From the Department of Molecular Biology and
Biotechnology, University of Sheffield, S10 2TN, United Kingdom, the
Facoltà di Scienze MM. FF. NN., Biotechnologie Vegetali,
Università di 37134 Verona, Italy, and the ¶ Section de
Biophysique des Protéines et des Membranes, Department de
Biologie/Cellulaire et Moleculaire Section de Biophysique des Proteines
et des Molecules F91191 & Unite de Recherche Associee 2096/CNRS,
CE-Saclay, France
Received for publication, April 12, 2001
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ABSTRACT |
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Resonance Raman excitation spectroscopy combined
with ultra low temperature absorption spectral analysis of the major
xanthophylls of higher plants in isolated antenna and intact thylakoid
membranes was used to identify carotenoid absorption regions and study
their molecular configuration. The major electronic transitions of the light-harvesting complex of photosystem II (LHCIIb) xanthophylls have been identified for both the monomeric and trimeric states of the
complex. One long wavelength state of lutein with a 0-0 transition at
510 nm was detected in LHCIIb trimers. The short wavelength 0-0 transitions of lutein and neoxanthin were located at 495 and 486 nm,
respectively. In monomeric LHCIIb, both luteins absorb around 495 nm,
but slight differences in their protein environments give rise to a
broadening of this band. The resonance Raman spectra of violaxanthin
and zeaxanthin in intact thylakoid membranes was determined. The broad
0-0 absorption transition for zeaxanthin was found to be located in the
503-511 nm region. Violaxanthin exhibited heterogeneity, having two
populations with one absorbing at 497 nm (0-0), 460 nm (0-1), and 429 nm (0-2), and the other major pool absorbing at 488 nm (0-0), 452 nm
(0-1), and 423 nm (0-2). The origin of this heterogeneity is discussed. The configuration of zeaxanthin and violaxanthin in thylakoid membranes
was different from that of free pigments, and both xanthophylls (notably, zeaxanthin) were found to be well coordinated within the
antenna proteins in vivo, arguing against the possibility of their free diffusion in the membrane and supporting our recent biochemical evidence of their association with intact oligomeric light-harvesting complexes (Ruban, A. V., Lee, P. J.,
Wentworth, M., Young, A. J., and Horton, P. (1999) J. Biol. Chem. 274, 10458-10465).
The light-harvesting antenna
(LHA)1 of higher plants binds
five types of xanthophylls: lutein, neoxanthin, violaxanthin,
zeaxanthin, and antheraxanthin. The last three constitute the
xanthophyll cycle, which has been suggested to participate in the
process of dissipation of excess excitation energy, giving rise to
nonphotochemical fluorescence quenching (1-3). Several important
questions concerning these xanthophylls remain unanswered: why is there
such a variety of xanthophyll types in antenna; what is the exact
molecular mechanism of zeaxanthin action in nonphotochemical
fluorescence quenching; where are xanthophylls located; and what is the
nature of their interaction with the protein and other pigments? LHA
consists of a number of pigment-protein complexes accommodating
different types and amounts of xanthophylls (5-8). The major and most
characterized LHA complex, the trimeric LHCIIb, binds 2 luteins, 1 neoxanthin, and between 0.1 and 1 violaxanthin per monomer (7, 9). The amount of bound violaxanthin was found to depend on the treatment during purification (9), as well as on plant growth conditions (10).
The two luteins of LHCIIb are thought to correspond to the two
carotenoid molecules located near the transmembrane helixes A and B in
the inner core of the complex (11), thus being tightly associated with
it and probably having a structural role. Site-directed mutagenesis
experiments have suggested that neoxanthin is associated with helix C
(12). Neoxanthin was found to have the highest affinity of binding to
the complex (9). In contrast, the binding affinity of violaxanthin to
the complex is the lowest, and it can be easily removed by various
treatments. This biochemical work has therefore established that a
large population of xanthophyll cycle carotenoids is peripherally bound
to LHA complexes.
The structure of the minor PSII antenna complexes as well as all LHCI
complexes is not known; therefore, it is not clear where xanthophyll
molecules are bound to them, what the nature of this binding is, or
what the carotenoid configuration is involved. The data on xanthophyll
stoichiometry in the minor PSII antenna are controversial (4, 6, 8) and
may well again reflect natural variation of xanthophyll ratios.
However, it is clear that unlike LHCIIb, the minor antenna PSII
complexes, CP24, CP26, and particularly CP29, contain at least one
strongly bound xanthophyll cycle carotenoid. The efficiency of
violaxanthin de-epoxidation It is clear that new methodologies are needed to identify, locate, and
analyze xanthophylls and their configuration and function in LHA
complexes. One powerful approach has been to reconstitute light-harvesting complexes from Lhcb polypeptides and pigment mixtures of varying composition. Site-directed mutagenesis of these
polypeptides has been used to determine the location and specificity of
carotenoid binding sites (12). The effects of carotenoid binding to
peripheral sites in LHA complexes has allowed investigation of their
role in energy dissipation (14-17). Along with the development of
these biochemical techniques there has been progress toward the
development of instrumental methods to analyze antenna xanthophylls.
