Functional Heterogeneityof Photosystem II in Domain Specific Regions of the Thylakoid Membrane of Spinach (Spinaciaoleracea L.) † John Veerman, ‡ Michael D. McConnell, ‡ Sergei Vasil’ev, ‡ FikretMamedov, § Stenbjo ¨rn Styring, § and Doug Bruce* ,‡ Department of Biology, Brock Uni ersity,St. Catharines, Ontario, L2S 3A1, Canada, and Departmentfor Photochemistry and Molecular Science, Molecular Biomimetics, Uppsala Uni ersity, Box 523, S-751 20, Uppsala, Sweden Recei edSeptember 21, 2006; Re ised Manuscript Recei ed January 17, 2007 ABSTRACT: A mild sonication and phase fractionation method has beenused to isolatefive regions of the thylakoid membrane inorder to characterize thefunctionallateral heterogeneityof photosynthetic reaction centers and light harvesting complexes. Low-temperature fluorescence and absorbance spectra, absorbance cross-section measurements, and picosecond time-resolved fluorescence decaykinetics were used to determine the relativeamounts of photosystem II (PSII) and photosystem I(PSI), todetermine the relative PSII antenna size, and to characterize theexcited-state dynamics of PSIand PSII in each fraction. Marked progressive increasesin the proportion of PSIcomplexes were observed in thefollowing sequence: grana core (BS), whole grana(B3), margins (MA), stroma lamellae (T3), and purified stromal fraction (Y100). PSII antenna size was drastically reduced in the margins of the grana stack and stroma lamellae fractions as compared to the grana. Picosecond time-resolved fluorescence decaykinetics of PSII were characterized by three exponential decay components in the grana fractions, and were found tohave only twodecay components with slower lifetimesin the stroma. Results are discussed in the frameworkofexisting models ofchloroplast thylakoid membrane lateral heterogeneity and the PSII repaircycle. Kinetic modeling of the PSII fluorescence decaykinetics revealed that PSII populationsin the stromaand grana margin fractions possess much slower primary charge separation rates and decreased photosynthetic efficiency when compared to PSII populationsin the grana stack. In the process of oxygenic photosynthesis plants utilize light energy to split water into molecular oxygen, protons, and electrons and produce both ATP and NADPHfor use in carbon fixation. This process requires photosystem I(PSI 1 ) and photosystem II (PSII) operating in tandem. PSIand PSII core complexes are large supramolecular pigment protein complexes containing 96 and 35 chloro- phyll a (Chl a) molecules (1, 2)and (3, 4), respectively. In higher plants both photosystems are associated withperiph- eralChl a and Chl b-containing antennacomplexes, known as light harvesting complex I (LHCI) and light harvesting complex II (LHCII). The PSII associated LHC family includesthe Chl binding proteins, CP24, CP26, and CP29. The PSIcore complex associates with four LHCI monomers, while two LHCII trimers and three monomers (CP24, CP26 and CP29) are coupled to each PSII core. Theassociation of theauxiliary antennae with the PSIcore complexhas generated a model of PSI LHCIwith a total of 167 Chl molecules (5, 6)and a Chl a/b ratioofabout 9. The PSII LHCII complex is characterized as possessing about 150 Chl molecules, with a Chl a/b ratioofabout 2.5 (7, 8). In addition to these basic structural units,higher plants produce variable amounts ofadditional LHCII serving to supplementthe light harvesting capacityof each photosystem. Theseadditional LHCII complexes canbe dynamically reallocatedbetween the two photosystems to optimize cooperation between them in a process denoted as state transitions, see ref 9 for review. Photosynthetic membranes ofchloroplasts consist of the appressed regions (grana stacks)and the unappressed regions (stroma lamellae). Grana stacks contain a preponderance of PSII and the stroma lamellae PSI(10 12). PSII populations localized in these two membrane areas are fundamentally different with respectto antenna size and photochemical activity. PSII complexesin the grana stack represent a highly active population with respectto donorand acceptor side electron transport, while those in the stroma lamellae have been observed tobe relatively inactive(13 15). A mild sonication and phase fractionation method has been developed to isolate several granaand stroma fractions (13, 14, 16);these membrane fractions are illustrated with respect to their location in chloroplast thylakoid membranesin Figure 1. Studies of these fractions have indicated thatinactivation of PSII with respectto oxygen evolution and forward electron transfer from Q A progressively increases with increasing distance fromthe granacore (13 15). Lateral heterogeneity † This work wassupported by a researchgrant from NSERC to D.B. Work in Sweden wassupported by the Swedish Research Council and the Swedish Energy Agency (S.S. and F.M.). * Corresponding author. E-mail: dbruce@brocku.ca. Fax: 905 688- 1855. Phone: 905 688-5550 ext 3826. ‡ Brock University. § UppsalaUniversity. 1 Abbreviations: PS, photosystem; RC, reaction center;Chl, chlo- rophyll; P680, primary electron donor in photosystem II; Q A ,primary quinoneelectron acceptor in photosystem II; DAS,decay-associated spectrum; F 0, the minimal fluorescence level associated with photo- chemically active or“open” reaction centers with an oxidizedprimary quinoneelectron acceptor, QA; FM, the maximallevel offluorescence associated with photochemically inactive or “closed” reaction centers with reducedprimary quinoneelectron acceptor, Q A . 3443 Biochemistry 2007, 46, 3443 3453 10.1021/bi061964r CCC: $37.00 © 2007 American ChemicalSociety Published on Web 02/16/2007