Carotenoid-binding sites of the major light-harvesting complex II of higher plants.

Recombinant light-harvesting complex II (LHCII) proteins with modified carotenoid composition have been obtained by in vitro reconstitution of the Lhcb1 protein overexpressed in bacteria. The monomeric protein possesses three xanthophyll-binding sites. The L1 and L2 sites, localized by electron crystallography in the helix A/helix B cross, have the highest affinity for lutein, but also bind violaxanthin and zeaxanthin with lower affinity. The latter xanthophyll causes disruption of excitation energy transfer. The occupancy of at least one of these sites, probably L1, is essential for protein folding. Neoxanthin is bound to a distinct site (N1) that is highly selective for this species and whose occupancy is not essential for protein folding. Whereas xanthophylls in the L1 and L2 sites interact mainly with chlorophyll a, neoxanthin shows strong interaction with chlorophyll b, inducing the hyperchromic effect of the 652 nm absorption band. This observation explains the recent results of energy transfer from carotenoids to chlorophyll b obtained by femtosecond absorption spectroscopy. Whereas xanthophylls in the L1 and L2 sites are active in photoprotection through chlorophyll-triplet quenching, neoxanthin seems to act mainly in (1)O(2)(*) scavenging.     

Single-molecule electron tunneling spectroscopy of the higher plant light-harvesting complex LHC II

Electronic spectroscopy of a single biological molecule is demonstrated with approximately 4 A spatial resolution. The light-harvesting complex II (LHC II), in the ground and photo-excited states, was studied using scanning tunneling microscopy and spectroscopy of intact Photosystem II complexes. Analysis of the spectra indicates that the main mechanisms of tunneling between the STM tip and the surface involve delocalized electronic states of the LHC II and local vibronic states associated with C=C, C=O, C-H, N-H, and O-H groups near the LHC II surface. Conduction within the bulk LHC II is then due to ohmic and hopping conduction as well as tunneling between amino acid residues. Light activation of LHC II occurs via a photoconductive rather than a photovoltaic mechanism. There is a dramatic light-induced increase in the electronic density of states indicating a light-induced enhancement of energy and electron delocalization which is important for the efficient and rapid transfer of excitation energy from LHC II to the Photosystem II reaction center.     

Chlorophyll a and carotenoid triplet states in light-harvesting complex II of higher plants.

Laser-flash-induced transient absorption measurements were performed on trimeric light-harvesting complex II to study carotenoid (Car) and chlorophyll (Chl) triplet states as a function of temperature. In these complexes efficient transfer of triplets from Chl to Car occurs as a protection mechanism against singlet oxygen formation. It appears that at room temperature all triplets are being transferred from Chl to Car; at lower temperatures (77 K and below) the transfer is less efficient and chlorophyll triplets can be observed. In the presence of oxygen at room temperature the Car triplets are partly quenched by oxygen and two different Car triplet spectral species can be distinguished because of a difference in quenching rate. One of these spectral species is replaced by another one upon cooling to 4 Ki demonstrating that at least three carotenoids are in close contact with chlorophylls. The triplet minus singlet absorption (T-S) spectra show maxima at 504-506 nm and 517-523 nm, respectively. In the Chl Qy region absorption changes can be observed that are caused by Car triplets. The T-S spectra in the Chl region show an interesting temperature dependence which indicates that various Car's are in contact with different Chl a molecules. The results are discussed in terms of the crystal structure of light-harvesting complex II.     

Configuration and dynamics of xanthophylls in light-harvesting antennae of higher plants. Spectroscopic analysis of isolated light-harvesting complex of photosystem II and thylakoid membranes

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).     

