Determination of relative chlorophyll binding affinities in the major light-harvesting chlorophyll a/b complex

The major light-harvesting complex (LHCIIb) of photosystem II can be reconstituted in vitro from its recombinant apoprotein in the presence of a mixture of carotenoids and chlorophylls a and b. By varying the chlorophyll a/b ratio in the reconstitution mixture, the relative amounts of chlorophyll a and chlorophyll b bound to LHCIIb can be changed. We have analyzed the chlorophyll stoichiometry in recombinant wild type and mutant LHCIIb reconstituted at different chlorophyll a/b ratios in order to assess relative affinities of the chlorophyll-binding sites. This approach reveals five sites that exclusively bind chlorophyll b. Another site exhibits a slight preference of chlorophyll b over chlorophyll a. The remaining six sites are filled preferentially with chlorophyll a but also tolerate chlorophyll b when this is offered at a large excess. Three of these chlorophyll a-affine sites could be assigned to distinct positions defined by the three-dimensional LHCIIb structure. Exclusive chlorophyll b sites complemented by chlorophyll a sites that are selective only to a certain extent are consistent with the observation that chlorophyll b but not chlorophyll a is essential for reconstituting stable LHCIIb. These data offer an explanation why a rather constant chlorophyll a/b ratio is observed in native LHCIIb despite the apparent promiscuity of some binding sites.     

Folding in vitro of light-harvesting chlorophyll a/b protein is coupled with pigment binding

The major light-harvesting chlorophyll a/b protein (LHCIIb) of the plant photosynthetic apparatus is able to self-organise in vitro. When the recombinant apoprotein, Lhcb1, is solubilised in the denaturing detergent sodium (or lithium) dodecylsulfate (SDS or LDS) and then mixed with chlorophylls and carotenoids under renaturing conditions, structurally authentic LHCIIb forms. Assembly of functional LHCIIb, as indicated by the establishment of energy transfer between complex-bound chlorophyll molecules, occurs in two apparent kinetic steps with time constants of 10 to 30 seconds and 50 to 300 seconds, depending on the reaction conditions. Here, we use circular dichroism (CD) in the far-UV range to monitor the folding of the LHCIIb apoprotein as it is complexed with pigments. The alpha-helix content in the protein's secondary structure increases in two apparent kinetic steps with time constants similar to those observed for the establishment of chlorophyll energy transfer. When the carotenoid concentration in the reaction mixture is reduced, the time constants of alpha-helix formation increase, as do those for the appearance of chlorophyll energy transfer. This indicates that both processes, pigment assembly and secondary structure formation, are tightly coupled. A substantial amount of alpha-helix is present in dodecylsulfate-solubilised LHCIIb apoprotein and appears to be distributed among various protein domains.     

Consecutive binding of chlorophylls a and b during the assembly in vitro of light-harvesting chlorophyll-a/b protein (LHCIIb)

The apoprotein of the major light-harvesting chlorophyll a/b complex (LHCIIb) is post-translationally imported into the chloroplast, where membrane insertion, protein folding, and pigment binding take place. The sequence and molecular mechanism of the latter steps is largely unknown. The complex spontaneously self-organises in vitro to form structurally authentic LHCIIb upon reconstituting the unfolded recombinant protein with the pigments chlorophyll a, b, and carotenoids in detergent micelles. Former measurements of LHCIIb assembly had revealed two apparent kinetic phases, a faster one (tau1) in the range of 10 s to 1 min, and a slower one (tau2) in the range of several min. To unravel the sequence of events we analysed the binding of chlorophylls into the complex by using time-resolved fluorescence measurements of resonance energy transfer from chlorophylls to an acceptor dye attached to the apoprotein. Chlorophyll a, offered in the absence of chlorophyll b, bound with the faster kinetics (tau1) exclusively whereas chlorophyll b, in the absence of chlorophyll a, bound predominantly with the slower kinetics (tau2). In double-jump experiments, LHCIIb assembly could be dissected into a faster chlorophyll a and a subsequent, predominantly slower chlorophyll b-binding step. The assignment of the faster and the slower kinetic phase to predominantly chlorophyll a and exclusively chlorophyll b binding, respectively, was verified by analysing the assembly kinetics with a circular dichroism signal in the visible domain presumably reflecting the establishment of pigment-pigment interactions. We propose that slow chlorophyll binding is confined to the exclusively chlorophyll b binding sites whereas faster binding occurs to the chlorophyll a binding sites. The latter sites can bind both chlorophylls a and b but in a reversible fashion as long as the complex is not stabilised by proper occupation of the chlorophyll b sites. The resulting two-step model of LHCIIb assembly is able to reconcile the highly specific binding sites containing either chlorophyll a or b, as seen in the recent crystal structures of LHCIIb, with the observation of promiscuous binding sites able to bind both chlorophyll a and b in numerous reconstitution analyses of LHCIIb assembly.     

