio of about 1:1. The two chromophores interact strongly, and light energy absorbed by the carotenoid is transferred to the retinal with a quantum efficiency of ∼40%. The antenna carotenoid extends the wavelength range of the collection of light for uphill 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 growth in a high–ionic strength environment (3), as do haloarchaea. S. ruber has a deep red color from salinixanthin, which constitutes nearly 100% of its carotenoid content. The chemical structure of salinixanthin is established, and it was proposed to provide protection from photodamage and stabilize the cell membrane, because both the polyene and the fatty acid parts of this carotenoid acyl glycoside were predicted to be immersed in the lipid bilayer (4). We report here that salinixanthin is not the only pigment in the S. ruber cell membrane and that heterotrophy is not the only source of energy for this organism. These bacteria contain an unusual retinal protein that uses salinixanthin to harvest light energy in a wider spectral range than is possible with retinal alone and then uses 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 a eukaryote (8), but with two chromophores. We term it here xanthorhodopsin. Its 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 (Fig. 1A), which is abolished by the protonophore carbonyl cyanide m-chlorophenyl-hydrazone (CCCP). When assayed in 1 M Na2SO4, these light-dependent pH changes are unaffected by added chloride ions. Thus, the vesicles contain an outward-directed light-driven proton pump, such as bacteriorhodopsin, and they lack a detectable amount of a chloride pump, such as halorhodopsin, that would produce chloride-dependent light-induced alkalinization in the presence of CCCP (11).

Fig. 1.

Light-induced proton transport, inhibition of respiration in S. ruber, their action spectra, and an estimation of the efficiency of energy transfer from carotenoid to retinal in xanthorhodopsin. (A) pH changes in a membrane vesicle suspension produced by illumination at 550 nm at pH 6.5 are shown in the absence and presence of 20 μM CCCP at a light intensity of 2 mW cm–2. (B) Kinetics of light-induced changes in the rate of oxygen uptake caused by photoinhibition of respiration in the cell suspension of S. ruber are shown in the absence and presence of 10 μM CCCP, 550 nm, 2 mW cm–2. a.u., arbitrary units. (C) Action spectra for photo-inhibition of the respiration by intact cells (open circles) and light-induced proton transport in membrane vesicles (solid circles), measured as in (B) and (A), respectively. (D) Estimated absorption spectrum of the complex and the contribution of carotenoid component to it and to the action spectrum are shown, as explained in the text. Spectrum 1: the calculated spectrum of xanthorhodopsin; spectrum 2: bacteriorhodopsin, 10 nm shifted to the blue and appropriately scaled to model the retinal component of xanthorhodopsin; spectra 3 and 4: carotenoid components of the absorption spectrum and the action spectrum, respectively, obtained by subtracting the contribution of the retinal component. The ratios of the integrals of the carotenoid and retinal components are ∼2.5 and 1 in the absorption spectrum and the action spectrum, respectively. The increase in the cross sections for light collection and the photoreaction from the carotenoid are thus 3.5-fold and 2-fold. OD, optical density.

The existence of observed light-driven proton transport suggested that Salinibacter might contain a bacterial rhodopsin. The wavelength dependence of the transport (action spectrum) was determined by two independent methods: measuring photoinhibition of respiration and measuring light-induced pH changes. The transmembrane electrochemical gradient produced by the light-driven proton pump inhibits respiration of intact S. ruber cells and results in a temporary increase of the ambient oxygen concentration (Fig. 1B). As expected, the rise in oxygen level is abolished by the CCCP. This method had been used for demonstrating the physiological function of bacteriorhodopsin in halobacterial cells (12, 13). The action spectrum is more complex than what would be expected for a retinal protein. It exhibits a shoulder at 560 nm, where a retinal chromophore would absorb, and sharp bands at 521 and 486 nm (Fig. 1C) that correspond to the maxima in the structured absorption spectrum of cell membranes (fig. S1) containing salinixanthin. Figure 1C shows that the action spectrum for light-induced pH changes in membrane vesicle suspensions agrees well with that of the inhibition of respiration. The action spectra indicate that this system depends on a complex of the retinal protein and a carotenoid and that light absorbed by both carotenoid and retinal is effective in proton transport. The contribution of these two components to the action spectrum is shown in Fig. 1D.

The presence of a retinal chromophore was confirmed by bleaching and reconstitution. Incubation of Salinibacter cell membranes with hydroxylamine produced a difference absorption spectrum that exhibited the broad minimum at 565 nm expected if retinal were released from a bacteriorhodopsin-like chromophore by this treatment (14), as well as the corresponding maximum at 364 nm from the retinal oxime produced (Fig. 2A). Additionally, the difference spectrum contains sharp depletion bands at 521, 487, and 457 nm. The latter three bands are near the absorption maxima of salinixanthin (fig. S1) but show remarkably greater spectral resolution similar to the action spectrum (Fig. 1C). Reconstitution of the membranes, which were washed free of hydroxylamine, with all-trans retinal resulted in the recovery of not only the broad band near 565 nm of the retinal chromophore but also sharp bands (Fig. 2B) that correspond to the negative peaks after bleaching. These sharp bands must originate from one or a few salinixanthin molecules that closely interact with the retinal protein and change their spectrum (mainly narrowing the vibronic bands) when the complex is formed.

