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.

Crystallographic structure of xanthorhodopsin, the light-driven proton pump with a dual chromophore

“…2 A), somewhat less than the 56 Ϯ 3°estimated from the polarization anisotropy of retinal fluorescence (4). The discrepancy may originate from the off-axis orientation of the transition moment, as in rhodopsin (24).…”

“…Because energy transfer is from the shortlived S 2 carotenoid level (4), there must be a short distance and favorable geometry between the 2 chromophores to account for its high (40-50%) efficiency. Close interaction of the 2 chromophores is indicated by dependence of the carotenoid conformation on the presence of the retinal in the protein (1,5,6) and spectral changes of the carotenoid during the photochemical transformations of the retinal (1), but, as for the proteorhodopsin family of proteins, no direct structural information has been available (4,7). Unexpectedly, the crystallographic structure of xanthorhodopsin we report here reveals not only the location of the antenna but also striking differences from the archaeal retinal proteins, bacteriorhodopsin and archaerhodopsin.…”

Carotenoid response to retinal excitation and photoisomerization dynamics in xanthorhodopsin

“…Perhaps the most relevant difference is that in XR the Schiff base counterion is an aspartate-histidine complex rather than an aspartate as in BR [2]. Sharing of a proton between the aspartate and the histidine would lead to an extended lifetime of the excited state in a fraction of XR pool at neutral pH and especially upon lowering the pH [3], as it does neutralization of Asp 85 in BR [31]. …”

“…The angle between the conjugated systems of the two chromophores is 46°, providing an arrangement suitable for efficient energy transfer. Indeed, SX-to-retinal energy transfer occurs with an efficiency of 40% as demonstrated by action spectra for oxygen uptake in respiring cells [1], fluorescence excitation spectra in XR-containing cell membranes [3], and by direct measurements of energy transfer by femtosecond transient absorption [4]. Thus, the single carotenoid serving as a light-harvesting pigment makes XR the simplest known antenna system.…”

“…Comparison of the S 2 lifetimes of SX in NaBH 4 -treated XR, which has unperturbed SX binding site, but prevents SX-to-retinal energy transfer[3], allowed to determine the energy transfer rate. The S 2 lifetime of 110 fs observed in NaBH 4 -treated XR was shortened to 66 fs in untreated XR, leading to a calculated energy transfer time of 165 fs and an energy transfer efficiency of 40% [4].…”