Early steps in the assembly of light-harvesting chlorophyll a/b complex: time-resolved fluorescence measurements

The light-harvesting chlorophyll a/b complex (LHCIIb) spontaneously assembles from its pigment and protein components in detergent solution. The formation of functional LHCIIb can be detected in time-resolved experiments by monitoring the establishment of excitation energy transfer from protein-bound chlorophyll b to chlorophyll a. To detect the possible initial steps of chlorophyll binding that may not yet give rise to chlorophyll b-to-a energy transfer, we have monitored LHCIIb assembly by measuring excitation energy transfer from a fluorescent dye, covalently bound to the protein, to the chlorophylls. In order to exclude interference of the dye with protein folding or pigment binding, the experiments were repeated with the dye bound to four different positions in the protein. Initial chlorophyll binding occurs at roughly the same rate as the establishment of chlorophyll b-to-a energy transfer, in the range of 10 s. However, under limiting chlorophyll concentrations, the binding of chlorophyll a clearly precedes that of chlorophyll b. The complex containing the apoprotein, carotenoids, and chlorophyll a but no chlorophyll b is biochemically unstable and therefore cannot be isolated. However, chlorophyll a binding into this weak complex is specific, as it does not occur with a C-terminal deletion mutant of Lhcb1 which still contains most chlorophyll-ligating amino acids but is unable to fold and assemble into functional LHCIIb. As a scenario for LHCIIb assembly in the thylakoid, we propose the initial formation of a labile Lhcb1-chlorophyll a-carotenoid complex that then becomes stabilized by the binding (or formation in situ) of chlorophyll b.     

Carotenoid binding sites in LHCIIb. Relative affinities towards major xanthophylls of higher plants.

The major light-harvesting complex of photosystem II can be reconstituted in vitro from its bacterially expressed apoprotein with chlorophylls a and b and neoxanthin, violaxanthin, lutein, or zeaxanthin as the only xanthophyll. Reconstitution of these one-carotenoid complexes requires low-stringency conditions during complex formation and isolation. Neoxanthin complexes (containing 30-50% of the all-trans isomer) disintegrate during electrophoresis, exhibit a largely reduced resistance against proteolytic attack; in addition, energy transfer from Chl b to Chl a is easily disrupted at elevated temperature. Complexes reconstituted in the presence of either zeaxanthin or lutein contain nearly two xanthophylls per 12 chlorophylls and are more resistant against trypsin. Lutein-LHCIIb also exhibits an intermediate maintenance of energy transfer at higher temperature. Violaxanthin complexes approach a xanthophyll/12 chlorophyll ratio of 3, similar to the ratio in recombinant LHCIIb containing all xanthophylls. On the other hand, violaxanthin-LHCIIb exhibits a low thermal stability like neoxanthin complexes, but an intermediate accessibility towards trypsin, similar to lutein-LHCIIb and zeaxanthin-LHCIIb. Binary competition experiments were performed with two xanthophylls at varying ratios in the reconstitution. Analysis of the xanthophyll contents in the reconstitution products yielded information about relative carotenoid affinities of three assumed binding sites. In lutein/neoxanthin competition experiments, two binding sites showed a strong preference (> 200-fold) for lutein, whereas the third binding site had a higher affinity (25-fold) to neoxanthin. Competition between lutein and violaxanthin gave a similar result, although the specificities were lower: two binding sites have a 36-fold preference for lutein and one has a fivefold preference for violaxanthin. The lowest selectivity was between lutein and zeaxanthin: two binding sites had a fivefold higher affinity for lutein and one has a threefold higher affinity to zeaxanthin.     

