Molecular adaptation of photoprotection: triplet states in light-harvesting proteins

The photosynthetic light-harvesting systems of purple bacteria and plants both utilize specific carotenoids as quenchers of the harmful (bacterio)chlorophyll triplet states via triplet-triplet energy transfer. Here, we explore how the binding of carotenoids to the different types of light-harvesting proteins found in plants and purple bacteria provides adaptation in this vital photoprotective function. We show that the creation of the carotenoid triplet states in the light-harvesting complexes may occur without detectable conformational changes, in contrast to that found for carotenoids in solution. However, in plant light-harvesting complexes, the triplet wavefunction is shared between the carotenoids and their adjacent chlorophylls. This is not observed for the antenna proteins of purple bacteria, where the triplet is virtually fully located on the carotenoid molecule. These results explain the faster triplet-triplet transfer times in plant light-harvesting complexes. We show that this molecular mechanism, which spreads the location of the triplet wavefunction through the pigments of plant light-harvesting complexes, results in the absence of any detectable chlorophyll triplet in these complexes upon excitation, and we propose that it emerged as a photoprotective adaptation during the evolution of oxygenic photosynthesis.     

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.     

An investigation into the ATP requirement for phosphorylation of thylakoid proteins and for the ATP-induced decrease in the yield of chlorophyll fluorescence in chloroplasts at different stages of development

The phosphorylation of thylakoid membrane polypeptides has been investigated in chloroplasts prepared from peas that had been grown under intermittent light and then exposed to between 4 and 48 h of continuous light. At 4 h, when the ratio of the total amount of labelling of a 9 kDa-polypeptide relative to light-harvesting chlorophyll protein (LHCP) polypeptides was much greater than 1, the affinity for ATP was found to be the same (S_0.5, approx. 100 μM) for both polypeptides. In contrast, in fully greened chloroplasts, when labelling of LHCP was much greater than that of the 9 kDa-polypeptide, the S_0.5 for ATP was 40 μM for LHCP and 500 μM for the 9 kDa-polypeptide. A correlation was observed during development between the affinity for ATP of the 9 kDa-species and its abundance relative to LHCP. It is suggested that these polypeptides compete for phosphorylation by the same protein kinase. Simultaneous assay of the ATP-induced fluorescence decrease at different ATP concentrations revealed a close correlation with LHCP labelling but not with labelling of the 9 kDa-polypeptide. This correlation held irrespective of which polypeptide was the major phosphoprotein.     

Developmental Loss of Photosystem II Activity and Structure in a Chloroplast-Encoded Tobacco Mutant, Lutescens-1

Lutescens-1, a tobacco mutant with a maternally inherited dysfunction, displayed an unusual developmental phenotype. In vivo measurement of chlorophyll fluorescence revealed deterioration in photosystem II (PSII) function as leaves expanded. Analysis of thylakoid membrane proteins by polyacrylamide gel electrophoresis indicated the physical loss of nuclear- and chloroplast-encoded polypeptides comprising the PSII core complex concomitant with loss of activity. Freeze fracture electron micrographs of mutant thylakoids showed a reduced density, compared to wild type, of the EF_s particles which have been shown previously to be the structural entity containing PSII core complexes and associated pigment-proteins. The selective loss of PSII cores from thylakoids resulted in a higher ratio of antenna chlorophyll to reaction centers and an altered 77 K chlorophyll fluorescence emission spectra; these data are interpreted to indicate functional isolation of light-harvesting chlorophyll a/b complexes in the absence of PSII centers. Examination of PSII reaction centers (which were present at lower levels in mutant membranes) by monitoring the light-dependent phosphorylation of PSII polypeptides and flash-induced O₂ evolution patterns demonstrated that the PSII cores which were assembled in mutant thylakoids were functionally identical to those of wild type. We conclude that the lutescens-1 mutation affected the correct stoichiometry of PSII centers, in relation to other membrane constituents, by disrupting the proper assembly and maintenance of PSII complexes in lutescens-1 thylakoid membranes.     

