Psiilhcii Supercomplexes

In grana membranes of green plants and green algae, a variable number of proteins of the LHC superfamily have been shown to associate with dimeric PSII core complexes to form the so-called PSII-LHC-II supercomplexes. Such complexes were first observed in electron microscopic images of grana membranes from spinach solubilised by the mild detergent P-dodecylmaltoside [16]. These rectangular supercomplexes, in the following denoted as C2S2 supercomplexes (see below), contain all, or almost all, PSII core proteins. Additionally, at each side, they contain one trimeric LHC-II complex consisting of the Lhcbl and Lhcb2 gene products, one CP29 monomer (the Lhcb4 gene product), and one CP26 monomer (the Lhcb5 gene product) [17]. EM micrographs of partially unfolding grana membranes clearly reveal the presence of rectangular supercomplexes in the membranes [18], indicating that the supercomplexes occur as such in the membranes and represent native organizations of PSII and LHC-II. A three-dimensional structure of this supercomplex was constructed by Nield and Barber based on a low-resolution structure from spinach obtained by cryoelectron microscopy and on the high-resolution crystal structures of the (cyanobacterial) PSII core, LHC-II and the extrinsic PsbP and PsbQ proteins - 19]. Very similar structures of PSII-LHC-II complexes were observed in the green alga Chlamydomonas reinhardtii [20] and the liverwort Marchantia polymorpha [21]. The C2S2 PSII-LHC-II supercomplex was also observed to be the organizational unit of PSII and LHC-II in a barley mutant lacking PSI [22] and was, therefore, suggested to form the unit in which the smallest possible amount of LHC complexes is bound to PSII in grana.

It was noted that the CP29-LHC-II-CP26 peripheral antenna structure needs a PSII core dimer for its binding. CP29 binds to one core monomer, CP26 to the other and LHC-II to both (Figure 6.1). This may explain why small supercomplexes of a PSII core monomer and one or two peripheral antenna proteins were never found. It was shown by analysis of mutants that at least one small PSII core protein enhances the binding of the CP29-LHC - II-CP26 unit. This protein is PsbZ, a small PSII protein with two transmemebrane a-helices. Mutants without PsbZ appeared to have strongly reduced CP26 levels and very unstable PSII-LHC-II supercomplexes [24].

In grana membranes, there are usually considerably more than two trimeric LHC-II complexes per PSII dimer, especially in low-light growing conditions. In addition, there is a third monomeric LHC protein (CP24, the Lhbc6 gene product). Two additional binding sites for trimeric LHC-II and one additional binding site for a monomeric LHC-II protein were, indeed, found after a very short and mild detergent treatment with a-dodecylmaltoside of PSII grana membranes from spinach [25-27]. The binding sites of the trimers were designated S, M, and L (for strongly, moderately and loosely bound LHC-II,) (Figure 6.1). In Arabidopsis

Figure 6.1 Model of the C2S2M2 PSII-LHC-II supercomplex of Arabidopsis thaliana and of the C2S2ML supercomplex of spinach. The atomic model of trimeric LHC-II has been used for fitting of the S-, M- and L-trimers. The C2S2M2 map iscurrently the best resolved supercomplex with a resolution of about 15A (adapted from [23]).

Figure 6.1 Model of the C2S2M2 PSII-LHC-II supercomplex of Arabidopsis thaliana and of the C2S2ML supercomplex of spinach. The atomic model of trimeric LHC-II has been used for fitting of the S-, M- and L-trimers. The C2S2M2 map iscurrently the best resolved supercomplex with a resolution of about 15A (adapted from [23]).

thaliana and Marchantia polymorpha, S and M trimers were found in the same position, but L trimers could not be detected [21, 28], In Chlamydomonas rein-hardtii, LHC-II binds only to the PSII core dimer at the S position, which is most likely due to the absence of a CP24 homologue in this organism. Based on an up-to-date supercomplex map (the C2S2M2 supercomplex map from Arabidopsis), the M trimer is rotationally shifted by about 25 ° compared to the S trimer. In plants, the S trimer consists predominantly of the Lhcbl and Lhcb2 gene products; the M trimer most likely consists of the Lhcbl and Lhcb3 gene products [29]. The additional binding of, at maximum, four LHC , II trimers at the rectangular C2S2 supercomplexes does not mean that all available LHC-II trimers are directly bound to PSII. In normal growth conditions, about half of all LHC , II trimers are not directly connected to PSII and occur in the membranes in LHC, II-only regions. A small number of these unconnected trimers have been shown to form supercomplexes of seven LHC-II trimers [30].