Random mutations directed to transmembrane and loop domains of the light-harvesting chlorophyll a/b protein: impact on pigment binding.

The major light-harvesting complex of photosystem II (LHCII) can be reconstituted in vitro by folding its bacterially expressed apoprotein, Lhcb, in detergent solution in the presence of chlorophylls and carotenoids. To compare the impact of alpha-helical transmembrane domains and hydrophilic loop domains of the apoprotein on complex formation and stability, we introduced random mutations into a segment of the protein comprising the stromal loop, the third (C-proximal) transmembrane helix, and part of the amphipathic helix in the C-terminal domain. The mutant versions of Lhcb were screened for the loss of their ability to form stable LHCII upon reconstitution in vitro. Most steps during the screening, including expression of the recombinant protein, its reconstitution with pigments, and the assay for complex formation by measuring energy transfer from chlorophyll b to chlorophyll a, were performed as one-vessel reactions on 96-well microtiter plates. This enabled us to screen several hundred mutant Lhcb versions. Mutants that had lost their ability to form stable LHCII carried between one and four amino acid exchanges. Among the single-point mutations, several were at positions in the C-proximal transmembrane helix, including an amino acid that is thought to be directly involved in chlorophyll binding. However, we also found four point mutations in the stromal loop domain that, in our assay, completely abolished the formation of stable LHCII. These data show that the stromal loop domain has a significant impact on LHCII formation and/or stability in vitro.     

The light-harvesting chlorophyll a/b complex can be reconstituted in vitro from its completely unfolded apoprotein

The major light-harvesting chlorophyll a/b protein (LHCIIb) of higher plants is one of the few membrane proteins that can be refolded in vitro. During folding, the apoprotein is assembled with pigments to form a structurally authentic and functional pigment--protein complex. All reconstitution procedures used so far include solubilization of the apoprotein in sodium dodecyl sulfate (SDS) where the protein adopts approximately half of its alpha-helical folding present in the native structure. This paper shows that this preformed alpha-helix is not a prerequisite for LHCIIb folding in vitro. The apoprotein can also be reconstituted starting from a solution in guanidinium hydrochloride (Gnd) where the protein contains no detectable helical structure. Reconstitution yields are somewhat lower in the Gnd than in the SDS procedure, but the reconstitution products exhibit very similar biochemical and spectroscopic properties. The kinetics of LHCIIb assembly, as assessed by time-resolved fluorescence measurements, are virtually the same in both reconstitution procedures. This demonstrates that the initiation of alpha-helix formation is not a rate-limiting step in LHCIIb apoprotein folding.     

The negatively charged amino acids in the lumenal loop influence the pigment binding and conformation of the major light-harvesting chlorophyll a/b complex of photosystem II

The major chlorophyll (Chl) a/b complexes of photosystem II (LHCIIb), in addition to their primary light-harvesting function, play key roles in the organization of the granal ultrastructure of the thylakoid membranes and in various regulatory processes. These functions depend on the structural stability and flexibility of the complexes. The lumenal side of LHCIIb is exposed to broadly variable pH environments, due to the build-up and decay of the pH gradient during photosynthesis. Therefore, the negatively charged amino acids in the lumenal loop might be of paramount importance for adjusting the structure and functions of LHCIIb. In order to clarify the structural roles of these residues, we investigated the pigment stoichiometries, absorption, linear and circular dichroism spectra of the reconstituted LHCIIb complexes, in which the negatively charged amino acids in the lumenal loop were exchanged to neutral ones (E94G, E107V and D111V). The mutations influenced the pigment binding and the molecular architecture of the complexes. Exchanging E94 to G destabilized the 3(10) helix in the lumenal loop structure and led to an acquired pH sensitivity of the LHCIIb structure. We conclude that these amino acids are important not only for pigment binding in the complexes, but also in stabilizing the conformation of LHCIIb at different pHs.     