Absorption, linear and circular dichroism, and triplet state
spectroscopies have been applied to identify their electronic
absorption bands and energy transfer pathways to and from chlorophyll
molecules (18-22). However, it remains difficult to make unambiguous
assignments for even simpler systems, such as those containing only
three xanthophylls, and impossible to make reasonable spectral
assignments in the Soret absorption region of whole thylakoid
membranes. Recently, we have combined optical absorption spectroscopy
with selective excitation resonance Raman spectroscopy in order to
identify the absorption transitions of lutein and neoxanthin in LHCIIb
trimers (23). Unlike previous work (18), which used LHCIIb containing
three types of carotenoids, we have prepared LHCII, which contained only lutein and neoxanthin, a more simple system. The characteristic Raman feature for neoxanthin and lutein, the The approaches mentioned above are susceptible to various artifacts and
limitations, such as the removal of lipids and pigments by the
detergents used in the isolation and reconstitution procedures and the
resulting alteration in protein conformation, xanthophyll binding
affinity, and xanthophyll environment. One way forward would be the
isolation of a more integrated LHA, in which pigments are less
perturbed by detergent treatment. For example, we have recently
isolated an oligomeric LHCIIb-enriched antenna complex using very mild
detergent treatment of PSII particles and unstacked thylakoid membranes
(9), and this preparation contained a higher proportion of violaxanthin
and zeaxanthin. However, the most ideal approach would be to analyze
carotenoid structure and dynamics in situ in intact
thylakoid membranes.
The aim of the work described in this paper is to further develop the
new spectroscopic methodologies to analyze xanthophylls, and most
importantly to make progress toward their application to more complex
systems including the whole thylakoid membrane. This approach not only
enables the determination of the electronic transitions of all
xanthophylls in vivo but also allows monitoring of their
conformational and configurational dynamics and binding within the
native LHA. To achieve this aim, we have first undertaken a systematic
search for the characteristic features of isolated carotenoids that are
found in the photosynthetic membrane, in order to use them for
constructing the resonance Raman profiles. This allowed absorption band
assignments not only for xanthophylls of isolated antenna complexes but
also violaxanthin and zeaxanthin of intact antennae in thylakoid
membranes. We have obtained for the first time the resonance Raman
spectra of violaxanthin and zeaxanthin in vivo. It is
concluded that zeaxanthin adopts a configuration that is likely to
reflect its well defined binding within the antenna, rather than a free
location in the membrane. This work establishes a new approach to the
study of complex carotenoid-containing systems and offers a broad range
of applications, from identification and assessment of xanthophyll
configuration in reconstituted/isolated complexes to in vivo
investigation of xanthophyll cycle carotenoids in order to establish
their role in photoprotective mechanisms.
Carotenoid samples were prepared as described in Ref. 24 by Dr.
Denise Phillip (John Moores University, Liverpool, United Kingdom). LHCIIb was prepared from dark-adapted spinach leaves using
isoelecrofocusing of PSII-enriched particles, as described in
Ref. 8. Purification of LHCIIb trimers and removal of violaxanthin was
carried out on a sucrose gradient (9). LHCIIb monomers were prepared by
phospholipase A2 treatment of LHCIIb trimers for 36 h in the
presence of 20 mM of CaCl2 at a chlorophyll
concentration of 0.5 mM, followed by purification on a
sucrose gradient as described previously (9). Intact thylakoid
membranes were obtained by the procedure described in Ref. 8. To induce
maximum violaxanthin de-epoxidation, thylakoids were incubated at room
temperature at a chlorophyll concentration of 200 µM for
2 h in a medium containing 5 mM
D-isoascorbate, 10 mM HEPES, and 10 mM sodium citrate at pH 5.5 with or without 5 mM Mg2+.
Absorption spectra were recorded on a Varian Cary E5 double-beam
scanning spectrophotometer; measurements at 4 K were performed using a helium bath cryostat (Utreks). Low temperature resonance Raman
spectra were obtained in a helium flow cryostat (Air Liquide, Paris,
France) using a Jobin-Yvon U1000 Raman spectrophotometer equipped with a liquid nitrogen-cooled charge-coupled devices detector (Spectrum One, Jobin-Yvon, Paris, France) as described in Ref.
25. Excitation was provided by Coherent Argon (Innova 100) and Krypton
(Innova 90) lasers (at 457.9, 476.5, 496.5, 488.0, 501.7, and
514.5 nm and at 528.7 and 413.1 nm, respectively) and a Liconix
helium-cadmium laser (at 441.6 nm). The choice of this wavelength range
was determined by the absorption profiles of the xanthophylls used.