Light-dependent, chilling effects on phosphorylation of thylakoid proteins and consequences for associated photochemical activities in maize

When maize ( Zea mays L. cv. LG11) leaves are exposed to low temperatures and high light modifications to both photosystem 2 (PS2) and the light-harvesting chlorophyll a/b protein complex associated with photosystem 2 (LHC2) occur. This study examines the consequences of these modifications for phosphorylation of LHC2 and PS2 polypeptides and the associated changes in electron transport. Maize leaves were chilled at 5°C for 6 h under photon flux densities of 1 500 and 250 μmol m^-2 s^-1. Thylakoids were then isolated from the leaves and their abilities to phosphorylate LHC2 and PS2 polypeptides and modify electron transport activities were determined. Measurements of chlorophyll fluorescence induction in the thylakoids were also made. Thylakoids isolated from leaves chilled under high light and from leaves kept in the ambient growth environment had similar phosphoprotein profiles. However, polypeptide phosphorylation in thylakoids from the chilled leaves did not produce a decrease in PS2 electron transport. Chilling leaves under low light produced a decrease in the ability of isolated thylakoids to phosphorylate PS2, but not LHC2, polypeptides, which was not associated with any change in the phosphorylation-induced decrease in PS2 electron transport. Chilling under high, but not low, light appears to produce changes in membrane organisation that do not affect the ability of the thylakoids to phosphorylate PS2 and LHC2 polypeptides, but which do prevent the phosphorylation-induced decrease in excitation energy transfer from LHC2 to PS2.     

Biosynthesis of the light-harvesting chlorophyll a/b protein. Control of messenger RNA activity by light

1. Antibodies raised against the 26000-Mr polypeptides of the light-harvesting chlorophyll a/b proteins of pea leaves specifically immunoprecipitated two 32000-Mr polypeptides synthesized when pea leaf poly(A)-containing RNA was translated in vitro. On the basis of immunochemical relatedness and by comparison of their partial tryptic digestion products, the 32000-Mr products formed in vitro are identified as precursors to the authentic polypeptides of the light-harvesting chlorophyll a/b complex. 2. The specificity of the immunoprecipitation permitted the development of an assay for the cellular levels of translationally active light-harvesting protein mRNA in plants exposed to different light regimes. Low levels of the mRNAs were detectable in dark-grown plants. Exposure to continuous illumination caused these levels to increase by at least ten-fold and led to the appearance of large quantities of the light-harvesting chlorophyll a/b complex. In plants exposed to intermittent illumination (2 min of white light every 2 h for 2 days), the light-harvesting complex did not accumulate, although levels of mRNA specifying the polypeptides of the complex were high (50% of those in continuously illuminated plants). 3. Messenger RNAs encoding the light-harvesting proteins were detected in polysomes of intermittently illuminated leaves. These polysomes were active in a wheat-germ 100 000 X g supernatant "run-off" system, to form light-harvesting protein precursors, under conditions when only nascent polypeptide chains initiated in vivo were elongated and terminated. These results demonstrate that the inability of intermittently illuminated leaves to accumulate the light-harvesting proteins is not due to a selective inhibition of the translation of the corresponding mRNAs. 4. Intermittently illuminated leaves were labelled with [35S]methionine in darkness, and incorporation of radioisotope into the light-harvesting proteins and their precursors was assayed immunologically. No pool of untransported or unprocessed 32000-Mr precursor polypeptides could be detected in the soluble fraction (cytoplasm and stroma). However, low levels of the mature 26000-Mr polypeptides were detected in the membrane fraction. It is concluded that the newly synthesized light-harvesting chlorophyll a/b protein fail to accumulate in intermittently illuminated leaves because they undergo rapid turnover. The site of light-harvesting protein breakdown is probably the thylakoid membrane, and the cause of breakdown is probably the absence of chlorophyll a and chlorophyll b molecules that are required for eventual stabilization of the proteins within the photosynthetic membrane.     