Single amino acids in the lumenal loop domain influence the stability of the major light-harvesting chlorophyll a/b complex

The major light-harvesting complex of photosystem II (LHCIIb) is one of the most abundant integral membrane proteins. It greatly enhances the efficiency of photosynthesis in green plants by binding a large number of accessory pigments that absorb light energy and conduct it toward the photosynthetic reaction centers. Most of these pigments are associated with the three transmembrane and one amphiphilic alpha helices of the protein. Less is known about the significance of the loop domains connecting the alpha helices for pigment binding. Therefore, we randomly exchanged single amino acids in the lumenal loop domain of the bacterially expressed apoprotein Lhcb1 and then reconstituted the mutant protein with pigments in vitro. The resulting collection of mutated recombinant LHCIIb versions was screened by using a 96-well-format plate-based procedure described previously [Heinemann, B., and Paulsen, H. (1999) Biochemistry 38, 14088-14093], enabling us to test several thousand mutants for their ability to form stable pigment-protein complexes in vitro. At least one-third of the positions in the loop domain turned out to be sensitive targets; i.e., their exchange abolished formation of LHCIIb in vitro. This confirms our earlier notion that the LHCIIb loop domains contribute more specifically to complex formation and/or stabilization than by merely connecting the alpha helices. Among the target sites, glycines and hydrophilic amino acids are more prominently represented than hydrophobic ones. Specifically, the exchange of any of the three acidic amino acids in the lumenal loop abolishes reconstitution of stable pigment-protein complexes, suggesting that ionic interactions with other protein domains are important for correct protein folding or complex stabilization. One hydrophobic amino acid, tryptophan in position 97, has been hit repeatedly in independent mutation experiments. From the LHCIIb structure and previous mutational analyses, we propose a stabilizing interaction between this amino acid and F195 near the C-proximal end of the third transmembrane helix.     

Decreasing the chlorophyll a/b ratio in reconstituted LHCII: structural and functional consequences.

Trimeric (bT) and monomeric (bM) light-harvesting complex II (LHCII) with a chlorophyll a/b ratio of 0.03 were reconstituted from the apoprotein overexpressed in Escherichia coli. Chlorophyll/xanthophyll and chlorophyll/protein ratios of bT complexes and 'native' LHCII are rather similar, namely, 0.28 vs 0. 27 and 10.5 +/- 1.5 vs 12, respectively, indicating the replacement of most chlorophyll a molecules with chlorophyll b, leaving one chlorophyll a per trimeric complex. The LD spectrum of the bT complexes strongly suggests that the chlorophyll b molecules adopt orientations similar to those of the chlorophylls a that they replace. The circular dichroism (CD) spectra of bM and bT complexes indicate structural arrangements resembling those of 'native' LHCII. Thermolysin digestion patterns demonstrate that bT complexes are folded and organized like 'native' trimeric LHCII. Surprisingly, in the bT complexes at 77 K, half of the excitations that are created on either chlorophyll b or xanthophyll are transferred to chlorophyll a. No or very limited triplet transfer from chlorophyll b to xanthophyll appears to take place. However, the efficiency of triplet transfer from chlorophyll a to xanthophyll is close to 100%, even higher than in 'native' LHCII at 77 K. It is concluded from the triplet-minus-singlet and CD results that the single chlorophyll a molecule that on the average is present in each bT complex binds preferably next to a xanthophyll molecule at the interface between the monomers.     

In vitro reconstitution of a light-harvesting gene product: deletion mutagenesis and analyses of pigment binding.