Fig. 1 displays absorption spectra of the four major xanthophylls that have been studied. The number of laser
excitation lines (indicated in Fig. 1 by dotted arrows) covers all of the 0-0 transitions and also part of the
electron-vibrational bands (0-1 and 0-2). It is likely that carotenoid
absorption maxima in vivo will be somewhat shifted from
those in organic solvent. However, from previous experience with LHCIIb
xanthophylls, we have found that pyridine provides an environment with
polarizability (n = 1.5092) much closer to that of
lipid and membrane protein environments than such commonly used
solvents as n-hexane (n = 1.3750) or ethanol
(1.3611). The amplitude of the resonance effect is proportional to the
square of the absorption probability. This offers an efficient
line-narrowing effect in the Raman excitation profiles compared with
absorption spectra, and the increased selectivity makes it possible to
resolve complex bandshapes.
Wavelength-selective Resonance Raman Spectroscopy of Isolated
Carotenoids--
Carotenoids are very efficient Raman scatterers and
exhibit a very strong resonance enhancement (26). Four main frequency regions have been observed, calculated, and assigned as follows:
The position of the Organization of Xanthophylls in LHCIIb: Lutein and
Neoxanthin--
Neoxanthin and lutein have very strong binding
affinities to LHCIIb compared with violaxanthin, which can be removed
by detergent (9). Therefore, it was possible to prepare LHCIIb free
from violaxanthin, containing only lutein and neoxanthin. The RR
spectra of this LHCIIb sample, after monomerization, were obtained, and the
The absorption spectrum of the LHCIIb trimer is known to contain a new
transition around 510 nm not found in monomers (18, 23) (see Fig.
4B). Although it was suggested to belong to violaxanthin (18), it was still present in preparations free from this carotenoid (23). Analysis of RR excitation profiles for Resonance Raman Spectroscopy of Thylakoid Membranes--
The Soret
absorption band of the whole thylakoid membrane is more complex than
that of LHCIIb in the carotenoid region (Fig. 5); this complexity arises from, among
other factors, the presence of extra carotenoids, violaxanthin, and
RR spectra were measured for the thylakoid membranes containing only
violaxanthin and those enriched with zeaxanthin. The spectra were
clearly different in the
An alternative approach to deconvolution of the spectrum is to
calculate difference RR spectra (de-epoxidized-minus-epoxidized) following normalization at the 1540 cm
An attempt was made to compare absolute RR amplitudes for violaxanthin-
and zeaxanthin-containing thylakoids on the same scale without using
this normalization procedure. First, exactly the same amount of
chlorophyll was used in each sample, and an average was calculated from
several replicates. Second, the small but reproducible signals from
chlorophyll at 1437, 1354, and 1327 cm
The
Thus, excitation at 488.0 nm is selective for violaxanthin, whereas
excitation above 500 nm (e.g. at 528.7 nm) is selective for
zeaxanthin. With this information, it is possible to explore the state
of violaxanthin and zeaxanthin in the thylakoid membrane, compared with
pigments dissolved in detergent/lipid micelles or in organic solvent.
In Figs. 8 and
9, RR difference spectra were obtained
for xanthophylls dissolved in pyridine (spectrum 1), in the free pigment fraction following detergent treatment of thylakoid
membranes (spectrum 2), and for thylakoid de-epoxidation treatment (spectrum 3) excited at 488.0 nm (Fig. 8) and
528.7 nm (Fig. 9). The spectra are (+Vio)-(+Zea) and (+Zea)-(+Vio) for 488.0 and 528.7 nm excitation, respectively. For 488.0 nm excitation, the thylakoid spectra in the
The thylakoid spectra for 528.7 nm excitation are again similar to the
spectra of isolated zeaxanthin, either in solvent or in detergent-lipid
mycelles (Fig. 9). The In this paper, we have demonstrated a new approach to the
characterization of higher plant xanthophylls using comparison of absorption band structure and resonance Raman excitation profiles. The
approach proved to be an effective methodology for identification and
monitoring of the molecular conformation and configuration of
carotenoids both in isolated pigment-protein complexes and, most
significantly, in intact thylakoid membranes. This method is based on
the identification of a number of characteristic fingerprints in the
resonance Raman spectrum for each carotenoid involved. Analysis of
isolated LHCIIb monomers and trimers containing only two types of
xanthophylls, lutein and neoxanthin, allowed the identification of
their corresponding absorption bands and molecular configuration.
In the monomeric state of LHCIIb, the configuration and environment of
the two LHCIIb luteins are very similar, both absorbing at 495 nm. The
broader full width at half maximum for the band at 495 nm in the
monomer spectrum compared with that of the trimer (Fig. 4) may suggest
that their maxima positions differ within this region and therefore
that their environment is also slightly different. In the trimer there
is a 510 nm band not found in the monomer; this band was assigned to a
red-shifted lutein in the former. Its intensity was significantly
reduced in the second derivative spectrum because of the much broader
FWHM compared with the short wavelength form of the pigment. In the
trimer, the 510 nm lutein undergoes significant twisting, as a result either of an influence of the protein or of interaction with the other
pigments or lipids present. It is not clear from our data whether this
lutein plays a specific role in, for example, stabilization of the
trimer and/or photoprotection of chlorophylls. However, these large
differences in environment of the two luteins are important from
structural and spectroscopic viewpoints (11, 18-21). This approach has
revealed the molecular dynamics of a bound xanthophyll as a function of
the state of oligomerization of the complex, and this for the first
time. These factors may (at least in part) be behind the existence of
monomeric (minor antenna), trimeric (major LHCIIb), and oligomeric
states of antenna proteins in vivo. In addition, the
methodology described here could be also used in the investigation of
the LHC reconstitution process, using various types of xanthophylls
(33, 34).