Function and evolution of grana

Chloroplasts are descended from cyanobacteria, and they retain many features of the cyanobacterial photosynthetic apparatus. However, land-plant chloroplasts have a strikingly different thylakoid membrane organization to that of cyanobacteria. Usually the two photosystems are laterally segregated; Photosystem II is concentrated in complex stacked-membrane structures known as grana. The function of grana has long been debated. Recent studies on membrane organization in chloroplasts, cyanobacteria and purple bacteria now offer a new perspective. I argue that grana allow the presence of a large light-harvesting antenna for Photosystem II, without excessively restricting electron transport. Other organisms solve this problem in different ways. Land plants evolved from macroalgae that were adapted to high light conditions; they evolved grana as a new solution to the problem of efficient photosynthesis in shade.     

Structures of proteins and cofactors: X-ray crystallography

Protein crystallography is the predominately used technique for the determination of the three-dimensional structures of proteins and other macromolecules. In this article, the methodology utilized for the measurement and analysis of the diffraction data from crystals is briefly reviewed. As examples of both the usefulness and difficulties of this technique, the determination of the structures of several photosynthetic pigment–protein complexes is described, namely, the reaction center from purple bacteria, photosystem I and photosystem II from cyanobacteria, the light-harvesting complex II from purple bacteria, and the FMO protein from green bacteria.     

PHYLOGENETIC DISTRIBUTION OF THE MAJOR DIATOM LIGHT-HARVESTING PIGMENT-PROTEIN DETERMINED BY IMMUNOLOGICAL METHODS 1

Monospecific, polyclonal antibodies raised against the apoprotein of the major light-harvesting pigment-protein of Phaeodactylum tricornutum Bohlin UTEX 646 were used to determine (1) whether this complex was common to the class Bacillariophyceae, whose members contain chlorophylls a and c and fucoxanlhin; (2) whether antigenically-related apoproteins were present in other chlorophyll c-containing groups, and (3) whether there was immunological homology with the light-hanvsting chlorophyll a/b protein of similar photosynthetic function in the Chlorophyta and vascular plants. We have used protein blotting techniques to show that antibodies against the two P. tricornutum light-harvesting complex polypeptides cross-reacted with one or two polypeptides of similar molecular weight (17–21 kD) in all ten diatom species examined, representing two orders and six families. No cross-reactivity was obtained with total membrane polypeptides from isolated representatives of three chromophyte algal divisions (Chrysophyta, Cryptophyta, Pyrrophyta), all of which contained chlorophyll c. No cross-reactivity was observed with membrane Polypeptides isolated from members of two classes of Chlorophte algae. These data suggest that the Bacillariophyceae may be monophyletic, and that the primary structure of the diatom light-harvesting complex is not closely related to pigment-protein complexes with similar function in other chlorophyll c-containing unicellular algal groups. Lastly, it may be possible to use the antibodies to the diatom light-harvesting polypeptides as specific markers for diatoms in natural phytoplankton assemblages.     

Accessory pigment fluorescence for quantitation of photosynthetic microbial populations.

The in vivo fluorescence of the primary accessory pigments in purple bacteria (carotenoids), green bacteria (bacteriochlorophyll), green algae (chlorophyll), and cyanobacteria (phycocyanin) was found to be a linear function of cell concentration over three of four orders of magnitude. The lowest cell concentrations detectable were 10(4) cells/ml for procaryotes and 10(3) cells/ml for eucaryotes.     

Photosystem II in Guard Cells of Vicia faba: Immunological Detection

Antibodies were raised against individual polypeptides of the oxygen-evolving photosystem II (PSII) complex from mesophyll chloroplasts of Vicia faba (Long Pod). These antibodies were used to probe immunologically for the presence of the main structural components of the PSII complex in guard cell chloroplasts, using both immunofluorescence microscopy and Western blotting. Immunofluorescence of epidermal peels with antibodies raised against the extrinsic 33 kilodalton polypeptide, as well as the 47 and the 44 kilodalton subunits and the light-harvesting chlorophyll a/b protein, resulted in intense fluorescence indicating the presence of these polypeptide components in guard cell chloroplasts. Results obtained with Western blot analysis showed that the relative amounts of the 33 kilodalton and light-harvesting complex protein polypeptides are between 60 and 80% of that found in mesophyll cells (on chlorophyll basis). These results provide evidence for the existence of structural components associated with PSII activity in guard cell similar to those of mesophyll chloroplasts.     