In some conditions, the PSII-LHC-II supercomplexes organize themselves into semi-crystalline arrays in grana membranes. The formation of regular lattices of PSII in rows is made possible by the rectangular (C 2 S2) or diamond-like shape (C2S2M2) of the supercomplexes. The way these supercomplexes organize themselves into larger complexes could be made visible by analysis of solubilised megacomplexes, dimers of PSII , LHC, 2 I supercomplexes. To date, six types of megacomplexes have been observed [29, 31], Details of the rows in grana membranes were visualized by image analysis of negatively stained specimens; it appeared that in most of the detected types of rows, the supercomplexes were connected to each other as in the megacomplexes [29]. In spinach, the majority of these rows were shown to consist of an asymmetric C2S2M repeating unit [32]. A few crystalline arrays consisting of a C [ S2 repeating unit were also observed. In membranes obtained from a PSI 4ess barley mutant, the crystalline arrays consisted exclusively of a C[ S2 repeating unit, consistent with the absence of CP24 and the M-trimer in this mutant [22]. In Arabidopsis membranes, only rows consisting of a C2S2M2 repeating unit were observed [28]. Isolated supercomplexes of Arabidopsis contained a larger number of occupied M binding sites than those of spinach [28] [ so, it seems that the binding of LHC-II at the M site is stronger in Arabidopsis than in spinach.

Analysis of supercomplexes isolated from Arabidopsis plants expressing antisense constructs to Lhcb gene products or from plants with a T-DNA knockout in an lhcb gene provided information of the specific roles in the supramolecular organization of PSII and LHC-II. Thus far, supercomplexes and crystalline arrays in grana membranes were analyzed from mutants lacking Lhcb4 (CP29), Lhcb5 (CP26), Lhcb6 (CP24), and Lhcb2 and Lhcbl (trimeric LHC-II).

No intact supercomplexes could be isolated from plants lacking CP29 and crystalline arrays were not detected [33]. This suggests that CP29 occupies a unique position in the PSII macrostructure and that its presence is essential for the formation of PSII [ LHC. II supercomplexes. In all other investigated mutants, both supercomplexes and crystalline arrays were observed. From plants lacking CP26, supercomplexes and crystalline arrays were observed with empty CP26 binding sites but with an otherwise normal appearance [33]. Because earlier investigations of PSII-LHC-II supercomplexes from spinach revealed many complexes with empty CP26 binding sites [26, 27], these results indicate that the presence of CP26 is not required for the formation of PSII-LHC-II macroorganization. Plants lacking CP24 only give rise to C2S2 supercomplexes and to crystalline arrays in membranes with the C2S2 supercomplex as a repeating unit [31]. This indicates that the presence of CP24 is required for the binding of LHC-II at the M site. In all mutants, the binding site appeared to be unique and could not be occupied by other monomeric proteins of the LHC family. The plants lacking CP29 or CP26 showed a rather normal photosynthetic performance [34], though in the field the fitness was lower [35]. Plants lacking CP24, however, showed a stronger phenotype [31], with decreased abilities of state transitions (see Section 6.2.3[ and rapidly reversible non-photochemical quenching (see Section 6.2). In particular, these plants lacked the so-called psi-type circular dichroism signal [36], reflecting long-range interactions between chlorophylls. The decrease of the signal indicates a change in the overall macrostructure and stresses the importance of the presence of the M trimer for proper photosynthetic functioning.

In plants with antisense constructs against Lhcb2, not only was the synthesis of Lhcb2 almost completely abolished, but also that of the strongly related Lhcb1 protein [34] [ In these plants, strongly increased levels of Lhcb5 and, to a lesser extent, Lhcb3 were observed, resulting in supercomplexes and crystalline arrays with trimers of Lhcb5 and Lhcb3 at the S and M sites [37, 38] [ This replacement is unique because expression of antisense constructs to other LHC-types of proteins, including those of PSI (see Section 6.2.2), did not lead to increased synthesis of other proteins, stressing the importance of the organization of PSII and LHC-II in supercomplexes.

LHC [ II does not only bind to PSII to form PSII [LHC-II supercomplexes, it is also primarily responsible for the formation of grana stacks in green plants. The stromal surface of LHC-II is not only very flat and overall negatively charged, but it also contains one domain of four positively charged residues. The interactions between the positively charged residues of one LHC-II with the negatively charged surface of an LHC-II in the opposing membrane, combined with the electrostatic screening of the negative surface with divalent cations, are probably the most important factors in determining the stacking of the thylakoid membranes [ 8]. Because of the stacking, the proteins with large extrinsic groups at the stromal surface, PSI and the ATP synthase complex, are excluded from the stacks, resulting in a spatial separation of PSI and PSII (Figure 6.2). This separation prevents spill-over of excitation energy from PSII to PSI and allows the formation of a very large light-harvesting antenna for PSII [39]. It is possible that interactions between the lumenal extensions of the extrinsic proteins of PSII involved in water oxidation in opposing membranes also influence the shape of the grana [29].

Supramolecular associations of PSII and other peripheral light-harvesting proteins of the LHC superfamily have not been studied until very recently when the first map of supercomplexes of a PSII core dimer and three or four monomeric Chl a/c proteins of the cryptophyte Rhodomonas CS24 became available [40]. The projection of the largest complex is shown in Figure 6.3 suggests that the four Chl a/c proteins are bound at similar positions as the CP24, CP29, one monomer of the S-LHC-II trimer and CP26 proteins in green plants.

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