Fig. 2.

Spectroscopic detection of the retinal protein of Salinibacter. (A) Curves 1 through 5 show the difference spectra upon illumination of a cell-membrane suspension at >550 nm in the presence of 0.2 M hydroxylamine, pH 7.2, for 4, 12, 20, 36, and 60 min at 20°C, respectively. The amplitude of the 487-nm difference band is 10 to 15% of the total carotenoid absorbance. (B) Curves 1 through 4 show the difference spectra measured 5, 15, 25, and 60 min after addition of all-trans retinal to the S. ruber cell membranes, which had been bleached with 0.2 M hydroxylamine.

The interaction of the two chromophores is modulated in the photocycle. Illumination of the Salinibacter membranes with green light at 175 K causes the formation of a photoproduct with a red-shifted retinal band, which is stable at this temperature but reconverts when illuminated by red light (Fig. 3A). This is behavior characteristic of the K photointermediate of bacteriorhodopsin (1517). The peculiarity of the difference spectrum of Salinibacter retinal protein is that besides the increase of absorbance near 610 nm from the red-shifted retinal chromophore, a set of three additional sharp bands appear at 448, 482, and 518 nm. These peaks originate from a small (∼2 nm) blue shift of the three main carotenoid bands. Perturbation of the carotenoid in the K-like photoproduct appears to be electrochromic and suggests close interaction with the retinal. Reversal of the changes of the carotenoid bands with red light (Fig. 3A), which is absorbed by the retinal but not by the carotenoid, is further evidence for interaction between the carotenoid and the retinal in the complex.

Fig. 3.

Absorption changes during the photocycle of xanthorhodopsin. (A) Light-induced absorption changes in a water-glycerol suspension of cell membranes at 175 K. Spectrum 1: illumination of membranes with 520-nm light; spectrum 2: subsequent illumination at >650 nm. (B) Kinetics of laser flash–induced absorption changes at selected characteristic wavelengths, as indicated, in membranes of Salinibacter solubilized with 0.15% dodecyl maltoside in 100 mM NaCl, pH 8.8, 20°C. The global fit indicates that kinetics include at least six components, with time constants of 7.5 μs, 35 μs, 280 μs, 1.3 ms, 11 ms, and 100 ms. (C) Transient difference spectra during the photocycle, measured at 10, 30, 60, 100, 160, and 250 ms (curves 1 through 6), after a 532-nm laser flash. Conditions and sample are as in (B).

The absorption changes during the photocycle at ambient temperature (Fig. 3, B and C) indicate the formation of intermediates similar to the K, L, M, N, and O photoproducts of bacteriorhodopsin (15) and proteorhodopsin (18). The absorption changes at 410 nm after flash photoexcitation reflect mainly the formation of the M intermediate with a deprotonated retinal Schiff base, as expected in a proton pump. The difference spectra associated with photointermediates also exhibit perturbation of the absorption bands of salinixanthin (Fig. 3C). The changes, arising on the millisecond time scale, involve band-broadening similar to the spectra after bleaching with hydroxylamine (Fig. 2A). Together with the spectrum of the K state (Fig. 3A), they suggest that the carotenoid senses both electrostatic changes and conformational shifts of the retinal and the protein during the photocycle.

Substantial purification of the complex was achieved by repeated incubation of Salinibacter membranes with 0.01% dodecyl maltoside, followed by centrifugation to recover the membranes. This procedure extracts most of the excess bulk carotenoids as well as nearly all other proteins (19), and the xanthorhodopsin then can be solubilized with 0.15% dodecyl maltoside. The solubilized xanthorhodopsin exhibits a slight blue shift of the retinal chromophore band but retains all the characteristic absorption bands of the membranes, and because of less light scattering, more spectroscopic details are revealed (fig. S2). It appears that after hydrolysis of the Schiff base, salinixanthin loses its rigid environment and acquires a broader, less-structured spectrum, similar to that of the bulk carotenoid (fig. S2). This broadening of the carotenoid absorption bands, which is responsible for the negative peaks in Fig. 2A, is revealed by second-derivative spectra that are better suited for resolving overlapping bands (fig. S3). In such spectra, the narrow carotenoid bands are more pronounced than are the wider band of the retinal chromophore. The overall shape of the carotenoid vibronic bands after bleaching resembles those before bleaching, but the bands shift slightly to the red and become wider (e.g., the width of the 486-nm band increases from ∼24 to 36 nm). Further, the shapes and the amplitude ratios of the difference bands after bleaching the sample enriched in xanthorhodopsin are the same as in a sample enriched in bulk salinixanthin. This strongly suggests that salinixanthin is a stoichiometric component of the retinal protein complex.