AB96, a gene encoding a Pisum sativum chlorophyll a/b binding protein [Coruzzi et al. (1983) J. Biol. Chem. 258, 1399-1402], can be expressed in Escherichia coli and reconstituted with pigments by the procedure described by Plumley and Schmidt [(1987) Proc. Natl. Acad. Sci. U.S.A. 84, 146-150]. Following purification by polyacrylamide gel electrophoresis, the reconstituted pigment-protein complex (CP2) is shown to have similar pigment-binding characteristics to native CP2 complexes isolated from thylakoid membranes. Therefore, the AB96 gene product contains binding sites for chlorophylls a and b and xanthophylls, all of which are necessary for optimal reconstitution in vitro. Absorption, fluorescence, and circular dichroism spectroscopy indicate that the pigments are oriented accurately and that chlorophylls a and b are adjoined for energy transfer. Studies with proteins produced after deletion mutagenesis of AB96 indicate that NH2-terminal amino acids 1-21 and COOH-terminal amino acids 219-228 do not play a role in pigment binding. In contrast, amino acids 50-57 and 204-212 (encompassing one of three conserved histidine residues) are essential for reconstitution. Residues near the presumed NH2- and COOH-terminal alpha-helix boundaries (22-49 and 213-218, respectively) affect the stability of reconstituted CP2 during electrophoresis at 4 degrees C. Correlation of diminished chlorophyll a binding with disappearance of a negative circular dichroism near 684 nm suggests that amino acids 213-218 near the COOH-terminal boundary of the third membrane-spanning helix affect the binding of some chlorophyll a molecules.     

Kinetic analysis of nonphotochemical quenching of chlorophyll fluorescence. 2. Isolated light-harvesting complexes

The chlorophyll fluorescence yield of purified photosystem II light-harvesting complexes can be lowered by manipulation of experimental conditions. In several important respects, this quenching resembles the nonphotochemical quenching observed in isolated chloroplasts and leaves, therefore providing a model system for investigating the underlying mechanism. A methodology based on the principles of enzyme kinetic analysis has already been applied to isolated chloroplasts, and this same experimental approach was used here with purified LHCIIb, CP26, and CP29. It was found that the kinetics of the decrease in fluorescence yield robustly fitted a second-order kinetic model with respect to time after induction of quenching. The second-order rate constant was dependent upon the complex that was analyzed, the detergent concentration, the solution pH, and the presence of exogenous xanthophyll cycle carotenoids. In contrast, the formation of an absorbance change at 683 nm that accompanies quenching displayed first-order kinetics. The reversal of quenching also displayed second-order kinetics. These data show that quenching results from a binary reaction, possibly arising between two chlorophyll molecules. On the basis of these data, a model for the regulation of nonphotochemical quenching based upon the allosteric control of the conformation of light-harvesting complexes by protonation and xanthophyll binding is presented.     

Carotenoid-to-chlorophyll energy transfer in recombinant major light-harvesting complex (LHCII) of higher plants. I. Femtosecond transient absorption measurements

The energy transfer kinetics from carotenoids to chlorophylls and among chlorophylls has been measured by femtosecond transient absorption kinetics in a monomeric unit of the major light-harvesting complex (LHCII) from higher plants. The samples were reconstituted complexes with different carotenoid contents. The kinetics was measured both in the carotenoid absorption region and in the chlorophyll Q(y) region using two different excitation wavelengths suitable for selective excitation of the carotenoids. Analysis of the data shows that the overwhelming part of the energy transfer from the carotenoids occurs directly from the initially excited S(2) state of the carotenoids. Only a small part (<20%) may possibly take an S(1) pathway. All the S(2) energy transfer from carotenoids to chlorophylls occurs with time constants <100 fs. We have been able to differentiate among the three carotenoids, two luteins and neoxanthin, which have transfer times of approximately 50 and 75 fs for the two luteins, and approximately 90 fs for neoxanthin. About 50% of the energy absorbed by carotenoids is initially transferred directly to chlorophyll b (Chl b), while the rest is transferred to Chl a. Neoxanthin almost exclusively transfers to Chl b. Due to various complex effects discussed in the paper, such as a specific coupling of Chl b and Chl a excited states, the percentage of direct Chl b transfer thus is somewhat lower than estimated by us previously for LHCII from Arabidopsis thaliana. (Connelly, J. P., M. G. Müller, R. Bassi, R. Croce, and A. R. Holzwarth. 1997. Biochemistry. 36:281). We can distinguish three different Chls b receiving energy directly from carotenoids. We propose as a new mechanism that the carotenoid-to-Chl b transfer occurs to a large part via the B(x) state of Chl b and to the Q(x) state, while the transfer to Chl a occurs only via the Q(x) state. We find no compelling evidence in favor of a substantial S(1) transfer path of the carotenoids, although some transfer via the S(1) state of neoxanthin can not be entirely excluded. The S(1) lifetimes of the two luteins were determined to be 15 ps and 3.9 ps. A detailed quantitative analysis and kinetic model of the processes described here will be presented in a separate paper.     