The application of RR spectroscopy to thylakoid membranes has revealed
important new information about the xanthophyll cycle carotenoids,
zeaxanthin and violaxanthin. The physiological role of these
carotenoids is unclear, as is their location in the thylakoid membrane.
Indirect measurements (correlating nonphotochemical fluorescence
quenching with de-epoxidation state) have suggested that xanthophyll
cycle carotenoids may be completely free in the thylakoid membrane and
can only move to a very small number of quenching centers in LHCII
proteins upon Ultra-low temperature absoprtion spectroscopy has also revealed new
information about these carotenoids. The zeaxanthin-minus-violaxanthin absorption spectrum of thylakoids had a complex structure. The heterogeneity within this spectrum suggested that violaxanthin exists
in two different populations. This suggestion is consistent with the
observation that violaxanthin was found to be differently bound to the
minor and major LHCII and LHCI complexes. It is possible that one
population corresponds to violaxanthin associated with the minor LHCII
or LHCI, whereas the other is peripherally bound to LHCIIb. An
alternative explanation is that the dual band structure of the
difference spectrum zeaxanthin minus violaxanthin originates from
excitonically coupled violaxanthin. In this case, the coupling energy
(V) will be within 180 cm It is interesting to mention that acidification did not cause
alteration in violaxanthin maximum positions, which one would anticipate in the case of Clearly, the application of spectroscopic methods to the analysis of
xanthophylls in isolated complexes and intact thylakoids provides a new
approach to understanding the structure and dynamics of these
molecules. In particular, the selectivity of the RR technique and its
freedom from the artifacts and problems normally associated with
absorption and fluorescence measurements on complex intact systems have
provided new information about violaxanthin and zeaxanthin in
vivo. In the future, we hope to apply a similar methodology to
whole leaves.
Acknowlegdements--
We acknowledge Dr. Denise Phillip and Prof.
Andrew Young for a kind gift of carotenoid samples.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
a prerequisite for the photoprotective
energy dissipation state in thylakoid
was found to be the reciprocal
of the relative affinity of binding of this xanthophyll to various
antenna components, suggesting that only loosely bound molecules are
accessible to the violaxanthin de-epoxidase (9). A similar tendency was
observed for the PSI antenna, which contains at least 50% tightly
bound violaxanthin that was not converted into zeaxanthin even under conditions favoring maximum de-epoxidation (9, 13). Therefore, it may
be argued that the "structural" violaxanthin molecules, which are
strongly coordinated within the minor LHA complexes, do not play a
role in the xanthophyll cycle.
1 maximum
position, was identified and used to build the Raman excitation
profiles for the isolated complex. This allowed not only an estimation of the energies for the three absorption bands (0-0, 0-1, and 0-2) but
also an observation of the dynamics of carotenoid configuration upon
oligomerization of the complex.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Room temperature absorption spectra of the
main thylakoid xanthophylls: zeaxanthin (Zea), lutein
(Lut), violaxanthin (Vio), and
neoxanthin (Neo) in pyridine. Dotted
arrows indicate positions of the laser lines used in resonance
Raman experiments, and solid arrows the three carotenoid
absorption transitions, 0-0, 0-1, and 0-2.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1 - C = C stretching vibrations;
2
- C-C stretches coupled either to C-H in-plane bending or
C-CH3 stretching;
3 - CH3
in-plane rocking vibrations;
4 - C-H out-of-plane
bending modes. In RR spectra of carotenoid molecules, the position of
1 varies according to the number of conjugated C=C bands
that these molecules possess, being higher in the case of shorter
conjugated chains (27). cis-Carotenoids also exhibit higher
1 frequencies than all-trans-carotenoids (28,
29). We have found this frequency to be in general unaffected by the
solvent type (pyridine, n-hexane, ethanol, and cyclohexane; data not shown), in agreement with the published data for
-carotene (30). Fig. 2 shows 488 nm excited RR
spectra of the four main LHC antenna xanthophylls and
-carotene, all
dissolved in pyridine. The different numbers of conjugated double bonds
in these carotenoids (from 9 to 11) and the 9-cis
conformation of neoxanthin give rise to a large
1
variability from zeaxanthin at 1524 cm
1 to
neoxanthin at 1533 cm
1. Other regions of the
RR spectra are also specific for certain xanthophylls. The
arrows in Fig. 2 show the characteristic frequencies for
neoxanthin in the
2 region at 1120, 1132, and 1203 cm
1, probably due to its 9-cis
conformation, and for violaxanthin in the
3 band at 1007 cm
1. The
4 region was very low
in intensity for all carotenoids, out-of-plane modes being formally
resonance-forbidden for fully planar molecules. However, they can
become significant under conditions in which the carotenoid undergoes
configurational rearrangements leading to twisting of the molecule,
due, for instance, to interaction with its environment. This situation
is not frequently found in solvent or detergent media, but it has been
observed in certain cases for carotenoids attached to antenna complexes
(26, 23). Molecular distortion of this kind requires energy, which can
be gained in the close contact with protein environments such as a
hydrophobic helix. Thus, the
4 region can be used as a
marker for pigment-protein interactions and environmental perturbations involving the carotenoid molecule.