The spectrum of the xanthorhodopsin complex (Fig. 1D, spectrum 1) was calculated from the spectrum of the partly purified sample (fig. S2) by satisfying the condition that the resolution of the vibronic bands should be the same as are those in the action spectrum (19). From the published extinction coefficients of the two chromophores, 240,000 M–1 cm–1 for salinixanthin (4) and 63,000 M–1 cm–1 for the retinal chromophore of bacteriorhodopsin (20), we estimate that the ratio of salinixanthin to retinal is 0.75:1. However, the extinction coefficient for most carotenoids with the same number of conjugated bonds as salinixanthin is lower, ∼180,000 M–1 cm–1 (19). With this number, the retinal-to-carotenoid ratio in the purified complex will be 1:1. Only the carotenoid bound in the complex functions as an antenna, because the spectra of bound and free carotenoid are different (fig. S2) and the action spectrum corresponds to the spectrum of the bound state (Fig. 1C). The action spectrum will consist of the retinal chromophore band plus the band of the bound carotenoid, multiplied by the efficiency of energy transfer to the retinal. Because the contribution of the carotenoid bands to the action spectrum is about 40% of their contribution to the absorption spectrum (Fig. 1D), we conclude that the efficiency of energy transfer is ∼40%. In photosynthetic organisms, the efficiency of energy transfer between carotenoids and chlorophyll is from 15% to 100% (9, 21, 22).

Mass spectrometry of the more intense of the two bands on a SDS–polyacrylamide gel from the purified sample (19) identified the gene product from a proteorhodopsin- or bacteriorhodopsin-like open reading frame (Fig. 4) in the Salinibacter genome (accession number NC_006812), but no other proteins were found in the sample that could bind retinal. Under these conditions, the expression of other retinal proteins, such as halorhodopsin identified in the genome (23), must be much less than that of xanthorhodopsin.

Fig. 4.

Alignment of the sequences of xanthorhodopsin (XR), proteorhodopsin (PR), and bacteriorhodopsin (BR). Xanthorhodopsin contains the functionally important residues known for retinal binding and proton transport, including homologs of Tyr57, Arg82, Asp85, Trp86, Asp96, Trp182, Glu194, Tyr185, Asp212, and Lys216, with numbering and helical segments (underlined) for bacteriorhodopsin. There are 30 residues (marked red) common to all three proteins (in two cases, they are Asp/Glu correspondences). The residue identities between xanthorhodopsin and bacteriorhodopsin (red plus purple) are somewhat more numerous (58 versus 46) than with proteorhodopsin (red plus yellow), but as in proteorhodopsin, the internal proton donor homologous to Asp96 is Glu. A set of four phenylalanine residues, located on what should be the side of helix E that faces toward the lipid bilayer, might be involved in binding the carotenoid (31).

For mechanistic reasons that have been thoroughly explored in bacteriorhodopsin (24), retinal must be the site of proton translocation in xanthorhodopsin also. The rationale for the carotenoid in the dual light-harvesting system is that it increases the cross section for light absorption and extends it to the spectral region, where the absorption of the retinal chromophore is low. Other functions might be the protection of the retinal protein from photo-oxidation by singlet oxygen, because carotenoids are known to be efficient deactivators of triplet states, and an increase of protein stability. These functions of carotenoids are common in chlorophyll-based photosynthetic systems, where they harvest light energy and transfer it to chlorophyll (21), but they have not been shown for retinal-based pumps or receptors (2527).

The discovery of a carotenoid antenna in a retinal protein raises an interesting question about energy transfer. The intense absorption bands of carotenoids are caused mainly by transition to the strongly allowed S2 state (21). This state is very short-lived (50 to 200 fs), because through internal conversion, it populates lower-lying excited states, including the S1 state with a much longer lifetime. S1 and S2 both participate in energy transfer to bacteriochlorophyll. However, the energy level of S1 in spirilloxanthin, a carotenoid with 13 conjugated bonds, similar to salinixanthin, is far below that of the 560-nm absorption band of the retinal chromophore (21). If the same is true for salinixanthin, energy transfer cannot be from the lower excited state S1. Could there be energy transfer from an excited state located between S2 and S1, as suggested for carotenoid-chlorophyll complexes (30)? If not, energy transfer will be only from S2 and rapid, implying close proximity and exact geometry of the donor and acceptor. The substantial changes of the carotenoid spectrum during its retinal-dependent formation of the complex and during the photocycle suggest that the binding site is specially “crafted” to accommodate the carotenoid molecule in a position optimal for energy transfer to the retinal. The xanthorhodopsin complex represents the simplest electrogenic pump with an accessory antenna pigment, and it might be an early evolutionary development in using energy transfer for energy capture.

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