retinaldehyde and salinixanthin

Retinal proteins play significant roles in light-induced protons/ions transport across the cell membrane. A recent studied retinal protein, gloeobacter rhodopsin (gR), functions as a proton pump, and binds the carotenoid salinixanthin (sal) in addition to the retinal chromophore. We have studied the interactions between the two chromophores as reflected in the circular dichroism (CD) spectrum of gR complex. gR exhibits a weak CD spectrum but following binding of sal, it exhibits a significant enhancement of the CD bands. To examine the CD origin, we have substituted the retinal chromophore of gR by synthetic retinal analogues, and have concluded that the CD bands originated from excitonic interaction between sal and the retinal chromophore as well as the sal chirality induced by binding to the protein. Temperature increase significantly affected the CD spectra, due to vanishing of excitonic coupling. A similar phenomenon of excitonic interaction lose between chromophores was recently reported for a photosynthetic pigment-protein complex (Nature Commmun, 9, 2018, 99). We propose that the excitonic interaction in gR is weaker due to protein conformational alterations. The excitonic interaction is further diminished following reduction of the retinal protonated Schiff base double bond. Furthermore, the intact structure of the retinal ring is necessary for obtaining the excitonic interaction.

Topics: Carotenoids; Cyanobacteria; Glycosides; Hydrogen-Ion Concentration; Models, Molecular; Molecular Structure; Protein Binding; Protein Conformation; Proton Pumps; Retina; Retinaldehyde; Rhodopsin; Rhodopsins, Microbial; Schiff Bases; Stereoisomerism; Temperature

The retinal proton pump xanthorhodopsin (XR) was recently found to function with an attached carotenoid light harvesting antenna, salinixanthin (SX). It is intriguing to discover if this departure from single chromophore architecture is singular or if it has been adopted by other microbial rhodopsins. In search of other cases, retinal protein encoding genes in numerous bacteria have been identified containing sequences corresponding to carotenoid binding sites like that in XR. Gloeobacter rhodopsin (GR), exhibiting particularly close homology to XR, has been shown to attach SX, and fluorescence measurements suggest SX can function as a light harvesting (LH) antenna in GR as well. In this study, we test this suggestion in real time using ultrafast transient absorption. Results show that energy transfer indeed occurs from S2 of SX to retinal in the GR-SX composite with an efficiency of ∼40%, even higher than that in XR. This validates the earlier fluorescence study, and supports the notion that many microbial retinal proteins use carotenoid antennae to harvest light.

Topics: Carotenoids; Cyanobacteria; Energy Transfer; Glycosides; Kinetics; Retinaldehyde; Rhodopsins, Microbial

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.

Topics: Bacterial Proteins; Binding Sites; Carotenoids; Circular Dichroism; Cyclohexenes; Glycosides; Molecular Conformation; Norisoprenoids; Retinaldehyde; Rhodopsins, Microbial

Xanthorhodopsin (xR) is a recently discovered retinal protein that contains, in addition to the retinal chromophore, a carotenoid (salinixanthin) absorbing at 456, 486, and 520 nm, which functions as a light-harvesting antenna. We have studied the interactions between the two chromophores by monitoring the absorbance and circular dichroism (CD) spectroscopies of artificial pigments derived from synthetic retinal analogues characterized by shifted absorption maxima. In addition, we have followed the binding process of the synthetic chromophores to the apomembrane of xR. We have revealed that the CD spectrum of xR originated mainly from the carotenoid chromophore without a significant contribution of the retinal chromophore. Because the binding process rate of these analogues is slower compared to all-trans retinal, it was possible to detect and analyze the major alterations in the CD spectrum. It was revealed that the main changes occur as a result of binding site occupation by the retinal chromophore and not because of the formation of the retinal-protein covalent bond.

Topics: Absorption; Bacterial Proteins; Binding Sites; Biomimetics; Carotenoids; Circular Dichroism; Glycosides; Molecular Conformation; Protein Binding; Proton Pumps; Retinaldehyde; Sphingobacterium; Stereoisomerism

We show that salinixanthin, the light-harvesting carotenoid antenna of xanthorhodopsin, can be reconstituted into the retinal protein from Gloeobacter violaceus expressed in Escherichia coli. Reconstitution of gloeobacter rhodopsin with the carotenoid is accompanied by characteristic absorption changes and the appearance of CD bands similar to those observed for xanthorhodopsin that indicate immobilization and twist of the carotenoid in the binding site. As in xanthorhodopsin, the carotenoid functions as a light-harvesting antenna. The excitation spectrum for retinal fluorescence emission shows that ca. 36% of the energy absorbed by the carotenoid is transferred to the retinal. From excitation anisotropy, we calculate the angle between the two chromophores as being ca. 50 degrees , similar to that in xanthorhodopsin. The results indicate that gloeobacter rhodopsin binds salinixanthin in a manner similar to that of xanthorhodopsin and suggest that it might bind a carotenoid also in vivo. In the crystallographic structure of xanthorhodopsin, the conjugated chain of the carotenoid lies on the surface of helices E and F, and the 4-keto ring is immersed in the protein at van der Waals distance from the ionone ring of the retinal. The 4-keto ring is in the space occupied by a tryptophan in bacteriorhodopsin, which is replaced by the smaller glycine in xanthorhodopsin and gloeobacter rhodopsin. Specific binding of the carotenoid and its light-harvesting function are eliminated by a single mutation of the gloeobacter protein that replaces this glycine with a tryptophan. This indicates that the 4-keto ring is critically involved in carotenoid binding and suggests that a number of other recently identified retinal proteins, from a diverse group of organisms, could also contain carotenoid antenna since they carry the homologous glycine near the retinal.

