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BBA - Bioenergetics (v.1706, #3)
Inactivation of the geranylgeranyl reductase (ChlP) gene in the cyanobacterium Synechocystis sp. PCC 6803 by Alexey V. Shpilyov; Vladislav V. Zinchenko; Sergey V. Shestakov; Bernhard Grimm; Heiko Lokstein (pp. 195-203).
Geranylgeranyl reductase catalyses the reduction of geranylgeranyl pyrophosphate to phytyl pyrophosphate required for synthesis of chlorophylls, phylloquinone and tocopherols. The gene chlP (ORF sll1091) encoding the enzyme has been inactivated in the cyanobacterium Synechocystis sp. PCC 6803. The resulting ΔchlP mutant accumulates exclusively geranylgeranylated chlorophyll a instead of its phytylated analogue as well as low amounts of α-tocotrienol instead of α-tocopherol. Whereas the contents of chlorophyll and total carotenoids are decreased, abundance of phycobilisomes is increased in ΔchlP cells. The mutant assembles functional photosystems I and II as judged from 77 K fluorescence and electron transport measurements. However, the mutant is unable to grow photoautotrophically due to instability and rapid degradation of the photosystems in the absence of added glucose. We suggest that instability of the photosystems in ΔchlP is directly related to accumulation of geranylgeranylated chlorophyll a. Increased rigidity of the chlorophyll isoprenoid tail moiety due to three additional CC bonds is the likely cause of photooxidative stress and reduced stability of photosynthetic pigment–protein complexes assembled with geranylgeranylated chlorophyll a in the ΔchlP mutant.
Keywords: Abbreviations; Chl; a; (phytylated) chlorophyll; a; Chl; a; gg; geranylgeranylated Chl; a; Chlide; a; chlorophyllide; a; ChlG; chlorophyll synthase; ChlP; geranylgeranyl reductase; chlP; gene encoding ChlP; DCBQ; 2,6-dichloro-; p; -benzoquinone; GGPP; geranylgeranyl pyrophosphate; Km/Km; r; kanamycin/kanamycin resistance; OD; optical density; ORF; open reading frame; PBS; phycobilisome; PC; phycocyanin; PPP; phytyl pyrophosphate; PSI, PSII; photosystem I, II; RC; reaction center; WT; wild typeChlorophyll biosynthesis; Geranylgeranyl reductase; Geranylgeranylated chlorophyll; Synechocystis; sp. PCC 6803
Fast oxidation of the primary electron acceptor under anaerobic conditions requires the organization of the photosynthetic chain of Rhodobacter sphaeroides in supercomplexes by Pierre Joliot; Anne Joliot; André Verméglio (pp. 204-214).
The kinetics of reoxidation of the primary acceptor Qa has been followed by measuring the changes in the fluorescence yield induced by a series of saturating flashes in intact cells of Rhodobacter sphaeroides in anaerobic conditions. At 0 °C, about half of Qa− is reoxidized in about 200 ms while reoxidation of the remaining fraction is completed in several seconds to minutes. The fast phase is associated with the transfer of ubiquinone formed at site Qo of the cytochrome bc1 complex while the slowest phase is associated with the diffusion of ubiquinone present in the membrane prior to the flash excitation. The biphasic kinetics of Qa− oxidation is interpreted assuming that the electron chain is organized in supercomplexes that associate two RCs and one cyt bc1 complex, which allows a fast transfer of quinone formed at the level of cyt bc1 complex to the RCs. In agreement with this model, the fast phase of Qa− reoxidation is inhibited by myxothiazol, a specific inhibitor of cyt bc1. The PufX-deleted mutant displays only the slowest phase of Qa− oxidation; it is interpreted by the lack of supramolecular organization of the photosynthetic chain that leads to a larger average distance between cyt bc1 and RCs.
Keywords: Abbreviations; AFM; atomic force spectroscopy; cyt; cytochrome; LH; light-harvesting; P; 870; primary electron donor; Q; a; , Q; b; primary and secondary electron acceptors; Rb.; Rhodobacter; RC; reaction center; UQ; ubiquinoneElectron transfer; Rb. sphaeroides; Reaction center; Cytochrome; bc; 1; complex; Supercomplex
Salt-induced redox-independent phosphorylation of light harvesting chlorophyll a/ b proteins in Dunaliella salina thylakoid membranes by Xian-De Liu; Yun-Gang Shen (pp. 215-219).