Stark spectroscopy of the light-harvesting complex II in different oligomerisation states

The electric field-induced absorption changes (Stark effect) of light-harvesting complex II (LHCII) in different oligomerisation states-monomeric, trimeric and aggregated-have been probed at 77 K. All the chlorophyll (Chl) a molecules exhibit electro-optic properties in the Q(y) absorption region characterized by a change in dipole moment /Deltamu-->/ =0.6+/-0.06D/f and polarizability, Tr(Deltaalpha;) approximately 55+/-5 A(3)/f(2) upon electronic excitation, which are similar to those of unbound monomeric Chl a, indicating the absence of strong delocalization of the excitations which would be expected in the presence of strong excitonic interactions. The Stark effect in the Chl b absorption region is significantly bigger with /Deltamu-->/ values of the order of 2.0+/-0.2 D/f and it is attributed to strong interactions with neoxanthin molecules. Clear oligomerisation-dependent differences are observed in the carotenoid region, mainly due to the appearance of a new xanthophyll absorption band at 509 in the spectra of trimers and oligomers. It is ascribed to some lutein molecules, in agreement with previous experimental observations. The electro-optic properties of these lutein molecules are significantly different from those of the other xanthophylls in LHCII, which do not exhibit such a big change in dipole moment upon electronic excitation (/Deltamu-->/ =14.6+/-2.0 D/f). Upon aggregation of LHCII some extra absorption appears on the red side of the main Chl a Q(y) absorption band. In contrast to an earlier suggestion [J. Phys. Chem., A 103 (1999) 2422], no indications are found for the charge-transfer character of the corresponding band. The assignments of the S(2) electronic transitions of neoxanthin and lutein in LHCII and possible origins of the Stark effect are discussed.     

Central-Cis isomers of lutein found in the major light-harvesting complex of Photosystem II (LHC IIb) of higher plants

Lutein (,-carotene-3,3-diol) is the major carotenoid of the light-harvesting systems of higher plants. Lutein was isolated at 4°C and in complete darkness from the bulk light-harvesting complex of Photosystem II of spinach (LHC IIb) and from BBY particles. Separation using normal-phase HPLC (with 2D detection) in comparison to the authentic isomers (prepared by iodine-sensitised isomerization) showed the presence of a number of geometrical isomers of this xanthophyll in PS II, namely all-trans (the major component); 13-cis, 13-cis and 15-cis-lutein. Iodine-sensitised photo-isomerization of all-trans lutein produced six geometrical isomers of lutein as determined by HPLC. The configuration of five of these isomers was determined by ¹H-NMR to be all-trans, 9-cis, 9-cis, 13-cis and 13-cis. In addition, small amounts of another isomer have been tentatively identified to be 15-cis lutein on the basis of its electronic absorption spectrum. The possible functional significance of the presence of cis-isomers of this carotenoid in LHC IIb is discussed.     

Fluorescence emission spectra of photosystem I, photosystem II and the light-harvesting chlorophyll a/b complex of higher plants

Fluorescence emission spectra excited at 514 and 633 nm were measured at -196 degrees C on dark-grown bean leaves which had been partially greened by a repetitive series of brief xenon flashes. Excitation at 514 nm resulted in a greater relative enrichment of the 730 nm emission band of Photosystem I than was obtained with 633 nm excitation. The difference spectrum between the 514 nm excited fluorescence and the 633 nm excited fluorescence was taken to be representative of a pure Photosystem I emission spectrum at -196 degrees C. It was estimated from an extrapolation of low temperature emission spectra taken from a series of flashed leaves of different chlorophyll content that the emission from Photosystem II at 730 nm was 12% of the peak emission at 694 nm. Using this estimate, the pure Photosystem I emission spectrum was subtracted from the measured emission spectrum of a flashed leaf to give an emission spectrum representative of pure Photosystem II fluorescence at -196 degrees C. Emission spectra were also measured on flashed leaves which had been illuminated for several hours in continuous light. Appreciable amounts of the light-harvesting chlorophyll a/b protein, which has a low temperature fluorescence emission maximum at 682 nm, accumulate during greening in continuous light. The emission spectra of Photosystem I and Photosystem II were subtracted from the measured emission spectrum of such a leaf to obtain the emission spectrum of the light-harvesting chlorophyll a/b protein at -196 degrees C.     

