Removal and reconstitution of the carotenoid antenna of xanthorhodopsin

Abstract

Salinixanthin, a C(40)-carotenoid acyl glycoside, serves as a light-harvesting antenna in the retinal-based proton pump xanthorhodopsin of Salinibacter ruber. In the crystallographic structure of this protein, the conjugated chain of salinixanthin is located at the protein-lipid boundary and interacts with residues of helices E and F. Its ring, with a 4-keto group, is rotated relative to the plane of the π-system of the carotenoid polyene chain and immobilized in a binding site near the β-ionone retinal ring. We show here that the carotenoid can be removed by oxidation with ammonium persulfate, with little effect on the other chromophore, retinal. The characteristic CD bands attributed to bound salinixanthin are now absent. The kinetics of the photocycle is only slightly perturbed, showing a 1.5-fold decrease in the overall turnover rate. The carotenoid-free protein can be reconstituted with salinixanthin extracted from the cell membrane of S. ruber. Reconstitution is accompanied by restoration of the characteristic vibronic structure of the absorption spectrum of the antenna carotenoid, its chirality, and the excited-state energy transfer to the retinal. Minor modification of salinixanthin, by reducing the carbonyl C=O double bond in the ring to a C-OH, suppresses its binding to the protein and eliminates the antenna function. This indicates that the presence of the 4-keto group is critical for carotenoid binding and efficient energy transfer.

Fig. 1

Reaction of membrane fraction containing salinixanthin not bound to xanthorhodopsin with 5 mM persulfate in the dark, pH 5.7. a Absorption spectra of suspension measured before (1) and after (2–6) addition of 5 mM persulfate and incubation for 1, 2, 4, 8 and 12 h. b Corresponding difference spectra “spectrum i minus spectrum 1,” where i = spectra 2–6 in a

Fig. 2

Bleaching (oxidation) of salinixanthin in suspension of S. ruber cell membranes containing xanthorhodopsin upon incubation with 5 mM ammonium persulfate in the dark at pH 5.7. a 1–5, absorption spectra measured before and after incubation for 1, 2, 3 and 10 h, respectively. b 1–4, absorption changes caused by incubation with ammonium persulfate for 1, 2, 3 and 10 h, respectively. c Kinetics of absorption changes at 1, 486 and 2, 600 nm. d Second derivatives of the absorption spectra before (1) and after (2) incubation with ammonium persulfate for 10 h

Fig. 3

Difference absorption spectra obtained upon illumination with 550–650 nm light of membranes containing xanthorhodopsin in the presence of 0.2 M hydroxylamine, pH 7.2: spectrum 1, membranes after bleaching of carotenoids with ammonium persulfate; spectrum 2, membranes without ammonium persulfate treatment. Spectra were normalized at 360 nm

Fig. 4

Effect of carotenoid bleaching on the CD spectrum of xanthorhodopsin. Spectrum 1, initial membranes containing xanthorhodopsin, pH 5.7; spectrum 2, after bleaching of carotenoids with ammonium persulfate

Fig. 5

Kinetics of laser flash-induced absorption changes in the sample after bleaching of salinixanthin with 5 mM persulfate, at pH 8.6 at three wavelengths: 1, 410; 2, 570; and 3, 620 nm

Fig. 6

Absorption changes accompanying reconstitution of salinixanthin into xanthorhodopsin. a Absorption spectra of 1, 2.5 μM xanthorhodopsin bleached with ammonium persulfate and solubilized in 0.15% DDM, 50 mM MES (pH 5.7), 100 mM NaCl; 2, 0.7 μM salinixanthin in 0.15% DDM, 50 mM MES (pH 5.7), 100 mM NaCl; and 3, after mixing samples 1 and 2. b 1–5, absorption spectra obtained upon consecutive additions of 0.7 μM of salinixanthin to 2.5 μM xanthorhodopsin. Conditions as in a. c 1–5, absorption changes observed after addition of salinixanthin to xanthorhodopsin bleached with persulfate, measured after 5, 10, 20, 60 and 90 min, respectively, in 0.15% DDM, 50 mM MES (pH 5.7); no salt added

Fig. 7

Restoration of the CD spectrum after reconstitution of xanthorhodopsin with salinixanthin. Spectrum 1, xanthorhodopsin after bleaching of the carotenoid component with persulfate and solubilization in 0.15% DDM; spectrum 2, salinixanthin in 0.15% DDM; spectrum 3, after reconstitution (mixing samples 1 and 2); spectrum 4, initial (unbleached) xanthorhodopsin solubilized in 0.15% DDM, 50 mM MES (pH 5.7), 100 mM NaCl, scaled to spectrum 3

Fig. 8

Comparison of spectral features upon reconstitution of xanthorhodopsin with salinixanthin and salinixanthol. a Absorption spectra in ethanol of 1, salinixanthin and 2, salinixanthol. b Absorption spectra in 0.15% DDM (pH 5.7) of 1, xanthorhodopsin after treatment with persulfate and solubilization; 2, reconstituted with salinixanthin and 3, mixed with salinixanthol. c CD spectra of samples 1–3 described in b. d Excitation spectra for emission at 720 nm for samples 1–3 described in b. Absorption of the samples was 0.3, 0.24 and 0.23 at the maximum, respectively. Contribution from an unknown fluorescing center (maximum emission at 610 nm, maximum in the excitation spectrum at 456 nm) was subtracted. Spectra are scaled to their maxima