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Fig. 2.
Resonance Raman spectra of the principal
thylakoid membrane carotenoids in pyridine, excited at 488.0 nm.
Vertical arrows indicate the characteristic features in the
2 and
3 regions and the variation in
1 frequency, as discussed in the text.
1 band for each carotenoid was
determined for all excitation lines used (Fig.
3A). Each additional double bond produces an ~3 cm
1 downshift of
1. Comparing lutein and neoxanthin, there is a difference of 8 cm
1 in the
1
position. This parameter can therefore be used to identify xanthophyll
absorption transitions in LHCII complexes containing these two types of
xanthophylls (see below). Features in the
2 and
3 regions can be used to construct additional excitation profiles to confirm band assignments. For example, the
1 frequencies for lutein and violaxanthin for some
excitation lines can be very close (only 2 cm
1 apart) (Fig. 3A). However,
additional analysis of the
3 region can be used to make
unambiguous assignments, because the corresponding frequency difference
between lutein and violaxanthin is at least 4 cm
1 (Fig. 3B).
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Fig. 3.
Dependence of
1 (A) and
3 (B) frequency on
excitation wavelength for the four main thylakoid xanthophylls and
-carotene. Nonlinear regression curves are
shown as dotted lines.
1 maximum position was analyzed as a function of the
excitation wavelength. Whereas this parameter was only slightly
variable with the resonance wavelength for isolated xanthophylls (Fig. 3), for LHCIIb monomers, it was strongly dependent upon it (Fig. 4A, open circles). For 457.9 and 488.0 nm excitations, the
1 position was close to
that of isolated neoxanthin, whereas for other excitation wavelengths,
it was similar to that of lutein. This indicates that neoxanthin
contributions dominate the RR spectra obtained with 457.9 and 488.0 nm
excitations. Fig. 4A also shows the second derivative of the
absorption spectrum of the LHCIIb monomer, which shows two maxima, at
457 and 486 nm. The 29-nm shift between these bands is in a good
agreement with that expected between 0-0 and 0-1 transitions of
xanthophylls. An additional diagnostic parameter was the relative
amplitudes of the neoxanthin-specific bands at 1203 cm
1 in the
2 region and at
1006 cm
1 in the
3 band (see
Figs. 2 and 3). Excitation profiles for both of these closely matched
the
1 excitation profile, confirming the assignment of
the 485 nm band to neoxanthin. We suggest that the 495 and 466 nm bands
observed in the absorption spectrum most likely originate from lutein
(the 476 nm band arises from chlorophyll b).
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Fig. 4.
1 dependence on
excitation wavelength for LHCIIb, as compared with its 4 K absorption
band structure in the Soret region. A,
1
wavelength dependence for LHCIIb monomer (open circles) and
for neoxanthin and lutein (solid curves, Neo and Lut,
respectively; nonlinear regression plots are taken from Fig.
3A). Dashed-dotted line, second derivative of the
LHCIIb monomer 4 K absorption spectrum. Also shown are the relative
intensities of the neoxanthin-specific 1203 and 1006 cm
1 bands for LHCIIb monomers (closed
circles and open triangles, respectively).
B, second derivative of the 4 K absorption spectrum of
LHCIIb trimer (dashed-dotted line). Solid line,
trimer-minus-monomer difference absorption spectrum for LHCIIb,
indicating the new trimer band at 510 nm.
1 in LHCIIb
trimers has been used as evidence that the 510 band belonged to lutein, because the
1 frequency for both 501.7 and 514.5 nm
excitations was very close to that of lutein (23). In the second
derivative spectrum (Fig. 4B), it is significantly smaller
than the 485 and 495 nm bands, which are suggested to arise from
neoxanthin and lutein, respectively. Because there are two luteins
bound to each LHCIIb, if the 495 nm band corresponds to the 0-1 absorption band of one lutein molecule, then the 510 nm band should
belong to that of the other one. Why then are their absorption
amplitudes in the second derivative spectrum so different? A possible
answer to this question is that the 510 nm band may have a smaller
extinction coefficient. However, a more likely explanation lies in the
observation that the 510 nm band is at least 70% broader than that at
495 nm. As the amplitude of peaks in a second derivative spectrum is
reciprocal to their bandwidth, this results in the relative amplitude
of the 510 nm component being lower. Indeed, in the trimer minus
monomer difference spectrum, the bandwidth of the band around 510 nm
reaches 18 nm (Fig. 4B, solid line).