Degradation of the main Photosystem II light-harvesting complex.

Many factors trigger the degradation of proteins, including changes in environmental conditions, genetic mutations, and limitations in the availability of cofactors. Despite the importance for viability, still very little is known about protein degradation and its regulation. The degradation of the most abundant membrane protein on Earth, the light-harvesting complex of Photosystem II (LHC II), is highly regulated under different environmental conditions, e.g. light stress, to prevent photochemical damage of the reaction center. However, despite major effort to identify the protease/proteases involved in the degradation of the apoproteins of LHC II the molecular details of this important process remain obscure. LHC II belongs to the family of chlorophyll a/b binding proteins (CAB proteins) and is located in the thylakoid membrane of the plant chloroplast. The results of biochemical experiments to isolate and characterize the protease degrading LHC II are summarized here and compared to our own recent finding indicating that a metalloprotease of the FtsH family is involved in this process.     

Stark effect measurements on monomers and trimers of reconstituted light-harvesting complex II of plants

The electric-field induced absorption changes (Stark effect) of reconstituted light-harvesting complex II (LHCII) in different oligomerisation states-monomers and trimers-with different xanthophyll content have been probed at 77 K. The Stark spectra of the reconstituted control samples, containing the xanthophylls lutein and neoxanthin, are very similar to previously reported spectra of native LHCII. Reconstituted LHCII, containing lutein but no neoxanthin, shows a similar electrooptical response in the Chl a region, but the Stark signal of Chl b around 650 nm amounts to at most approximately 25% of that of the control samples. We conclude that neoxanthin strongly modifies the electronic states of the nearby Chl b molecules causing a large electrooptical response at 650 nm stemming from one or more Chls b in the control samples. Ambiguities about the assignment of several bands in the Soret region [Biochim. Biophys. Acta 1605 (2003) 83] are resolved and the striking difference in electric field response between the two lutein molecules is confirmed. The Stark effect in the carotenoid spectral region in both control and neoxanthin-deficient samples is almost identical, showing that the neoxanthin Stark signal is small and much less intense than the lutein Stark signal.     

Lateral mobility of the light-harvesting complex in chloroplast membranes controls excitation energy distribution in higher plants

Chloroplast thylakoid protein phosphorylation produces changes in light-harvesting properties and in membrane structure as revealed by freeze-fracture electron microscopy. Protein phosphorylation resulted in an increase in the 77 °K fluorescence signal at 735 nm relative to that at 685 nm. In addition, a decrease in connectivity between Photosystem II centers (PS II) and a dynamic quenching of the room temperature variable fluorescence was observed upon phosphorylation. Accompanying these fluorescence changes was a 23% decrease in the amount of stacked membranes. Microscopic analyses indicated that 8.0-nm particles fracturing on the P-face moved from the stacked into the unstacked regions upon phosphorylation. The movement of the 8.0-nm particles was accompanied by the appearance of chlorophyll b and 25 to 29 kD polypeptides in isolated stroma lamellae fractions. We conclude that phosphorylation of a population of the light-harvesting chlorophyll $\text{a}\text{b}$ protein complexes (LHC) in grana partitions causes the migration of these pigment proteins from the PS II-rich appressed membranes into the Photosystem I (PS I) enriched unstacked regions. This increases the absorptive cross section of PS I. In addition, we suggest that the mobile population of LHC functions to interconnect PS II centers in grana partitions; removal of this population of LHC upon phosphorylation limits PS II → PS II energy transfer and thereby favors spillover of energy from PS II to PS I.     