Folding, assembly, and stability of the major light-harvesting complex of higher plants, LHCII, in the presence of native lipids

The influence of thylakoid lipids on the association kinetics and thermal stability of the major light-harvesting complex of photosytem II (LHCII) has been studied in vitro. The apoprotein, light-harvesting chlorophyll a/b-binding protein (Lhcb1), can be refolded and complexed with pigments in detergent solution even in the absence of lipids. Two thylakoid lipids, phosphatidyl glycerol and digalactosyl diacylglycerol, are known to interact specifically with LHCII in vivo. Here we show that both of these lipids, as well as monogalactosyl diacylglycerol, stabilize reconstituted LHCII toward thermal denaturation. Two slow kinetic phases are connected with the establishment of energy transfer between chlorophyll b and chlorophyll a and, thus, are thought to reflect the formation of the pigment-protein complex with tightly coupled chlorophylls. The lipids studied here all have the same effect on the rate of complex assembly in vitro and slow these two kinetic phases by the same degree. Both kinetic phases also slow when reactant concentrations are decreased, suggesting that the corresponding reaction step(s) involve(s) pigment binding.     

Exchange of pigment-binding amino acids in light-harvesting chlorophyll a/b protein

Four amino acids in the major light-harvesting chlorophyll (Chl) a/b complex (LHCII) that are thought to coordinate Chl molecules have been exchanged with amino acids that presumably cannot bind Chl. Amino acids H68, Q131, Q197, and H212 are positioned in helixes B, C, A, and D, respectively, and, according to the LHCII crystal structure [Kühlbrandt, W., et al. (1994) Nature 367, 614-621], coordinate the Chl molecules named a(5), b(6), a(3), and b(3). Moreover, a double mutant was analyzed carrying exchanges at positions E65 and H68, presumably affecting Chls a(4) and a(5). All mutant proteins could be reconstituted in vitro with pigments, although the thermal stability of the resulting mutant versions of recombinant LHCII varied significantly. All complexes reconstituted with the mutant proteins contained fewer chlorophyll molecules per two lutein molecules than complexes reconstituted with the wild-type protein. However, the chlorophyll-binding amino acids could not be unambiguously assigned to binding either chlorophyll a or b, as in most cases more than one chlorophyll molecule was lost due to the mutation. The changes in Chl stoichiometries suggest that in LHCII some chlorophyll positions can be filled with either Chl a or b. Only some of the point mutations in LHCII affected the ability of the apoprotein to assemble into trimeric LHCII upon insertion into isolated thylakoid membranes. Among these were exchanges of H68 with either F or L, suggesting that the stability of the LHCII trimer significantly depends on this amino acid or the Chl molecule named a(5) that is attached to it and is located close to the center of the trimeric complex. The ion pair bridge between E65 and R185 in LHCII does not appear to be essential for the proper folding of the protein.     

The negatively charged amino acids in the lumenal loop influence the pigment binding and conformation of the major light-harvesting chlorophyll a/b complex of photosystem II

The major chlorophyll (Chl) a/b complexes of photosystem II (LHCIIb), in addition to their primary light-harvesting function, play key roles in the organization of the granal ultrastructure of the thylakoid membranes and in various regulatory processes. These functions depend on the structural stability and flexibility of the complexes. The lumenal side of LHCIIb is exposed to broadly variable pH environments, due to the build-up and decay of the pH gradient during photosynthesis. Therefore, the negatively charged amino acids in the lumenal loop might be of paramount importance for adjusting the structure and functions of LHCIIb. In order to clarify the structural roles of these residues, we investigated the pigment stoichiometries, absorption, linear and circular dichroism spectra of the reconstituted LHCIIb complexes, in which the negatively charged amino acids in the lumenal loop were exchanged to neutral ones (E94G, E107V and D111V). The mutations influenced the pigment binding and the molecular architecture of the complexes. Exchanging E94 to G destabilized the 3₁₀ helix in the lumenal loop structure and led to an acquired pH sensitivity of the LHCIIb structure. We conclude that these amino acids are important not only for pigment binding in the complexes, but also in stabilizing the conformation of LHCIIb at different pHs.     