Topics: Amino Acid Substitution; Bacteroidetes; Binding Sites; Carotenoids; Circular Dichroism; Cyanobacteria; Fluorescence Polarization; Glycosides; Hydroxylamine; Molecular Conformation; Protein Binding; Recombinant Proteins; Retinaldehyde; Rhodopsins, Microbial; Schiff Bases; Spectrometry, Fluorescence; Spectrophotometry

The cell membrane of Salinibacter ruber contains xanthorhodopsin, a light-driven transmembrane proton pump with two chromophores: a retinal and the carotenoid, salinixanthin. Action spectra for transport had indicated that light absorbed by either is utilized for function. If the carotenoid is an antenna in this protein, its excited state energy has to be transferred to the retinal and should be detected in the retinal fluorescence. From fluorescence studies, we show that energy transfer occurs from the excited singlet S(2) state of salinixanthin to the S(1) state of the retinal. Comparison of the absorption spectrum with the excitation spectrum for retinal emission yields 45 +/- 5% efficiency for the energy transfer. Such high efficiency would require close proximity and favorable geometry for the two polyene chains, but from the heptahelical crystallographic structure of the homologous retinal protein, bacteriorhodopsin, it is not clear where the carotenoid can be located near the retinal. The fluorescence excitation anisotropy spectrum reveals that the angle between their transition dipole moments is 56 +/- 3 degrees . The protein accommodates the carotenoid as a second chromophore in a distinct binding site to harvest light with both extended wavelength and polarization ranges. The results establish xanthorhodopsin as the simplest biological excited-state donor-acceptor system for collecting light.

Topics: Bacterial Proteins; Bacteroidetes; Carotenoids; Energy Transfer; Fluorescence Polarization; Glycosides; Protein Conformation; Retinaldehyde; Rhodopsin; Rhodopsins, Microbial; Spectrum Analysis, Raman

In xanthorhodopsin, a retinal protein-carotenoid complex of Salinibacter ruber, the carotenoid salinixanthin functions as a light-harvesting antenna in supplying additional excitation energy for retinal isomerization and proton transport. Another retinal protein, archaerhodopsin, has been shown to contain a carotenoid, bacterioruberin, but without an antenna function. We report here that the binding site confers a chiral geometry on salinixanthin in xanthorhodopsin and confirm that the same is true for bacterioruberin in archaerhodopsin. Cell membranes containing these rhodopsins exhibit CD spectra with sharp positive bands in the visible region where the carotenoids absorb, and in the case of xanthorhodopsin a negative band at 536 nm, as well as bands in the UV region. The carotenoid in ethanol has very weak optical activity in the visible region of the spectrum. Denaturation of the opsin upon deprotonation of the Schiff base at pH 12.5 eliminates the induced CD bands in both proteins. In one of these proteins, but not in the other, the carotenoid binding site depends entirely on the retinal. Hydrolysis of the retinal Schiff base of xanthorhodopsin with hydroxylamine eliminates the induced CD bands of salinixanthin. In contrast, hydrolysis of the Schiff base in archaerhodopsin does not abolish the CD bands of bacterioruberin. Thus, consistent with its antenna function, the carotenoid binding site interacts closely with the retinal only in xanthorhodopsin, and this interaction is the major source of the CD bands. In this protein, protonation of the counterion with a decrease in pH from 8 to 5 causes significant changes in the CD spectrum. The observed spectral features suggest that binding of salinixanthin in xanthorhodopsin involves the cyclohexenone ring of the carotenoid and its conformational heterogeneity is restricted.

Topics: Archaeal Proteins; Bacteroidetes; Carotenoids; Circular Dichroism; Glycosides; Halobacteriaceae; Hydrogen-Ion Concentration; Hydroxylamine; Light; Retinaldehyde; Rhodopsins, Microbial; Schiff Bases

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 approximately 40%. The antenna carotenoid extends the wavelength range of the collection of light for uphill transmembrane proton transport.

Topics: Amino Acid Sequence; Bacteroidetes; Carotenoids; Energy Transfer; Glycosides; Hydrogen-Ion Concentration; Hydroxylamine; Light; Light-Harvesting Protein Complexes; Mass Spectrometry; Molecular Sequence Data; Oxygen Consumption; Proton Pumps; Retinaldehyde; Rhodopsins, Microbial; Spectrophotometry, Ultraviolet; Spectrum Analysis