-carotene. However, it is possible to do comparative spectroscopic
studies on the membranes by replacing violaxanthin with zeaxanthin by
activation of the xanthophyll cycle. Activation of de-epoxidation in
thylakoids yielded about 80% replacement of violaxanthin with
zeaxanthin. The 4 K absorption spectrum changes significantly after
de-epoxidation of violaxanthin. In Fig. 5, arrows indicate a
decrease in 488 and 460 nm regions and the appearance of new bands at
505 and 476 nm. This is consistent with the room temperature difference spectra-illuminated-minus-dark, measured on leaves (31, 32). However,
the spectrum recorded at 4 K reveals more structure. The second
derivative of the difference spectrum (+Zea)-(+Vio) resolves a complex
picture (Fig. 5B). The three characteristic negative bands
show a doublet structure at 4 K: 488/497, 452/460, and 429/423 nm. The
doublet structure may arise from the two populations of violaxanthin
(i.e. integrally bound or peripheral), both of which were
de-epoxidized. There is also complexity in the positive bands, and the
503 and 511 nm positive bands may correspond to zeaxanthin 0-0 transitions. However, the de-epoxidation process could have caused
changes in antenna conformation, which could then affect other
xanthophylls, altering their absorption parameters and giving rise to
the complexity of the spectra shown in Fig. 5. Therefore, more evidence
is required to identify the origin of the absorption changes observed
upon violaxanthin de-epoxidation.
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Fig. 5.
A, 4 K absorption spectra of
thylakoid membranes containing violaxanthin (+Vio)
and following 80% conversion to zeaxanthin (+Zea).
(+Zea)-(+Vio) represents the corresponding difference
spectrum. Vertical arrows indicate characteristic negative
and positive bands in the difference spectrum. B, second
derivative of the (+Zea)-(+Vio) difference spectrum, revealing the
doublet nature of the spectrum (note that the spectrum has been
inverted).
1 region, the position of
1 maximum being 3 cm
1
downshifted in thylakoids containing zeaxanthin (Fig.
6, circles). It was found that
the RR spectra of thylakoids was much wider than the spectrum of LHCIIb
(Fig. 6A, broken line). This is most likely to be due to a
more complex structure of thylakoid RR because of additional
contributions of violaxanthin, zeaxanthin,
-carotene, and small
amounts of antheraxanthin. A deconvolution of this region using the
1 spectra of isolated carotenoids (Fig. 6A, solid
lines) provides evidence to support this view. The spectrum for
violaxanthin-containing thylakoids can be explained as the sum of
lutein, violaxanthin, neoxanthin, and
-carotene (Fig.
6A). The
1 regions for
-carotene and
zeaxanthin are almost identical and are added together in Fig.
6B; again, a good fit to the thylakoid spectrum was
obtained. Thus, the downshift in the
1 position after
de-epoxidation can be explained by an increase in the low-frequency
zeaxanthin/
-carotene signal (around 1522 cm
1) and a decrease in the violaxanthin band
at 1528 cm
1. Lutein (1526 cm
1) and neoxanthin (1534 cm
1) bands remained almost unchanged after
de-epoxidation. The domination of the spectrum by lutein is explained
by neoxanthin being further out of resonance from 501.7 nm excitation
compared with lutein, because it absorbs at 485 nm, whereas lutein
absorbs at 495 nm (see Fig. 1 and discussion above).
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Fig. 6.
1 region of the resonance Raman
spectra of thylakoid membranes containing violaxanthin (A)
and upon 80% conversion to zeaxanthin (B), excited at 501.7 nm. Open circles represent the original spectra.
Broken line, spectrum for LHCIIb, excited at 488.0 nm.
L, V, N, Z, and
indicate spectra of isolated
lutein, violaxanthin, neoxanthin, zeqaxanthin, and
-carotene,
respectively, used to fit the thylakoid spectra. Only the amplitudes
have been varied to obtain the best fit using SigmaPlot software. As
the spectra in this region are very similar for zeaxanthin and
-carotene (see Figs. 2 and 3), only one trace was used to represent
both carotenoids for zeaxanthin-containing membranes (B).
The modeled spectra matched the empirical data very closely and can be
seen occasionally as dotted curves. C,
(+Zea)-(+Vio) difference spectra for thylakoids at various
excitation wavelengths. Original spectra were normalized at 1540 cm
1 (indicated by a vertical dotted
line).
1
region, where contribution of the zeaxanthin and violaxanthin signals
is very low (see Fig. 6, A and B). Fig.
