The Binding of Xanthophylls to the Bulk Light-harvesting Complex of Photosystem II of Higher Plants

Phillip, Denise; Hobe, Stephan; Paulsen, Harald; Molnár, Péter; Hashimoto, Hideki; Young, Andrew

doi:10.1074/jbc.m202002200

Cited by 54 publications

(29 citation statements)

References 34 publications

(58 reference statements)

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“…This is in accordance with earlier observations that carotenoids are required for the formation of LHCIIb in vitro (8,9) with lutein being the most efficiently stabilizing carotenoid (22)(23)(24). Unexpectedly, the fluorescent dye monitor detected specific Chl a binding to Lhcb1 in the absence of Chl b, although it had been noted earlier that Chl b is an absolute requirement for stable Lhcb1-pigment complex formation in vitro (21,22) and stable insertion of Lhcb1 in etioplast membranes (25).…”

Section: Discussionsupporting

confidence: 78%

Early Steps in the Assembly of Light-harvesting Chlorophyll a/b Complex

Horn

Paulsen

2004

Journal of Biological Chemistry

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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.The major light-harvesting chlorophyll (Chl) 1 a/b complex (LHCIIb) greatly enhances the efficiency of photosynthesis by enlarging the absorption cross-section of photosystem (PS) II and, under some conditions, also of PS I. The structure of LHCIIb has been known in near-atomic detail since 1994 (1) and recently has been further resolved to 2.7 Å (2). Much less detail is known about some steps in the biogenesis of LHCIIb.The apoprotein, Lhcb1-3, is synthesized in its precursor form in the cytoplasm, imported into the plastid, and finally inserted into the thylakoid membrane via the signal recognition particle pathway (3, 4). It is unclear when and even where the protein becomes complexed with pigments. The signal recognition particle complex presumably keeps Lhcb1-3 in an unfolded and thus nonpigmented form until it is delivered to the thylakoid; on the other hand, Hoober and Eggink (5) proposed, based on observations in the green alga Chlamydomonas reinhardtii, that LHCIIb assembly takes place in the envelope membrane. We do not know how the Chls are delivered to their apoproteins, whether the last steps in their biogenetic pathway take place in the immediate neighborhood of the site of assembly or whether the pigments are transported there via some carrier (6, 7). All we can safely assume is that Chl molecules do not freely diffuse in the thylakoid membrane because this...

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Section: Discussionsupporting

confidence: 78%

Early Steps in the Assembly of Light-harvesting Chlorophyll a/b Complex

Horn

Paulsen

2004

Journal of Biological Chemistry

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“…This finding is consistent with a recent report that 3-hydroxy-␤-end groups, rather than ␤-carotene-derived xanthophylls per se, are the minimal requirements for the binding of xanthophylls to LHC II in vitro (Phillip et al, 2002). Hydroxylated ␤-rings (monohydroxy or dihydroxy xanthophylls) were able to promote the in vitro assembly of LHC II by facilitating the correct folding of LHC II proteins.…”

Section: Total Hydroxylation Activity and The Synthesis Of Specific Xsupporting

confidence: 82%

Functional Analysis of β- and ε-Ring Carotenoid Hydroxylases in Arabidopsis

et al. 2003

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Lutein and zeaxanthin are dihydroxy xanthophylls that are produced from their corresponding carotene precursors by the action of ␤ -and -ring carotenoid hydroxylases. Two genes that encode ␤ -ring hydroxylases ( ␤ -hydroxylases 1 and 2) have been identified in the Arabidopsis genome and are highly active toward ␤ -rings but only weakly active toward -rings. A third distinct activity required for -ring hydroxylation has been defined by mutation of the LUTEIN1 ( LUT1 ) locus, but LUT1has not yet been cloned. To address the individual and overlapping functions of the three Arabidopsis carotenoid hydroxylase activities in vivo, T-DNA knockout mutants corresponding to ␤ -hydroxylases 1 and 2 ( b1 and b2 , respectively) were isolated and all possible hydroxylase mutant combinations were generated. ␤ -Hydroxylase single mutants do not exhibit obvious growth defects and have limited impact on carotenoid composition relative to the wild type, suggesting that the encoded proteins have a significant degree of functional redundancy in vivo. Surprisingly, the b1 b2 double mutant, which lacks both known ␤ -hydroxylase enzymes, still contains significant levels of ␤ -carotene-derived xanthophylls, suggesting that additional ␤ -ring hydroxylation activity exists in vivo. The phenotype of double and triple hydroxylase mutants indicates that at least a portion of this activity resides in the LUT1 gene product. Despite the severe reduction of ␤ -carotene-derived xanthophylls (up to 90% in the lut1 b1 b2 triple mutant), the double and triple hydroxylase mutants still contain at least 50% of the wild-type amount of hydroxylated ␤ -rings. This finding suggests that it is the presence of minimal amounts of hydroxylated ␤ -rings, rather than minimal amounts of specific ␤ -carotene-derived xanthophylls, that are essential for light-harvesting complex II assembly and function in vivo. The carotenoid profiles in wild-type seeds and the effect of single and multiple hydroxylase mutations are distinct from those in photosynthetic tissues, indicating that the activities of each gene product differ in the two tissues. Overall, the hydroxylase mutants provide insight into the unexpected overlapping activity of carotenoid hydroxylases in vivo.