Light-induced changes in the chemical bond structure of light-harvesting complex II probed by FTIR spectroscopy

Light-harvesting complex II (LHC-II) regulates the light energy distribution between photosystem I and II in plants. This process is mediated by phosphorylation of the LHC-II protein, which depends on the oxidation state of photosynthetic electron carriers. In addition to this regulatory mechanism, it has recently been proposed that light can directly induce a conformational change in isolated LHC-II. To provide biophysical evidence for such a conformational change in the protein, we studied infrared absorbance changes in isolated LHC-II upon exposure to light flashes. Compared to the signals obtained with other proteins that exhibit well-characterized conformational changes, the signal in the LHC-II difference spectra is very weak. The position of the difference bands coincides with the main IR absorption bands of chlorophyll. We conclude that there are no detectable light-induced changes in the LHC protein structure and attribute the observed IR signals to light-induced chlorophyll degradation.     

Heat stress induces an aggregation of the light-harvesting complex of photosystem II in spinach plants

Whole spinach (Spinacia oleracea) plants were subjected to heat stress (25 degrees C-50 degrees C) in the dark for 30 min. At temperatures higher than 35 degrees C, CO2 assimilation rate decreased significantly. The maximal efficiency of photosystem II (PSII) photochemistry remained unchanged until 45 degrees C and decreased only slightly at 50 degrees C. Nonphotochemical quenching increased significantly either in the absence or presence of dithiothreitol. There was an appearance of the characteristic band at around 698 nm in 77 K fluorescence emission spectra of leaves. Native green gel of thylakoid membranes isolated immediately from heat-stressed leaves showed that many pigment-protein complexes remained aggregated in the stacking gel. The analyses of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting demonstrated that the aggregates were composed of the main light-harvesting complex of PSII (LHCIIb). To characterize the aggregates, isolated PSII core complexes were incubated at 25 degrees C to 50 degrees C in the dark for 10 min. At temperatures over 35 degrees C, many pigment-protein complexes remained aggregated in the stacking gel of native green gel, and immunoblotting analyses showed that the aggregates were composed of LHCIIb. In addition, isolated LHCII was also incubated at 25 degrees C to 50 degrees C in the dark for 10 min. LHCII remained aggregated in the stacking gel of native green gel at temperatures over 35 degrees C. Massive aggregation of LHCII was clearly observed by using microscope images, which was accompanied by a significant increase in fluorescence quenching. There was a linear relationship between the formation of LHCII aggregates and nonphotochemical quenching in vivo. The results in this study suggest that LHCII aggregates may represent a protective mechanism to dissipate excess excitation energy in heat-stressed plants.     

The binding of Xanthophylls to the bulk light-harvesting complex of photosystem II of higher plants. A specific requirement for carotenoids with a 3-hydroxy-beta-end group

The pigment composition of the light-harvesting complexes (LHCs) of higher plants is highly conserved. The bulk complex (LHCIIb) binds three xanthophyll molecules in combination with chlorophyll (Chl) a and b. The structural requirements for binding xanthophylls to LHCIIb have been examined using an in vitro reconstitution procedure. Reassembly of the monomeric recombinant LHCIIb was performed using a wide range of native and nonnative xanthophylls, and a specific requirement for the presence of a hydroxy group at C-3 on a single beta-end group was identified. The presence of additional substituents (e.g. at C-4) did not interfere with xanthophyll binding, but they could not, on their own, support reassembly. cis isomers of zeaxanthin, violaxanthin, and lutein were not bound, whereas all-trans-neoxanthin and different chiral forms of lutein and zeaxanthin were incorporated into the complex. The C-3 and C-3' diols lactucaxanthin (a carotenoid native to many plant LHCs) and eschscholtzxanthin (a retro-carotenoid) both behaved very differently from lutein and zeaxanthin in that they would not support complex reassembly when used alone. Lactucaxanthin could, however, be bound when lutein was also present, and it showed a high affinity for xanthophyll binding site N1. In the presence of lutein, lactucaxanthin was readily bound to at least one lutein-binding site, suggesting that the ability to bind to the complex and initiate protein folding may be dependent on different structural features of the carotenoid molecule. The importance of carotenoid end group structure and ring-to-chain conformation around the C-6-C-7 torsion angle of the carotenoid molecule in binding and complex reassembly is discussed.     