6C displays a number of such difference spectra for the
1 region. The vertical dotted line indicates
the normalization point, where the difference was always zero. It was
found that the shape of the spectrum was strongly dependent on
excitation wavelength. With 528.7, 514.5, and 501.7 nm excitation, an
almost symmetrical positive band was found, but with 496.5 and
particularly 488 nm excitation, there was a rather asymmetric decrease
in the high frequency region of
1 due to a decrease at
1530 cm
1. These data may be explained by the
positive band arising from zeaxanthin and the negative one from
violaxanthin, the wavelength dependence of the spectrum arising from
differential excitation of these two pigments. Indeed, when the
positions of the maxima and minima were plotted as a function of
excitation wavelength (Fig.
7A), it was found that all
components gave a
1 value either close to that for
violaxanthin (around 1530 cm
1) or zeaxanthin
(1520 cm
1); even the wavelength dependence of
each matched that of the isolated pigments in vitro (Fig.
7A, full lines; taken from Fig. 3A). Furthermore,
the relative extent of increase in
1 intensity around
1520 cm
1 matched the two positive bands in
the low temperature difference spectrum de-epoxidized minus epoxidized
(Fig. 7B). Therefore, it is concluded that the transitions
at 505-510 and 476 nm belong to the 0-0 and 0-1 bands of zeaxanthin.
On the other hand, the wavelength dependence for the intensity of the
negative band around 1530 cm
1 indicates that
488 and 460 nm absorption bands arise from violaxanthin. Even the 497 nm second derivative band shown in Fig. 5 may also belong to
violaxanthin, because excitation at 496.5 nm produced a relative
decrease in the
1 intensity in the violaxanthin region. Unfortunately, the limited number of excitation lines available did not
allow resolution of the 452 and 460 nm bands, but their distance from
the 488 and 497 nm transitions suggests that they arise from 0-1 vibrational satellites.
View larger version (31K):
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Fig. 7.
A, resonance Raman excitation profiles
of the 1 maxima (open circles) and minima
(closed circles) of the difference spectra in Fig.
6C. Solid lines are nonlinear regression plots of
1 wavelength dependence for violaxanthin
(Vio) and zeaxanthin (Zea) taken from Fig. 3.
B, resonance Raman excitation profiles for the normalized
1 amplitudes of positive (open circles) and
negative (closed circles) bands of the difference spectra
from Fig. 6C. Dashed line is the difference
spectrum taken from Fig. 5.
I
1 was calculated as the
positive/negative amplitude divided by the amplitude of the
1 in the spectrum of thylakoid membranes containing
violaxanthin only. C, resonance Raman excitation profiles
for the
1 amplitude in spectra for thylakoid membranes
containing violaxanthin (closed circles) and zeaxanthin
(open circles). Spectra were normalized to the chlorophyll
bands around 1437, 1354, and 1327 cm
1.
Triangles represent the reduced difference spectrum
((+Zea)-(+Vio))/(+Vio). D, resonance Raman excitation
profiles for the 1003: 1006 cm
1 ratio in the
3 region for violaxanthin-containing (closed
circles) and zeaxanthin-containing (open circles)
thylakoid membranes. Inset represents
3 RR
regions of isolated zeaxanthin (solid line) and violaxanthin
(dotted line). Error bars represent the standard
deviation calculated from the data obtained in four independent
experiments.
1 were
used for normalization. Both methods gave very similar results. As
expected, the intensity of the
1 band was dependent upon
excitation wavelength (Fig. 7C). The replacement of
violaxanthin by zeaxanthin caused significant changes in the excitation
profiles, particularly the decrease in intensity with 488 nm excitation
and increases above 496.5 nm in the de-epoxidized thylakoids. The
calculated difference spectrum (+Zea)-(+Vio) clearly showed the
positive changes at 476.5 and 496.5 nm and the negative band at 488 nm, again similar to the shape of the corresponding absorption difference spectrum. This confirms that the band at 488 nm absorption is in a good
resonance with 488.0 excitation. The formation of zeaxanthin enhances
the RR signal above 500 nm, where zeaxanthin 0-0 absorption resonates
with the 501.7, 514.5, and 528.7 nm lines.
3 region of the RR spectrum was also investigated in
the thylakoid samples. The
3 for zeaxanthin is located
at 1003 cm
1, whereas for violaxanthin it is
shifted down to 1007 cm
1 (see Fig.
3B and Fig. 7D, inset). Therefore, the 1003/1007
cm
1 amplitude ratio monitors the relative
contribution of these xanthophylls. The dependence of this ratio upon
excitation wavelength was determined (Fig. 7D). The lowest
value of the ratio was found to be at 488 nm excitation, consistent
with this resonance arising from violaxanthin. The ratio increased upon
de-epoxidation for all excitations used, consistent with an increase in
zeaxanthin content, whereas the larger differences between
de-epoxidized and epoxidized samples observed for 514.5 and 528.7 nm
excitation result from selective excitation of the
zeaxanthin present.