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“…In the case of L1/L2 a hydroxyl group at C3 on the ␤-end group turned out to be important (8). In contrast to Lu and Vx, which both exhibit an all-trans configuration of the extended polyene chain, Nx in photosynthetic tissue of chlorophyll b-containing organisms is present in its 9Ј-cis conformation (9).…”

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confidence: 99%

Assembly of the Major Light-harvesting Chlorophyll-a/b Complex

Hobe

Trostmann

Raunser

et al. 2006

Journal of Biological Chemistry

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The major light-harvesting chlorophyll-a/b complex in most higher plants contains three carotenoids, lutein, neoxanthin, and violaxanthin. How these pigments are assembled into the complex during its biogenesis is largely unknown. Here we show that neoxanthin but not lutein can dissociate from the fully assembled complex. Its equilibrium binding constant in a detergent system (0.1% n-dodecyl-␤-D-maltoside) was determined to be > 10 6 M ؊1 . Neoxanthin insertion into light-harvesting chlorophyll-a/b complex prefolded from overexpressed apoprotein (Lhcb1*2 from Pisum sativum) in the presence of chlorophylls a, b, and lutein as the sole carotenoid is kinetically controlled by an activation energy barrier of ϳ120 kJ mol ؊1 . This is the first thermodynamic and kinetic description of a binding equilibrium between a non-covalently bound pigment of the photosynthetic apparatus and its protein complex. Dissociation of neoxanthin from the major light-harvesting chlorophyll-a/b complex upon temperature increase is discussed in terms of providing a readily available substrate pool for synthesizing abscisic acid as part of a heat and drought stress response.The photosynthetic apparatus in the thylakoid membrane of green plants contains light-harvesting complexes enhancing its capacity to absorb light quanta that are then converted into chemical potential by the reaction centers. The major light-harvesting complex of photosystem II, LHCIIb, 2 is assembled in its trimeric form, equipped with 14 chlorophylls and 4 xanthophylls per apoprotein (1, 2). The xanthophylls consist of two molecules of lutein (Lu), one neoxanthin (Nx), and one violaxanthin (Vx), bound to the sites L1/L2, N1, and V1, respectively. L1 and L2 are located close to the center of the complex, stabilizing a superhelix of protein helices I and III, whereas N1 and V1 are located more peripherally.The specificity of xanthophyll binding sites has been addressed both by analyzing xanthophyll biosynthetic mutants (see below) and by employing recombinant in vitro systems. In the latter approach, complexes were formed by having different carotenoids compete for the various binding sites. Lu and Nx were found to bind to the L1/L2 and N1 sites, respectively, with high specificity (3, 4). As long as Lu is provided for binding to L1/L2, the N1 site does not bind any of the all-trans carotenoids Lu or Vx. Zx was shown to be a strong competitor for Lu, whereas Vx competes to a lesser extent. Consistently, when Lu is omitted completely from the refolding experiment, both Zx and Vx support complex formation by binding to L1/L2, although this increasingly compromises the complex stability. Moreover, Zx and Vx in L1/L2 compromise the binding specificity of N1, prompting its partial occupation with Vx. Xanthophyll biosynthetic mutants of Arabidopsis thaliana revealed a flexible response of the aggregational state of LHCIIb as well as of pigmentation of particular binding sites: double mutants lacking epoxycarotenoids as well as Lu exhibit a complete loss of trimeric LHCIIb with...

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The Binding of Xanthophylls to the Bulk Light-harvesting Complex of Photosystem II of Higher Plants

Cited by 54 publications

References 34 publications

Early Steps in the Assembly of Light-harvesting Chlorophyll a/b Complex

Early Steps in the Assembly of Light-harvesting Chlorophyll a/b Complex

Functional Analysis of β- and ε-Ring Carotenoid Hydroxylases in Arabidopsis

Assembly of the Major Light-harvesting Chlorophyll-a/b Complex

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