This study investigated the regulation of the major light harvesting chlorophyll a/ b protein (LHCII) phosphorylation in Dunaliella salina thylakoid membranes. We found that both light and NaCl could induce LHCII phosphorylation in D. salina thylakoid membranes. Treatments with oxidants (ferredoxin and NADP) or photosynthetic electron flow inhibitors (DCMU, DBMIB, and stigmatellin) inhibited LHCII phosphorylation induced by light but not that induced by NaCl. Furthermore, neither addition of CuCl2, an inhibitor of cytochrome b6 f complex reduction, nor oxidizing treatment with ferricyanide inhibited light- or NaCl-induced LHCII phosphorylation, and both salts even induced LHCII phosphorylation in dark-adapted D. salina thylakoid membranes as other salts did. Together, these results indicate that the redox state of the cytochrome b6 f complex is likely involved in light- but not salt-induced LHCII phosphorylation in D. salina thylakoid membranes.
Keywords: Abbreviations; DBMIB; 2,5-dibromo-3-methyl-6-isoprophyl-p-benzoquinone; DCMU; 3-(3,4-dichlorophenyl)-1,1-dimethylurea; FeCy; ferricyanide; Fd; ferredoxin; LHCII; light harvesting chlorophyll; a; /; b; binding proteins; PSI; photosystem I; PSII; photosystem II Dunaliella salina; Thylakoid membrane; LHCII phosphorylation; NaCl; Redox state; Cytochrome; b; 6; f; complex
Sequential assembly of photosynthetic units in Rhodobacter sphaeroides as revealed by fast repetition rate analysis of variable bacteriochlorophyll a fluorescence by Michal Koblízek; Joseph D. Shih; Seth I. Breitbart; Emma C. Ratcliffe; Zbigniew S. Kolber; C. Neil Hunter; Robert A. Niederman (pp. 220-231).
The development of functional photosynthetic units in Rhodobacter sphaeroides was followed by near infra-red fast repetition rate (IRFRR) fluorescence measurements that were correlated to absorption spectroscopy, electron microscopy and pigment analyses. To induce the formation of intracytoplasmic membranes (ICM) (greening), cells grown aerobically both in batch culture and in a carbon-limited chemostat were transferred to semiaerobic conditions. In both aerobic cultures, a low level of photosynthetic complexes was observed, which were composed of the reaction center and the LH1 core antenna. Interestingly, in the batch cultures the reaction centers were essentially inactive in forward electron transfer and exhibited low photochemical yields FV/ FM, whereas the chemostat culture displayed functional reaction centers with a rather rapid (1–2 ms) electron transfer turnover, as well as a high FV/ FM of ∼0.8. In both cases, the transfer to semiaerobiosis resulted in rapid induction of bacteriochlorophyll a synthesis that was reflected by both an increase in the number of LH1–reaction center and peripheral LH2 antenna complexes. These studies establish that photosynthetic units are assembled in a sequential manner, where the appearance of the LH1–reaction center cores is followed by the activation of functional electron transfer, and finally by the accumulation of the LH2 complexes.
Keywords: Abbreviations; bc; 1; complex; ubiquinol-cytochrome; c; 2; oxidoreductase; BChl; a; bacteriochlorophyll; a; CM; cytoplasmic membrane; IRFRR; infra-red fast repetition rate; F; 0; minimal fluorescence yield; F; M; maximal fluorescence yield; F; V; variable fluorescence (difference between; F; 0; and; F; M; ); ICM; intracytoplasmic membrane; LH1; core light-harvesting complex; LH2; peripheral light-harvesting complex; p; energy transfer between photosynthetic units; P; 870; reaction center bacteriochlorophyll; a; dimer; PSII; photosystem II; σ; functional absorption cross-sectionMembrane development; Light-harvesting complex; Photosynthetic membrane; Fast repetition rate fluorescence; Reaction center kinetics
pH dependence of charge transfer between tryptophan and tyrosine in dipeptides by Steven Y. Reece; JoAnne Stubbe; Daniel G. Nocera (pp. 232-238).
Time-resolved absorption spectroscopy has been employed to study the directionality and rate of charge transfer in W–Y and Ac-W–Y dipeptides as a function of pH. Excitation with 266-nm nanosecond laser pulses produces both W⋅ (or [⋅WH]+, depending on pH) and Y⋅. Between pH 6 and 10, W⋅ to was found to oxidize Y with kX⋅=9.0×104 s−1 and 1.8×104 s−1 for the W–Y and Ac-W–Y dipeptide systems, respectively. The intramolecular charge transfer rate increases as the pH is lowered over the range 6>pH>2. For 10− (Y−, tyrosinate anion), with a rate constant of kX⋅=1.2×105 s−1. The dependence of charge transfer directionality between W and Y on pH is important to the enzymatic function of several model and natural biological systems as discussed here for ribonucleotide reductase.
