Excitation energy-transfer and the relative orientation of retinal and carotenoid in xanthorhodopsin

Abstract

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.

FIGURE 1

Chemical structure of salinixanthin. From Lutnaes et al. (19).

FIGURE 2

Absorption, fluorescence, and excitation spectra of xanthorhodopsin. Effect of retinal removal with hydroxylamine. (A) Absorption spectrum of cell membranes of Salinibacter ruber containing xanthorhodopsin (at pH 5.5) before (spectrum 1) and after (spectrum 2) hydrolysis of the retinal Schiff base with hydroxylamine. The treatment converts the covalently bound retinal to the retinal oxime, whose absorption maximum is shifted from 563 nm to 367 nm, and the sharp carotenoid vibronic bands broaden. (B) Fluorescence emission of xanthorhodopsin, pH 5.5, excitation at 560 nm (spectrum 1) and 520 nm (spectrum 2). Spectrum 3, after hydroxylamine treatment and removal of the retinal oxime, excitation at 560 nm. Spectrum 4, spectrum 1 normalized to the amplitude for 520 nm excitation, by multiplying by 1.7. (C) Comparison of the xanthorhodopsin fluorescence bands at pH 8 (spectrum 1) and pH 5.5 (spectrum 2) with that of bacteriorhodopsin, pH 6 (spectrum 3). Excitation: 560 nm, 8 nm bandwidth. Absorbances of the samples at 560 nm were 0.29, 0.32, and 0.33, respectively. A small contribution from the Raman scattering of water was subtracted. (D) Fluorescence excitation spectra for the emission of xanthorhodopsin, sampled at 720 nm, at pH 8 (spectrum 1) and pH 5.5 (spectrum 2). Excitation beam bandwidth 4 nm.

FIGURE 3

Fluorescence of salinixanthin chromophore of xanthorhodopsin. (A) Fluorescence emission of xanthorhodopsin, excitation at 470 nm. Spectrum 1, measured emission; curves 2–4, Gaussian fits to three short-wavelength peaks. The residual contains mostly light scattering, and is not shown. Spectrum 5, sum of bands 2–4 (estimated emission from salinixanthin). Spectrum 6, measured emission as for spectrum 1, but after hydroxylamine bleaching, which removes the retinal and broadens the carotenoid peaks (see Fig. 2 A, spectrum 2). (B) Correction of fluorescence spectra for the contribution of xanthorhodopsin Raman band by deconvolution into Gaussian bands. Spectra 1 and 2 (dashed), measured emission spectra of S. ruber membranes in 20 mM MES, pH 5.5, produced by excitation at 485 and 490 nm, respectively. Note the shift of the sharp band at 522 nm in spectrum 1 to 527 nm in spectrum 2. Spectra 3–7, deconvoluted component spectra. Bands 3–5, Gaussians that fit the fluorescence bands of salinixanthin in xanthorhodopsin (maxima at 528, 563, and 602 nm, bandwidths, 26, 32, and 32 nm, and relative amplitudes 0.003, 0.0045, and 0.0015, respectively); spectrum 6, sum of the three Gaussian bands (spectra 3–5), assumed to be the carotenoid fluorescence spectrum. Spectrum 7, Raman scattering band (519 nm, bandwidth 14 nm, amplitude 0.0033). Excitation bandwidth 4 nm, emission monochromator bandwidth 8 nm. (C) Absorption and fluorescence spectra of the antenna salinixanthin in xanthorhodopsin. Spectrum 1, absorption spectrum (from Fig. 5 B, spectrum 1) plotted as the ratio A/ν); spectrum 2, fit of the fluorescence emission spectrum of the antenna carotenoid with a sum of three Gaussians (curve 6 in Fig. 3 B), divided by ν³. Spectrum 3 theoretical fluorescence spectrum (F_t/ν³) obtained as the mirror image of A/ν. A/ν and F/ν³ are expected to obey the mirror image rule most closely (31).

FIGURE 4

Effect of reduction of the retinal Schiff base with sodium borohydride on the absorption spectrum of xanthorhodopsin and the fluorescence intensity of salinixanthin. (A) Absorption spectra of (1) cell membrane fraction containing xanthorhodopsin, pH 8.5, 100 mM NaCl; (2) after reduction of the retinal Schiff base with sodium borohydride. (B) Second derivatives of the spectra in panel A (multiplied by −1) showing that the sharp carotenoid bands at 521, 486, and 456 nm did not change their shape on retinal Schiff base reduction. (C) Fluorescence spectra: (1) initial, pH 8.5; 2, treated with NaBH₄. (D) Second derivatives (multiplied by −1) of the fluorescence spectra of: (1) initial sample; (2) after treatment with NaBH₄. The maxima correspond to salinixanthin fluorescence bands.

