Investigating the Early Stages of Photosystem II Assembly in Synechocystis sp. PCC 6803

Abstract

Biochemical characterization of intermediates involved in the assembly of the oxygen-evolving Photosystem II (PSII) complex is hampered by their low abundance in the membrane. Using the cyanobacterium Synechocystis sp. PCC 6803, we describe here the isolation of the CP47 and CP43 subunits, which, during biogenesis, attach to a reaction center assembly complex containing D1, D2, and cytochrome b 559 , with CP47 binding first. Our experimental approach involved a combination of His tagging, the use of a D1 deletion mutant that blocks PSII assembly at an early stage, and, in the case of CP47, the additional inactivation of the FtsH2 protease involved in degrading unassembled PSII proteins. Absorption spectroscopy and pigment analyses revealed that both CP47-His and CP43-His bind chlorophyll a and ␤-carotene. A comparison of the low temperature absorption and fluorescence spectra in the Q Y region for CP47-His and CP43-His with those for CP47 and CP43 isolated by fragmentation of spinach PSII core complexes confirmed that the spectroscopic properties are similar but not identical. The measured fluorescence quantum yield was generally lower for the proteins isolated from Synechocystis sp. PCC 6803, and a 1-3-nm blue shift and a 2-nm red shift of the 77 K emission maximum could be observed for CP47-His and CP43-His, respectively. Immunoblotting and mass spectrometry revealed the co-purification of PsbH, PsbL, and PsbT with CP47-His and of PsbK and Psb30/ Ycf12 with CP43-His. Overall, our data support the view that CP47 and CP43 form preassembled pigment-protein complexes in vivo before their incorporation into the PSII complex.Photosystem II (PSII) 3 is the light-driven water:plastoquinone oxidoreductase of oxygenic photosynthesis, responsible for producing most of the oxygen in the atmosphere (1). It is located in the thylakoid membrane of chloroplasts and cyanobacteria and is a multisubunit lipoprotein complex composed of both intrinsic and extrinsic proteins. Crystal structures of dimeric PSII protein complexes isolated from the thermophilic cyanobacteria Thermosynechococcus elongatus (2-5) and Thermosynechococcus vulcanus (6, 7) have revealed the organization of the 20 subunits within each monomeric complex and the positions of the various cofactors. These include 35 chlorophyll (Chl) a molecules, two pheophytin a molecules, 12 carotenoids, two heme molecules, one non-heme iron, two calcium ions, two chloride ions, three plastoquinones, 25 lipids, and the Mn 4 Ca cluster, which catalyzes water oxidation (4).We are interested in understanding how PSII is assembled from its individual components. Current models suggest a stepwise assembly in both cyanobacteria and chloroplasts involving distinct intermediates (8 -10). However, as with other membrane protein complexes, detailed analysis of PSII assembly complexes is hindered by their low abundance in the membrane, and until now, early assembly intermediates of PSII have not been isolated and biochemically characterized.Here, we describe the isolation of the CP47 and ...

Discovery of a Chlorophyll Binding Protein Complex Involved in the Early Steps of Photosystem II Assembly in Synechocystis

“…The subunit composition of the RCII* complex corresponds well to the latest PSII structural models (Ferreira et al, 2004;Guskov et al, 2009;Umena et al, 2011) since the presence of most other small subunits and lumenal PSII proteins is dependent on the antenna chlorophyll-proteins CP47 and CP43. These two chlorophyll-proteins attach to the RCII complex as separate modules, together with neighboring small subunits and pigments, later in the process of PSII assembly (Boehm et al, 2011;Komenda et al, 2012a). In agreement with a previous report (Komenda et al, 2008), RCII* also contained the Ycf48 assembly factor ( Figure 1A).…”

“…PSII assembly is initiated through interaction of the D1 and D2 modules to form a RC intermediate, termed the RCII complex. The CP47 module (Boehm et al, 2011) is then attached to RCII forming a CP43-less complex, called RC47 (Boehm et al, 2012a). Oxygen-evolving monomeric and dimeric PSII are formed after addition of the CP43 module (Boehm et al, 2011), attachment of the luminal extrinsic proteins, and light-driven assembly of the oxygen-evolving Mn 4 CaO 5 complex (Komenda et al, 2008;Nixon et al, 2010).…”

Thylakoid FtsH Protease Contributes to Photosystem II and Cytochromeb 6 fRemodeling inChlamydomonas reinhardtiiunder Stress Conditions

“…In cyanobacteria, FtsH has mainly been studied for its role in D1 degradation (reviewed in Nixon et al, 2010;Komenda et al, 2012) and the removal of other damaged or unassembled PSII proteins Boehm et al, 2011). In one study, FtsH was also shown to contribute to the regulation of PSI amounts (Mann et al, 2000).…”

The ribosome-bound protein Pam68 promotes insertion of chlorophyll into the CP47 subunit of Photosystem II

“…Remarkably, the PsbH 117 subunit was hardly detectable even by specific antibodies, despite a high level of CP47 118 protein in the elution. PsbH is a component of CP47m (Boehm et al, 2011); hence the 119 absence of PsbH in the Pam68.f pull-down indicates that the association of CP47 with Pam68 120 is an early event that occurs before the attachment of PsbH to CP47. 121…”