Two-dimensional crystals of the photosystem II reaction center complex from higher plants

By detergent treatment of isolated photosynthetic membranes from maize chloroplasts, we have prepared two-dimensional crystals of the photosystem II complex. Two distinct crystal forms are produced by this treatment. Analysis of Fourier transforms of the crystals shows that each crystal type is formed from two inverted layers. Within the rectangular 17.8 x 26.7 nm unit cell of each layer is a tetrameric structure enclosing a two-fold symmetry axis, a result implying that the basic structural unit of photosystem II is dimeric. Tris-washing, which removes proteins associated with the oxygen-evolving apparatus from the inner surface of the photosynthetic membrane, causes a distinct change in the structure of these tetramers and reveals a dimeric core complex which may be directly associated with the photosystem II machinery.     

Single molecule fluorescence spectroscopy of pH sensitive oligonucleotide switches.

Several authors demonstrated that an oligonucleotide based pH-sensitive construct can act as a switch between an open and a closed state by changing the pH. To validate this process, specially designed fluorescence dye-quencher substituted oligonucleotide constructs were developed to probe the switching between these two states. This paper reports on bulk and single molecule fluorescence investigations of a duplex-triplex pH sensitive oligonucleotide switch. On the bulk level, only a partial quenching of the fluorescence is observed, similarly to what is observed for other published switches and is supposed to be due to intermolecular interactions between oligonucleotide strands. On the single molecule level, each DNA-based nanometric construct shows a complete switching. These observations suggest the tendency of the DNA construct to associate at high concentration.     

The effect of amphiphilic peptide surfactants on the light-harvesting complex II

The peptide surfactants are amphiphilic peptides which have a hydrophobic tail and a hydrophilic head, and have been reported to stabilize and protect some membrane proteins more effectively than conventional surfactants. The effects of a class of peptide surfactants on the structure and thermal stability of the photosynthetic membrane protein lightharvesting complex II (LHCII) in aqueous media have been investigated. After treatment with the cationic peptide surfactants A₆K, V₆K₂, I₅K₂ and I₅R₂, the absorption at 436 nm and 470 nm decreased and the absorption at 500–510 nm and 684–690 nm increased. Moreover, the circular dichroism (CD) signal intensity in the Soret region also decreased significantly, indicating the conformation of some chlorophyll (Chl) a, Chl b, and the xanthophyll molecules distorted upon cationic peptide surfactants treatment. The anionic peptide surfactants A₆D and V₆D₂ had no obvious effect on the absorption and CD spectra. Except for A₆D, these peptides all decreased the thermal stability of LHCII, indicating that these peptides may reconstitute protein into a less stable conformation. In addition, the cationic peptide surfactants resulted in LHCII aggregation, as shown by sucrose gradient ultracentrifugation and fluorescence spectra.     

Structural and functional heterogeneity in the major light-harvesting complexes of higher plants

The major light-harvesting complex (LHC IIb) of higher plants plays a crucial role in capturing light energy for photosynthesis and in regulating the flow of energy within the photosynthetic apparatus. Multiple isoforms of the protein bind chlorophyll and xanthophyll chromophores, but it is commonly believed that the pigment-binding properties of different LHC IIb complexes are conserved within and between species. We have investigated the structure and function of different LHC IIb complexes isolated from Arabidopsis thaliana grown under different light conditions. LHC IIb isolated from low light-grown plants shows increased amounts of the Lhcb2 gene product, increased binding of chlorophyll a, and altered energy transfer characteristics. We suggest that Lhcb2 specifically binds at least one additional chlorophyll a compared to the Lhcb1 gene product, and that differences in the functioning of LHC IIb from high and low light-grown plants are a direct consequence of the change in polypeptide composition. We show that changes in LHC IIb composition are accompanied by changes in photosynthetic function in vivo and discuss the possible functional significance of LHC IIb heterogeneity.