Movement of a sub-population of the light harvesting complex (LHCII) from grana to stroma lamellae as a consequence of its phosphorylation

Phosphorylation in vitro of the light-harvesting chlorophyll $\text{a}\text{b}$ protein complex associated with Photosystem II (LHC_II) resulted in the lateral migration of a subpopulation of LHC_II from the grana to the stroma lamellae. This movement was characterized by a decrease in the chlorophyll $\text{a}\text{b}$ ratio and an increase in the 77 K fluorescence emission at 681 nm in the stroma lamellae following phosphorylation. Polyacrylamide gel electrophoresis indicated that the principal phosphoproteins under these conditions were polypeptides of 26–27 kDa. These polypeptides increased in relative amount in the stroma lamellae and decreased in the grana during phosphorylation. Pulse/chase experiments confirmed that the polypeptides were labelled in the grana and moved to the stroma lamellae in the subsequent chase period. A fraction at the phospho-LHC_II, however, was unable to move and remained associated with the grana fraction. LHC_II which moved out into the stroma lamellae effectively sensitized Photosystem I (PS I), since the ability to excite fluorescence emission at 735 nm (at 77 K) by chlorophyll b was increased following phosphorylation. These data support the ‘mobile antenna’ hypothesis proposed by Kyle, Staehelin and Arntzen (Arch. Biochem. Biophys. (1983) 222, 527–541) which states that the alterations in the excitation-energy distribution induced by LHC_II phosphorylation are, in part, due to the change in absorptive cross-section of PS II and PS I, resulting specifically from the movement of LHC_II antennae chlorophylls from the PS-II-enriched grana to the PS-I-enriched stroma lamellae.     

The structure and function of bacterial light-harvesting complexes

The harvesting of solar radiation by purple photosynthetic bacteria is achieved by circular, integral membrane pigment-protein complexes. There are two main types of light-harvesting complex, termed LH2 and LH1, that function to absorb light energy and to transfer that energy rapidly and efficiently to the photochemical reaction centres where it is trapped. This mini-review describes our present understanding of the structure and function of the purple bacterial light-harvesting complexes.     

On the origin of photosynthesis as inferred from sequence analysis

Sequence alignments between membrane-spanning segments of pheophytin-quinone-type photosynthetic reaction centers, FeS-type photosynthetic reaction centers, core chlorophyll-proteins of PS II, chlorophyll t a/t b-containing antenna proteins of plants and light-harvesting complexes of purple bacteria led us to postulate a large common ancestral pigment-carrying protein with more than 10 membrane spans. Its original function as a UV-protector of the primordial cell is discussed. It is conceivable that a purely dissipative photochemistry started still in the context of the UV-protection. We suggest that mutations causing the t loss of certain porphyrin-type pigments led to the acquisition of redox cofactors and paved the way for a gradual transition from dissipative to productive photochemistry.     

Immunological evidence for apoproteins of the light-harvesting chlorophyll-protein complex in a mutant of barley lacking chlorophyll b

A barley mutant lacking chlorophyll b and the pigmented light-harvesting chlorophyll-protein of photo-system 2 is shown by several criteria to contain functional apoproteins of the light-harvesting complex. 1. Electrophoretic comparison of thylakoid polypeptide patterns, and the effects of trypsin treatment on these, suggests that the mutant contains several polypeptides equivalent in mobility to those of the wild-type complex. 2. An antibody monospecific for the light-harvesting complex agglutinated both wild-type and mutant thylakoids. 3. 'Western blot' immunoelectrophoretic analysis indicated that of four distinct subunits of the light-harvesting complex in the wild-type thylakoids, three are detectable in the mutant. 4. As in wild-type lamellae at least one of the light-harvesting complex polypeptides is phosphorylated by the endogenous protein kinase. The results are considered in terms of a general role for the light-harvesting complex polypeptides in membrane appression and the regulation of excitation energy distribution within thylakoids.     