1,
2 and
3 regions matches very closely that for isolated
violaxanthin in pyridine and for the free pigment fraction. This
clearly identifies the RR difference spectrum for thylakoids as the
violaxanthin spectrum with characteristic violaxanthin features at
1529, 1184, 1213, and 1006 cm
1 and adds
strength to the assertion that the 488 nm band originates from
violaxanthin. The
4 region for the thylakoid spectrum
exhibits one sharp transition at 949 cm
1 and
another at 962 cm
1, whereas the structure of
this region for the spectrum of the isolated pigment, either in solvent
or in detergent/lipid micelles, is almost absent, and intensity is
reduced. The presence of features in the
4 region
suggests that violaxanthin in vivo is distorted, most likely
due to its binding to antenna complexes.
View larger version (28K):
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Fig. 8.
Four main regions of the xanthophyll
resonance Raman spectrum, excited at 488 nm. 1 is the
isolated violaxanthin spectrum. 2 is a difference between
Raman spectrum of the free pigment fraction, containing violaxanthin,
and the spectrum for the fraction containing zeaxanthin (see
text). 3 is the difference between the Raman spectrum of
thylakoid membranes enriched in violaxanthin and the spectrum for
membranes enriched in zeaxanthin. Original spectra were normalized to
the chlorophyll bands at 1437, 1354, and 1327 cm 1.
View larger version (27K):
[in a new window]
Fig. 9.
Four main regions of the xanthophyll
resonance Raman spectrum excited at 528.7 nm. 1 is the
isolated zeaxanthin spectrum. 2 is a difference between
Raman spectrum of the free pigment fraction, containing zeaxanthin, and
the spectrum for the fraction containing violaxanthin (see text).
3 is the difference between the Raman spectrum of thylakoid
membranes enriched in zeaxanthin and the spectrum for membranes
enriched in violaxanthin. Original spectra were normalized to the
chlorophyll bands at 1437, 1354, and 1327 cm 1.
1 position around 1522 cm
1 and
3 maximum at 1003 cm
1 are identical in all three spectra. The
2 regions of zeaxanthin in pyridine and the thylakoid
spectrum are also similar apart from the downshift of the 1190 cm
1 band to 1185 cm
1. This downshift is also present in the
spectrum of the free pigment fraction. The
4 region for
the RR difference spectra of thylakoid membranes is strongly enhanced
and clearly structured compared with that for isolated zeaxanthin. For
the free pigment fraction, this region is slightly enhanced but less
structured in comparison to the thylakoid membrane spectrum. This
suggests that in vivo zeaxanthin is in a well defined environment.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
pH formation (35). Some data on isolated LHCII have
suggested that only the minor LHCII can bind violaxathin and zeaxanthin
tightly and that therefore these must be the functional proteins in
nonphotochemical fluorescence quenching (36). In contrast, we have
suggested that all of these carotenoids are bound, mostly to peripheral
sites on each of the proteins. The strongly associated violaxanthin
molecules located within minor complexes were found to be inaccessible
to the de-epoxidase enzyme and therefore unlikely to be involved in
photoprotection (9, 13). Therefore, we have suggested that the
peripheral violaxanthin and zeaxanthin molecules play the key role in
photoprotective energy dissipation. In this study, we have been able to
obtain the first data on the state of the xanthophyll cycle carotenoids in the thylakoid membrane. The more structured RR spectrum for both
violaxanthin and zeaxanthin in the thylakoid membrane compared with
those free in solution indicate that both of these carotenoids are in
fact in well coordinated environments in the thylakoid membrane, almost
certainly bound to protein and not free to move. Zeaxanthin appeared to
be more distorted than violaxanthin. Indeed, the
4
region is more structured and intense for zeaxanthin than violaxanthin,
suggesting that the former is in a tight association with the protein,
consistent with estimations of binding affinities to LHCII proteins
(9).
1 taking
into account the Davidov's splitting (E) of approximately 360 cm
1 (E = 2·V). In the case of a moderate transition dipole moment and a parallel orientation of two interacting violaxanthin molecules, they should be located within 0.5-0.7 nm of each other (37, 38).
pH-induced detachment of violaxanthin from
antenna to undergo de-epoxidation. Resonance Raman spectra were also
found to be unaffected (data not shown). This indicates that
acidification has no immediate effect on the state of violaxanthin.
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FOOTNOTES |
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* This work was supported by Grant 50/C11581 from United Kingdom Biotechnology and Biological Sciences Research Council.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed. Tel.: 44-1142224244; Fax: 44-1142728697; E-mail: a.ruban@sheffield.ac.uk.
** Supported by Training and Mobility of Researchers Grant ERBFMBICT983497.
Published, JBC Papers in Press, April 30, 2001, DOI 10.1074/jbc.M1032632010
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ABBREVIATIONS |
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The abbreviations used are: LHA, light-harvesting antenna; RR, resonance Raman; PSII, photosystem II; LHC, light-harvesting complex; (+Zea)-(+Vio), difference spectrum for zeaxanthin and violaxanthin.
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