Reconstitution of gloeobacter rhodopsin with echinenone: role of the 4-keto group

Abstract

In previous work, we reconstituted salinixanthin, the C(40)-carotenoid acyl glycoside that serves as a light-harvesting antenna to the light-driven proton pump xanthorhodopsin, into a different protein, gloeobacter rhodopsin expressed in Escherichia coli, and demonstrated that it transfers energy to the retinal chromophore [Imasheva, E. S., et al. (2009) Biochemistry 48, 10948]. The key to binding of salinixanthin was the accommodation of its ring near the retinal β-ionone ring. Here we examine two questions. Do any of the native Gloeobacter carotenoids bind to gloeobacter rhodopsin, and does the 4-keto group of the ring play a role in binding? There is no salinixanthin in Gloeobacter violaceous, but a simpler carotenoid, echinenone, also with a 4-keto group but lacking the acyl glycoside, is present in addition to β-carotene and oscillol. We show that β-carotene does not bind to gloeobacter rhodopsin, but its 4-keto derivative, echinenone, does and functions as a light-harvesting antenna. This indicates that the 4-keto group is critical for carotenoid binding. Further evidence of this is the fact that salinixanthol, an analogue of salinixanthin in which the 4-keto group is reduced to hydroxyl, does not bind and is not engaged in energy transfer. According to the crystal structure of xanthorhodopsin, the ring of salinixanthin in the binding site is turned out of the plane of the polyene conjugated chain. A similar conformation is expected for echinenone in the gloeobacter rhodopsin. We suggest that the 4-keto group in salinixanthin and echinenone allows for the twisted conformation of the ring around the C6-C7 bond and probably is engaged in an interaction that locks the carotenoid in the binding site.

Figure 1

Chemical structures of salinixanthin, salinixanthol, echinenone, and β-carotene.

Figure 2

Reconstitution of gloeobacter rhodopsin expressed in E. coli with echinenone. A. Absorption spectra in 0.02% DM, 25 mM MOPS, pH 7.2, 0.1 M NaCl of: 1, echinenone, 4 µM; 2, gloeobacter rhodopsin, 4 µM; 3, the spectrum taken 3 min after mixing; 4, after 24 h incubation. B. Absorption changes after addition of echinenone to gloeobacter rhodopsin and incubation at room temperature for: 2, 4, 8, 16 and 24 hours (spectra 1 through 5, respectively). C. Difference spectra of absorption changes after addition of 4 µM echinenone and incubation for 24 hours with: 1, gloeobacter rhodopsin, wild type; 2, its G178W mutant.

Figure 3

A. Absorption spectra of echinenone: 1, bound to the protein (obtained as a difference between spectra 4 and 2 in Figure 2A); 2, in ethanol, normalized at 485 nm maximum. B. Second derivatives of the spectra shown in panel A, d²A/dλ², multiplied by −1.

Figure 4

A. Circular dichroism spectra of: 1, echinenone in ethanol; 2, gloeobacter rhodopsin, 4 µM in 0.02 % DDM, 25 mM MOPS, 0.1 NaCl, pH 7.2; 3; after reconstitution with echinenone. B. Comparison of the CD spectra of gloeobacter rhodopsin reconstituted with: 1, echinenone; 2, salinixanthin; 3, salinixanthol; 4, β-carotene. The spectra were scaled to the same amount of rhodopsin (4 µM). Spectrum 2 was adapted from (23).

Figure 5

Absorption changes accompanying hydrolysis of the retinal Schiff base with hydroxylamine, 0.2 M, pH 7.2. A. Spectra of: 1, gloeobacter rhodopsin reconstituted with echinenone immediately after addition of 0.2 M hydroxylamine, 2 through 6, 10, 30, 60, 120, 360 min later; B. Difference “spectrum i minus spectrum 1”.

Figure 6

Excitation spectra for fluorescence emission of the retinal chromophore at 720 nm for: A. 1, gloeobacter rhodopsin; 2, as 1 but reconstituted with echinenone; 3, as 1 but with β-carotene added. Carotenoid/retinal ratio was 1:1; concentration of rhodopsin, close to 4 µM. pH 4.2 Absorbance of the samples in the maximum are between 0.1 and 0.2. B. 1, the G178W mutant, pH 4.2; 2, after reconstitution with echinenone (28 hours after addition). 3, Echinenone in 0.02% DDM (same concentration as in 2). C. 1, gloeobacter rhodopsin; 2, after reconstitution of gloeobacter rhodopsin with salinixanthin; 3, after mixing of gloeobacter rhodopsin with salinixanthol. Bandwidth of excitation beam, 8 nm.

Figure 7

A. Absorption spectra of: 1, gloeobacter rhodopsin, 2 µM in 0.02% DDM, pH 7.2, 0.1 M NaCl; 2, 2 µM of β-carotene added; 3, after 1 hour; 4, 5 hours later. B. Difference spectra: 1, “3 minus 2”; 2, “4 minus 3”.

Figure 8

Reaction of gloeobacter rhodopsin, with β-carotene added, with 0.2 M of hydroxylamine, pH 7.2 in the dark. A. Absorption spectra; 1, immediately after addition of hydroxylamine; 2 through 7, measured 10, 30, 60, 90, 150 and 240 min after addition; B. 1, through 6, absorption changes “spectrum i minus spectrum 1” where i are spectra 2 through 7 in panel A.