Identification of Carotenoids in Photosynthetic Proteins: Xanthophylls of the Light Harvesting Antenna - Carotenoids: Physical, Chemical, and Biological Functions and Properties - page 131

region for Arabidopsis plants kept in a high-light environment to induce the maximum formation of
zeaxanthin and NPQ ( light ). The control was plants placed in the dark for 10 min after illumination
( dark ). The corresponding resonance Raman spectra for these two states displays a clear difference.
The Raman spectrum of leaves from the light environment and therefore with both, zeaxanthin and
NPQ, revealed a relative increase in the intensity and a small upshift of the 535 nm band in com-
parison to the spectrum measured on leaves possessing zeaxanthin but no NPQ (Figure 7.11). The
light-minus-dark difference spectrum shows a n 1 maximum blueshifted toward 1520 cm −1 , which
is near the i ngerprint frequency of zeaxanthin (Figure 7.4). Remarkably, the difference spectra of
the NPQ4 mutant, which lacks the large part of NPQ, is almost nonexistent, (Figure 7.11a). These
observations produced the i rst evidence that the 535 nm band belongs to zeaxanthin. Estimations
based on the comparison of absorption and resonance Raman changes associated with NPQ have
allowed us to conclude that only about two zeaxanthin molecules per PSII are involved in the for-
mation of 535 nm band (Ruban et al., 2002b). Such a strong redshift of the absorption spectrum is
explained by the formation of J-type dimers of zeaxanthin, which have a 0-0 band in 530-535 nm
region (Figure 7.2). In the NPQ-associated resonance Raman spectrum, the n 1 amplitude becomes
negative for excitation wavelength below 500 nm (Ruban et al., 2002b). This observation suggests
that 535 nm zeaxanthin band has been formed from some short-wavelength forms of this pigment,
absorbing at 500-510 nm.
Several models can be suggested to explain the mechanism of zeaxanthin dimer formation
in NPQ. One is based on the assumption that after deepoxidation, zeaxanthin remains bound to
the same domain as violaxanthin. The aggregation of LHCII could force interactions between
stroma-facing zeaxanthin molecules situated on the two interacting trimers (Figure 7.11b) producing
J-type associates with 535 nm absorption. The latter serve as a good indicator for the conformational
antenna alterations leading to NPQ. As to whether the J-type aggregate can play a direct role in the
chlorophyll l uorescence quenching remains to be investigated. The fact that the 535 nm absorb-
ing zeaxanthin displays a typical resonance Raman spectrum for nonradical all- trans carotenoid
(Ruban et al., 2002b) suggests that this xanthophyll cannot be involved in the radical-type quench-
ing proposed for NPQ by Holt and coauthors (Holt at al., 2005).
The other model explaining the origin of 535 nm absorbing zeaxanthin involves PSII subunit S,
PsbS protein, which controls the dynamic range of NPQ by sensing the proton gradient and organiz-
ing the PSII antenna (Horton and Ruban, 2000; Li et al., 2000; Kiss et al., 2007). Isolated PsbS was
found to bind zeaxanthin and shift its 0-0 maximum toward 523-536 nm region (Aspinal et al., 2002).
The n 4 in the Raman spectrum of PsbS-bound zeaxanthin possesses a similar structure to that of
535 nm absorbing zeaxanthin identii ed in NPQ. Circular dichroism measurements revealed the for-
mation of a J-type dimer. The absorption of aromatic residues of the protein, mainly phenylalanine,
was also strongly redshifted (Aspinal et al., 2002). This coni rms the binding of zeaxanthin to PsbS.
Nevertheless, the question of whether or not the zeaxanthin binds to PsbS in vivo during NPQ still
remains controversial (Bonente et al., 2007). Alternatively, it is possible that the 535 nm signal arises
from a heterogenic interaction between a PsbS-bound zeaxanthin and a LHCII-bound zeaxanthin.
7.9 MOLECULAR ORIGINS OF THE RESONANCE RAMAN TWISTING
MODES OF ANTENNA XANTHOPHYLLS
The n 4 region enhancement and structure in the resonance Raman spectra of xanthophylls reviewed
in this chapter shows that it can be used for the analysis of carotenoid-protein interactions.
Figure 7.8 summarizes the spectra for all four major types of LHCII xanthophylls. Lutein 2 pos-
sesses the most intense and well-resolved n 4 bands. The spectrum for zeaxanthin is very similar to
that of lutein with a slightly more complex structure. This similarity correlates with the structural
similarity between these pigments. It is likely that they are both similarly distorted. The richer
structure of zeaxanthin spectrum may be explained by the presence of the two l exible b-end rings