Isolation and Biochemical Characterization of Monomeric and Dimeric Photosystem II Complexes from Spinach and Their Relevance to the Organisation of Photosystem II In vivo

Abstract

Membranes enriched in photosystem TI were isolated from spinach and further solubilised using n-octyl 8-D-glucopyranoside (OctGlc) and n-dodecyl P-D-maltoside (DodClc,). The OctGlc preparation had high rates of oxygen evolution and when subjected to size-exclusion HPLC and sucrose density gradient centrifugation, in the presence of DodGlc,, separated into dimeric (430 kDa), monomeric (236 kDa) photosystem I1 cores and a fraction containing photosystem I1 light-harvesting complex (Lhcb) proteins. The dimeric core fraction was more stable, contained higher levels of chlorophyll, 8-carotene and plastoquinone per photosystem I1 reaction centre and had a higher oxygen-evolving activity than the monomeric cores. Their subunit composition was similar (CP43, CP47, DI, D2, cytochrome b 559 and several lower-molecular-mass components) except that the level of 33-kDa extrinsic protein was lower in the monomeric fraction. Direct solubilisation of photosystem-11-enriched membranes with DodGlc,, followed by sucrose density gradient centrifugation, yielded a super complex (700 kDa) containing the dimeric form of the photosystem I1 core and Lhcb proteins: Lhcbl, Lhcb2, Lhcb4 (CP29), and LhcbS (CP26). Like the dimeric and monomeric photosystem I1 core complexes, the photosystem 11-LHCII complex had lost the 23-kDa and 17-kDa extrinsic proteins, but maintained the 33-kDa protein and the ability to evolve oxygen. It is suggested. with a proposed model, that the isolated photosystem 11-LHCII super complex represents an in vivn organisation that can sometimes form a lattice in granal membranes K K~V O K~S :dimer; photosynthesis; photosystem 11; spinach; structure.Photosystem I1 (PSII) is a pigment-protein complex embedded in the thylakoid membrane of higher plants, algae, and cyanobacteria. By utilizing sunlight, it catalyses the splitting of water into protons, electrons, and molecular oxygen. This is the most strongly oxidizing reaction known to occur in biology. The primary photochemical process driving this highly oxidizing reaction takes place in the reaction centre of PSII which, when isolated, consists of the D1 and D2 subunits, cytochrome b 559 (cyt b 559), and the psbl gene product (Nanba and Satoh, 1987;Barber et al., 1987). The reaction centre proteins are closely associated with two other chlorophyll-a-binding proteins (CP47 and CP43). as well as the oxygen-evolving complex (OEC) composed of a four-atom cluster of manganese and the 33-kDa PsbO extrinsic protein (Ikeuchi et al., 1985). Unlike cyanobacteria, higher plants and algae have two additional extrinsic proteins associated with the OEC which have apparent molecular masses of 23 kDa and 17 kDa (PsbP and PsbQ proteins, respectively) (Murata and Miyao, 1985). Higher plants and green algae also have chlorophyll-ah-binding antenna systems that transfer excitation energy to the PSII reaction centre rather than phycobilisomes, which function in the same way in red algae and cyanobacteria (Jansson, 1994). The chlorophyll-nlh-binding antenna consists of LHCII (Lhcbl, b2, a...

Isolation of a highly active PSII‐LHCII supercomplex from thylakoid membranes by a direct method

“…The above results and the Chl a/b ratio of band C are in agreement with previous studies of a PSII-LHCII supercomplex isolated from BBYs [5,6,8], indicating band C to be a PSII-LHCII supercomplex. However, in our case this form of the PSII-LHCII supercomplex contained the 16-kDa as well as the 23-kDa and 33-kDa OEC proteins at high levels.…”

Light-Harvesting in Photosystem II

“…The LHCII trimers differ somewhat in composition. The S trimer is composed of the products of the Lhcb1 and Lhcb2 genes and the M trimer in addition also contains the product of the Lhcb3 gene (Hankamer et al 1997). Ordered arrays of C2S2, C2S2M, and C2S2M2 have been observed in membranes of different plants (Boekema et al 2000;Daum et al 2010;Yakushevska et al 2001;Kouril et al 2011).…”

Structure of a photosystem II supercomplex isolated from Prochloron didemni retaining its chlorophyll a/b light-harvesting system

“…ents generated by the freeze-thawing technique (20) containing 50 mM Mes-NaOH (pH 6.0), 500 mM betaine, 20 mM CaCl 2 , 2.5 mM MgCl 2 , and 0.03% ␤-dodecyl maltoside and ultracentrifuged at 90,000 ϫ g for 16 h at 4°C (SW28 Beckman rotor). The procedure was similar to that used previously for isolating PSI supercomplexes from Synechocystis PCC 6803 (14,15) and Prochlorococcus SS120 (17).…”

“…Western blotting was conducted by using a published procedure (20) with Abs raised to the PSII reaction center PsbA (D1) protein of Synechocystis 6803 and to the reaction center proteins of PSI (PsaA) of Chlamydomonas reinhardtii (kindly provided by P. Nixon, Imperial College). Polypeptides separated by SDS͞PAGE were transferred to poly(vinylidene difluoride) membrane before identification by N-terminal sequencing conducted by J.…”