Keywords: Tyrosine; Tryptophan; Radical; Proton-coupled electron transfer; Ribonucleotide reductase
Non-photochemical quenching of chlorophyll a fluorescence by oxidised plastoquinone: new evidences based on modulation of the redox state of the endogenous plastoquinone pool in broken spinach chloroplasts by Pierre Haldimann; Merope Tsimilli-Michael (pp. 239-249).
Twenty-five years ago, non-photochemical quenching of chlorophyll fluorescence by oxidised plastoquinone (PQ) was proposed to be responsible for the lowering of the maximum fluorescence yield reported to occur when leaves or chloroplasts were treated in the dark with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), an inhibitor of electron flow beyond the primary quinone electron acceptor (QA) of photosystem (PS) II [C. Vernotte, A.L. Etienne, J.-M. Briantais, Quenching of the system II chlorophyll fluorescence by the plastoquinone pool, Biochim. Biophys. Acta 545 (1979) 519-527]. Since then, the notion of PQ-quenching has received support but has also been put in doubt, due to inconsistent experimental findings. In the present study, the possible role of the native PQ-pool as a non-photochemical quencher was reinvestigated, employing measurements of the fast chlorophyll a fluorescence kinetics (from 50 μs to 5 s). The about 20% lowering of the maximum fluorescence yield FM, observed in osmotically broken spinach chloroplasts treated with DCMU, was eliminated when the oxidised PQ-pool was non-photochemically reduced to PQH2 by dark incubation of the samples in the presence of NAD(P)H, both under anaerobic and aerobic conditions. Incubation under anaerobic conditions in the absence of NAD(P)H had comparatively minor effects. In DCMU-treated samples incubated in the presence of NAD(P)H fluorescence quenching started to develop again after 20–30 ms of illumination, i.e., the time when PQH2 starts getting reoxidised by PS I activity. NAD(P)H-dependent restoration of FM was largely, if not completely, eliminated when the samples were briefly (5 s) pre-illuminated with red or far-red light. Addition to the incubation medium of HgCl2 that inhibits dark reduction of PQ by NAD(P)H also abolished NAD(P)H-dependent restoration of FM. Collectively, our results provide strong new evidence for the occurrence of PQ-quenching. The finding that DCMU alone did not affect the minimum fluorescence yield F0 allowed us to calculate, for different redox states of the native PQ-pool, the fractional quenching at the F0 level ( Q0) and to compare it with the fractional quenching at the FM level ( QM). The experimentally determined Q0/ QM ratios were found to be equal to the corresponding F0/ FM ratios, demonstrating that PQ-quenching is solely exerted on the excited state of antenna chlorophylls.
Keywords: Abbreviations; Chl; chlorophyll; DCMU; 3-(3,4-dichlorophenyl)-1,1-dimethylurea; F; M; maximum fluorescence yield; F; 0; minimum fluorescence yield; I and J; intermediate steps in the Chl; a; fluorescence transient appearing between; F; 0; and; F; M; PQ; plastoquinone; PQH; 2; plastoquinol; PS; photosystem; Q; A; primary quinone electron acceptor of PS II; RC; reaction centreChlorophyll fluorescence quenching; Mercury; NAD(P)H; Photosystem II; Plastoquinone; Thylakoid
Methylviologen and dibromothymoquinone treatments of pea leaves reveal the role of photosystem I in the Chl a fluorescence rise OJIP by Gert Schansker; Szilvia Z. Tóth; Reto J. Strasser (pp. 250-261).
The effects of dibromothymoquinone (DBMIB) and methylviologen (MV) on the Chl a fluorescence induction transient (OJIP) were studied in vivo. Simultaneously measured 820-nm transmission kinetics were used to monitor electron flow through photosystem I (PSI). DBMIB inhibits the reoxidation of plastoquinol by binding to the cytochrome b6/ f complex. MV accepts electrons from the FeS clusters of PSI and it allows electrons to bypass the block that is transiently imposed by ferredoxin-NADP+-reductase (FNR) (inactive in dark-adapted leaves). We show that the IP phase of the OJIP transient disappears in the presence of DBMIB without affecting Fm. MV suppresses the IP phase by lowering the P level compared to untreated leaves. These observations indicate that PSI activity plays an important role in the kinetics of the OJIP transient. Two requirements for the IP phase are electron transfer beyond the cytochrome b6/ f complex (blocked by DBMIB) and a transient block at the acceptor side of PSI (bypassed by MV). It is also observed that in leaves, just like in thylakoid membranes, DBMIB can bypass its own block at the cytochrome b6/ f complex and donate electrons directly to PC+ and P700+ with a donation time Ï„ of 4.3 s. Further, alternative explanations of the IP phase that have been proposed in the literature are discussed.