FIGURE 5

Fluorescence excitation spectra of the retinal chromophore: comparison with absorption spectra and estimation of the efficiency of energy transfer. (A) Spectrum 1, absorption spectrum of xanthorhodopsin, pH 5.5. Spectrum 2, excitation spectrum for emission at 720 nm measured at magic angle (excitation with vertically polarized light (0°) and emission measured with polarizer set at 54.7°, which eliminates effects from preferential excitation). Excitation bandwidth 4 nm, emission bandwidth 32 nm. Spectrum 3, best fit of the excitation spectrum with retinal and carotenoid components in the absorption spectrum and with an efficiency for energy transfer of 0.49, determined as described in the text. (B) Spectra of the components of the excitation and absorption spectra: (1) carotenoid component in the absorption spectrum after subtraction of the retinal component and nonbound carotenoid; (2) carotenoid component in the excitation spectrum; (3) retinal component in the absorption and excitation spectra. (C) The ratio of the spectra of carotenoid components in the excitation and absorption spectra (spectrum 2 divided by spectrum 1 in Fig. 5 B). This ratio is the quantum efficiency of energy transfer, φ_m, from the carotenoid to the retinal.

FIGURE 6

Anisotropy of the excitation of retinal fluorescence. (A) Fluorescence excitation spectra (for 720-nm emission) measured under parallel and perpendicular polarization of excitation and emission beams. Spectrum 1, both beams vertically polarized (I_vv); spectrum 2, the polarizer in the emission beam set horizontally (I_vh). The first and second subscripts stand for polarization of the excitation and emission beams, respectively. (B) Spectrum of excitation anisotropy, R(λ), and its fit as a sum of two components, from retinal and carotenoid. Spectrum 1, excitation anisotropy; spectrum 2, fit of the anisotropy by the sum of two components, from the retinal and the carotenoid in xanthorhodopsin, R_r and R_bCar, multiplied by the fractional absorbances of the two chromophores and the quantum efficiency of energy transfer (for the carotenoid), R(λ) = R_rf_r(λ) + R_bCarf_bCar(λ)φ_m. From the fit, the anisotropies for the retinal and carotenoid components were determined to be R_r = 0.38, R_bCar = −0.04, respectively. (C) Fluorescence excitation anisotropy of bacteriorhodopsin. Dashed line is fit of experimental data with a constant, equal to 0.38.

FIGURE 7

Suggested scheme of energy transfer pathway from salinixanthin to the retinal chromophore of xanthorhodopsin. Light is absorbed by the carotenoid in the transitions from the ground state level S₀ to vibrational sublevels of the second excited state S₂ (), 486 nm is the main maximum. The transitions to the first excited state of the carotenoid S₁ () are not allowed for symmetry reasons (see most recent review (6)), but this state is populated in internal S₂-to-S₁ conversion. The carotenoid fluorescence with maxima at 529, 564, and 595–600 nm and the energy transfer to the S₁ state of retinal originate from the S₂ state. In analogy with bacteriorhodopsin, the retinal absorption band of xanthorhodopsin (∼560 nm at pH 8, and 563 nm at pH 5.5) will be from transition to the Franck-Condon levels of the S₁ state, which is also an acceptor of the energy transferred from salinixanthin. Relaxation of the excited retinal and its environment occurs on a timescale of 100–250 fs (40,69) and leads to a lower-energy excited state (state I) with a lifetime of ∼0.5 ps (58), from which the photoreaction and most of the fluorescence emission take place (39,69). Dashed arrows stand for internal conversion of excitation to heat. This is a minimal model. From analogy with other carotenoids (6,82,83) and bacteriorhodopsin (70), one might expect that both chromophores exhibit more excited singlet state levels than shown in this scheme. At least one “dark,” symmetry forbidden state () could be present between the S₁ (2¹Ag⁻) and S₂ () of the carotenoid. The “dark” S₂ (Ag⁻) state of the retinal chromophore (55) might be involved in the isomerization process starting from the I state (–73).