The small CAB-like proteins of Synechocystis sp. PCC 6803 bind chlorophyll

The large family of light-harvesting-like proteins contains members with one to four membrane spanning helices with significant homology to the chlorophyll a/b-binding antenna proteins of plants. From structural as well as evolutionary perspective, it is likely that the members of this family bind chlorophylls and carotenoids. However, undisputable evidence is still lacking. The cyanobacterial small CAB-like proteins (SCPs) are one-helix proteins with compelling similarity to the first and third transmembrane helix of LHCII (LHCIIb) including the chlorophyll-binding motifs. They have been proposed to act as chlorophyll-carrier proteins. Here, we analyze the in vivo absorption spectra of single scp deletion mutants in Synechocystis sp. PCC 6803 and compare the in vitro pigment binding ability of the SCP pairs ScpC/D and ScpB/E with the one of LHCII and a synthetic peptide containing the chlorophyll-binding motif (Eggink LL, Hoober JK (2000) J Biol Chem 275:9087-9090). We demonstrate that deletion of scpB alters the pigmentation in the cyanobacterial cell. Furthermore, we are able to show that chlorophylls and carotenoids interact in vitro with the pairs of ScpC/D and ScpB/E, demonstrated by fluorescence resonance energy transfer and circular dichroism.     

Regulation of chlorophyll apoprotein expression and accumulation. Requirements for carotenoids and chlorophyll.

Chlorophyll apoprotein accumulation and expression were examined in mutants of Chlamydomonas reinhardtii blocked at specific steps of carotenoid or chlorophyll synthesis. In the absence of carotenoids: 1) apoproteins of the core and light-harvesting complexes of photosystem I (CCI and LHCI, respectively) and photosystem II (CCII and LHCII, respectively) do not accumulate; 2) mRNAs for the CCI, CCII, and LHCII apoproteins accumulate to normal levels; and 3) synthesis of the chlorophyll apoproteins is differentially affected, or in some cases, not affected. In the absence of chlorophylls: 1) the apoproteins fail to accumulate; 2) mRNA levels for CCI and CCII apoproteins are relatively unchanged; 3) levels of LHCII apoprotein mRNA, but not rates of LHCII mRNA synthesis, are reduced in a light-dependent chlorophyll-synthesis mutant (ya12); and 4) synthesis of chlorophyll apoproteins is differentially affected or not affected in the case of several chloroplast-encoded apoproteins. These results demonstrate a direct role for carotenoids as well as chlorophylls in the stabilization of certain chlorophyll apoproteins and, for others, possibly in their translation. The data also indicate a role for chlorophyll synthesis in the stability of LHCII mRNA.     

Pigment binding of photosystem I light-harvesting proteins

Light-harvesting complexes (LHC) of higher plants are composed of at least 10 different proteins. Despite their pronounced amino acid sequence homology, the LHC of photosystem II show differences in pigment binding that are interpreted in terms of partly different functions. By contrast, there is only scarce knowledge about the pigment composition of LHC of photosystem I, and consequently no concept of potentially different functions of the various LHCI exists. For better insight into this issue, we isolated native LHCI-730 and LHCI-680. Pigment analyses revealed that LHCI-730 binds more chlorophyll and violaxanthin than LHCI-680. For the first time all LHCI complexes are now available in their recombinant form; their analysis allowed further dissection of pigment binding by individual LHCI proteins and analysis of pigment requirements for LHCI formation. By these different approaches a correlation between the requirement of a single chlorophyll species for LHC formation and the chlorophyll a/b ratio of LHCs could be detected, and indications regarding occupation of carotenoid-binding sites were obtained. Additionally the reconstitution approach allowed assignment of spectral features observed in native LHCI-680 to its components Lhca2 and Lhca3. It is suggested that excitation energy migrates from chlorophyll(s) fluorescing at 680 (Lhca3) via those fluorescing at 686/702 nm (Lhca2) or 720 nm (Lhca3) to the photosystem I core chlorophylls.