Keywords: Chl; a; fluorescence; OJIP-transient; 820-nm transmission; DBMIB; Methylviologen; Pisum sativum
Supercomplexes of IsiA and Photosystem I in a mutant lacking subunit PsaL by Roman Kouřil; Nataliya Yeremenko; Sandrine D'Haene; Gert T. Oostergetel; Hans C.P. Matthijs; Jan P. Dekker; Egbert J. Boekema (pp. 262-266).
The cyanobacterium Synechocystis PCC 6803 grown under short-term iron-deficient conditions assembles a supercomplex consisting of a trimeric Photosystem I (PSI) complex encircled by a ring of 18 IsiA complexes. Furthermore, it has been shown that single or double rings of IsiA with up to 35 copies in total can surround monomeric PSI. Here we present an analysis by electron microscopy and image analysis of the various PSI–IsiA supercomplexes from a Synechocystis PCC 6803 mutant lacking the PsaL subunit after short- and long-term iron-deficient growth. In the absence of PsaL, the tendency to form complexes with IsiA is still strong, but the average number of complete rings is lower than in the wild type. The majority of IsiA copies binds into partial double rings at the side of PsaF/J subunits rather than in complete single or double rings, which also cover the PsaL side of the PSI monomer. This indicates that PsaL facilitates the formation of IsiA rings around PSI monomers but is not an obligatory structural component in the formation of PSI–IsiA complexes.
Keywords: Photosystem I; IsiA; PsaL; Electron microscopy
Kinetics of excitation trapping in intact Photosystem I of Chlamydomonas reinhardtii and Arabidopsis thaliana by Janne A. Ihalainen; Ivo H.M. van Stokkum; Krzysztof Gibasiewicz; Marta Germano; Rienk van Grondelle; Jan P. Dekker (pp. 267-275).
We measured picosecond time-resolved fluorescence of intact Photosystem I complexes from Chlamydomonas reinhardtii and Arabidopsis thaliana. The antenna system of C. reinhardtii contains about 30–60 chlorophylls more than that of A. thaliana, but lacks the so-called red chlorophylls, chlorophylls that absorb at longer wavelength than the primary electron donor. In C. reinhardtii, the main lifetimes of excitation trapping are about 27 and 68 ps. The overall lifetime of C. reinhardtii is considerably shorter than in A. thaliana. We conclude that the amount and energies of the red chlorophylls have a larger effect on excitation trapping time in Photosystem I than the antenna size.
Keywords: Abbreviations; β-DM; n; -dodecyl-β,; d; -maltoside; PSI-LHCI; Photosystem I-light-harvesting complex I particle; DAS; decay-associated spectra; SAS; species-associated spectraPhotosystem I; Light-harvesting complex I; Fluorescence; Excitation dynamics
The effect of outer antenna complexes on the photochemical trapping rate in barley thylakoid Photosystem II by Enrico C.M. Engelmann; Giuseppe Zucchelli; Flavio M. Garlaschi; Anna Paola Casazza; Robert C. Jennings (pp. 276-286).
We have investigated the previous suggestions in the literature that the outer antenna of Photosystem II of barley does not influence the effective photosystem primary photochemical trapping rate. It is shown by steady state fluorescence measurements at the F0 fluorescence level of wild type and the chlorina f2 mutant, using the chlorophyll b fluorescence as a marker, that the outer antenna is thermally equilibrated with the core pigments, at room temperature, under conditions of photochemical trapping. This is in contrast with the conclusions of the earlier studies in which it was suggested that energy was transferred rapidly and irreversibly from the outer antenna to the Photosystem II core. Furthermore, the effective trapping time, determined by single photon counting, time-resolved measurements, was shown to increase from 0.17±0.017 ns in the chlorina Photosystem II core to a value within the range 0.42±0.036–0.47±0.044 ns for the wild-type Photosystem II with the outer antenna system. This 2.5–2.8-fold increase in the effective trapping time is, however, significantly less than that expected for a thermalised system. The data can be explained in terms of the outer antenna increasing the primary charge separation rate by about 50%.
Keywords: Antenna effect; Chlorina f2; Photosystem II; Thylakoid; Trapping time