Bacteriochlorophyll-protein complexes of aerobic bacteria,Erythrobacter longus and Erythrobacter species OCh 114

Bacteriochlorophyll(Bchl)-protein complexes were isolated from obligate aerobic bacteria, Erythrobacter longus and Erythrobacter species OCh 114. The apparent molecular weights, absorption spectra and polypeptide compositions of the light-harvesting complexes were, in general, similar to those of the light-harvesting Bchl-protein complexes of purple photosynthetic bacteria. The reaction center complexes of these bacteria also showed similar properties to those of the purple bacteria except for slightly altered polypeptides. However, the following characteristic features of the light-harvesting systems were found in these aerobic bacteria. Major carotenoids were not bound to the Bchl-protein complex in E. longus. In Erythrobacter sp. OCh 114, a new type of Bchl-protein complex which showed a single absorption band in the near infrared region at 806 nm was obtained. The reaction center of strain OCh 114 was associated with a c-type cytochrome.Abbreviations Bchl bacteriochlorophyll a - RC reaction center - SDS sodium dodecylsulfate - PAGE polyacrylamide gel electrophoresis     

The amino acid sequence of the polypeptide segment which regulates membrane adhesion (grana stacking) in chloroplasts

A positively charged amino acid sequence, located on the NH2 terminus of the polypeptides of the chlorophyll a/b light harvesting complex, stabilizes thylakoid membrane adhesion. Threonine residues in this segment are the site of light-induced, reversible phosphorylation; this covalent modification results in changes in excitation-energy distribution in chloroplast membranes. Removal of the positively charged peptide by treatment with trypsin or chemical modification of amino acids in the sequence disrupts thylakoid adhesion and inhibits regulation of excitation-energy distribution. Purified preparations of the chlorophyll a/b light harvesting complex consist of 2 major polypeptides of 27 and 26 kDa and 2 minor polypeptides of 29 and 25 kDa (based upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Trypsin treatment of the isolated chlorophyll proteins decreases the apparent molecular mass of the 27- and 26-kDa polypeptides by 1-1.5 kDa and releases 3 peptides; [Lys, Arg], Ser-Ala-Thr-Thr-Lys-Lys, and Ser-Ala-Thr-Thr-Lys. These peptides probably form the overlap sequence, [Lys, Arg]-Ser-Ala-Thr-Thr-Lys-Lys. The polypeptides of the chlorophyll a/b light-harvesting complex were separated by isoelectric focusing into 5 chlorophyll protein fractions which had isoelectric points between 4.0 and 4.55. The 27-kDa polypeptides had an isoelectric point of 4.3, and bound 11 chlorophyll molecules/polypeptide.     

The cation-dependence of the degree of protein phosphorylation-induced unstacking of pea thylakoids

Experiments are presented to show that the phosphorylation of the light-harvesting chlorophyll $\text{a}\text{b}\text{-}\text{protein}$ complex (LHC) induces structural reorganisation within the thylakoid membrane in response to the introduction of additional negative surface charges. The effect of cations of different valency on chlorophyll fluorescence measurements indicates that LHC-phosphorylation-induced reorganisation involves a change in the electrostatic screening capability of the added cation. At intermediate levels of cations (e.g., 1 or 2 mM Mg²⁺), which substantially stack non-phosphorylated membranes, it was found that membrane phosphorylation caused considerable unstacking as monitored by light scattering and electron microscopy. Concomitant with this was a large decrease in chlorophyll fluorescence indicative of randomisation of chlorophyll protein complexes which would result in an increase in energy transfer between the photosystems as well as an absorption cross-section change. At higher concentrations (e.g., above 5 mM Mg²⁺) a persistent ATP-induced decrease in chlorophyll fluorescence has been attributed to the displacement of charged phosphorylated LHC from the appressed granal to the non-appressed stromal lamellae, thus decreasing the absorption cross-section of Photosystem II. Under these circumstances only a small degree of unstacking was detected by light scattering and measurements of the percentage of thylakoid length which is stacked to form grana. However, when considered on a surface area basis, the structural changes observed can qualitatively account for the magnitude of the chlorophyll fluorescence quenching due to the lateral diffusion of LHC.