Characterization of the Ultraviolet-B Absorption Band of Carotenoids Using Solvent-dependent Shifts in Steady-State and Transient Absorption Spectra.

The versatile functions of carotenoids in biological systems are associated with the extended π-electron conjugation system. Strong visible absorption resulting from the optically allowed S2 (1Bu+) state and the low-lying optically forbidden S1 (2Ag-) state examined. Carotenoids also exhibit an absorption band in the ultraviolet-B region; however, the origin of this band (hereafter referred to as Suv state) is not well characterized. The Suv state is a candidate for the destination level of the well-known S1 → Sn transient absorption; however, an obvious energy mismatch has been observed. In this study, we examined the steady-state and picosecond transient absorption spectra of lycopene in various solvents. The Suv absorption of carotenoids with diverse conjugation lengths was also examined. The dependence of the energies on solvent polarizability and conjugation length revealed that both Suv and Sn are the "second" Bu+ state. The absorption spectrum for lycopene at 200 K revealed an additional vibrational band, which may be the vibrational origin of the S0 → Suv band. Considering the slow vibrational relaxation of the 2Ag- state, the S1 → Sn transition may represent the 2Ag- (v = 1) → 2Bu+ (v = 0) transition, and the energetic contradiction can be resolved.

Structure-based validation of recombinant light-harvesting complex II.

Light-harvesting complex II (LHCII) captures sunlight and dissipates excess energy to drive photosynthesis. To elucidate this mechanism, the individual optical properties of pigments in the LHCII protein must be identified. In vitro reconstitution with apoproteins synthesized by Escherichia coli and pigment-lipid mixtures from natural sources is an effective approach; however, the local environment surrounding each pigment within reconstituted LHCII (rLHCII) has only been indirectly estimated using spectroscopic and biochemical methods. Here, we used cryo-electron microscopy to determine the 3D structure of the rLHCII trimer and found that rLHCII exhibited a structure that was virtually identical to that of native LHCII, with a few exceptions: some C-terminal amino acids were not visible, likely due to aggregation of the His-tags; a carotenoid at the V1 site was not visible; and at site 614 showed mixed occupancy by both chlorophyll a and b molecules. Our observations confirmed the applicability of the in vitro reconstitution technique.

Discovery of a novel siphonaxanthin biosynthetic precursor in Codium fragile that accumulates only by exposure to blue-green light.

Photosynthetic organisms adapt to a variety of light conditions. Codium fragile, a macrosiphonous green alga, binds a unique carbonyl carotenoid, siphonaxanthin, to its major photosynthetic light-harvesting complexes, allowing it to utilize dim blue-green light for photosynthesis. Here, we describe the absolute chemical structure of a novel siphonaxanthin biosynthetic precursor, 19-deoxysiphonaxanthin, that accumulates specifically in the photosynthetic antenna only when cultivated under blue-green light. The action spectra of pigment accumulation suggest that siphonaxanthin biosynthesis is regulated by a specific wavelength profile. The results provide clues to a new acclimation mechanism to withstand hours of intense light at low tide and why siphonous algae have been growing invasively on the world's coasts for more than a century.

Wavelength-Dependent Optical Response of Single Photosynthetic Antenna Complexes from Siphonous Green Alga Codium fragile.

The siphonaxanthin-siphonein-Chl-a/b-protein (SCP) complex from the siphonous green alga Codium fragile is the major light-harvesting complex (LHC) of these alga and is highly homologous to that of green plants (trimeric pigment-protein complex, LHCII). Interestingly, we find remarkable differences in the spectral response from individual SCP complexes when excited at 561 and 639 nm. While excitation in the green spectral range reproduces the common LHCII-like emission features for most of the complexes, excitation in the red spectral range yields a red-shifted emission and a significant reduction of the fluorescence decay time. We hypothesize that the difference in spectral response of SCP to light in the green and red spectral ranges can be associated with the adaption of the algae to their natural habitat under water, where sudden intensity changes are diminished, and excess light features a red-enhanced spectrum that comes at tidal timings.

Preprocess dependence of optical properties of ensembles and single siphonaxanthin-containing major antenna from the marine green alga Codium fragile.

The siphonaxanthin-siphonein-Chl-a/b-protein (SCP) is the light-harvesting complex of the marine alga Codium fragile. Its structure resembles that of the major light-harvesting complexes of higher plants, LHC II, yet it features a reversed Chl a:Chl b ratio and it accommodates other variants of carotenoids. We have recorded the fluorescence emission spectra and fluorescence lifetimes from ensembles and single SCP complexes for three different scenarios of handling the samples. While the data obtained from ensembles of SCP complexes yield equivalent results, those obtained from single SCP complexes featured significant differences as a function of the sample history. We ascribe this discrepancy to the different excitation intensities that have been used for ensemble and single complex spectroscopy, and conclude that the SCP complexes undergo an aging process during storage. This process is manifested as a lowering of energetic barriers within the protein, enabling thermal activation of conformational changes at room temperature. This in turn leads to the preferential population of a red-shifted state that features a significant decrease of the fluorescence lifetime.

Structural insights into blue-green light utilization by marine green algal light harvesting complex II at 2.78 Å.

Light-harvesting complex II (LHCII) present in plants and green algae absorbs solar energy to promote photochemical reactions. A marine green macroalga, Codium fragile, exhibits the unique characteristic of absorbing blue-green light from the sun during photochemical reactions while being underwater owing to the presence of pigment-altered LHCII called siphonaxanthin-chlorophyll a/b-binding protein (SCP). In this study, we determined the structure of SCP at a resolution of 2.78 Å using cryogenic electron microscopy. SCP has a trimeric structure, wherein each monomer containing two lutein and two chlorophyll a molecules in the plant-type LHCII are replaced by siphonaxanthin and its ester and two chlorophyll b molecules, respectively. Siphonaxanthin occupies the binding site in SCP having a polarity in the trimeric inner core, and exhibits a distorted conjugated chain comprising a carbonyl group hydrogen bonded to a cysteine residue of apoprotein. These features suggest that the siphonaxanthin molecule is responsible for the characteristic green absorption of SCP. The replaced chlorophyll b molecules extend the region of the stromal side chlorophyll b cluster, spanning two adjacent monomers.

Pigment structure in the light-harvesting protein of the siphonous green alga Codium fragile.

The siphonaxanthin-siphonein-chlorophyll-a/b-binding protein (SCP), a trimeric light-harvesting complex isolated from photosystem II of the siphonous green alga Codium fragile, binds the carotenoid siphonaxanthin (Sx) and/or its ester siphonein in place of lutein, in addition to chlorophylls a/b and neoxanthin. SCP exhibits a higher content of chlorophyll b (Chl-b) than its counterpart in green plants, light-harvesting complex II (LHCII), increasing the relative absorption of blue-green light for photosynthesis. Using low temperature absorption and resonance Raman spectroscopies, we reveal the presence of two non-equivalent Sx molecules in SCP, and assign their absorption peaks at 501 and 535 nm. The red-absorbing Sx population exhibits a significant distortion that is reminiscent of lutein 2 in trimeric LHCII. Unexpected enhancement of the Raman modes of Chls-b in SCP allows an unequivocal description of seven to nine non-equivalent Chls-b, and six distinct Chl-a populations in this protein.