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1.
The ATP-dependent insertion of Mg2+ into protoporphyrin IX is the first committed step in the chlorophyll biosynthetic pathway. The reaction is catalyzed by magnesium chelatase, which consists of three gene products: BchI, BchD, and BchH. The BchI and BchD subunits belong to the family of AAA+ proteins (ATPases associated with various cellular activities) and form a two-ring complex with six BchI subunits in one layer and six BchD subunits in the other layer. This BchID complex is a two-layered trimer of dimers with the ATP binding site located at the interface between two neighboring BchI subunits. ATP hydrolysis by the BchID motor unit fuels the insertion of Mg2+ into the porphyrin by the BchH subunit. In the present study, we explored mutations that were originally identified in semidominant barley (Hordeum vulgare L.) mutants. The resulting recombinant BchI proteins have marginal ATPase activity and cannot contribute to magnesium chelatase activity although they apparently form structurally correct complexes with BchD. Mixing experiments with modified and wild-type BchI in various combinations showed that an exchange of BchI subunits in magnesium chelatase occurs during the catalytic cycle, which indicates that dissociation of the complex may be part of the reaction mechanism related to product release. Mixing experiments also showed that more than three functional interfaces in the BchI ring structure are required for magnesium chelatase activity.  相似文献   

2.
The FIGL-1 (fidgetin like-1) protein is a homolog of fidgetin, a protein whose mutation leads to multiple developmental defects. The FIGL-1 protein contains an AAA (ATPase associated with various activities) domain and belongs to the AAA superfamily. However, the biological functions and developmental implications of this protein remain unknown. Here, we show that the AAA domain of the Caenorhabditis elegans FIGL-1 protein (CeFIGL-1-AAA), in clear contrast to homologous AAA domains, has an unusually high ATPase activity and forms a hexamer in solution. By determining the crystal structure of CeFIGL-1-AAA, we found that the loop linking helices α9 and α10 folds into the short helix α9a, which has an acidic surface and interacts with a positively charged surface of the neighboring subunit. Disruption of this charge interaction by mutagenesis diminishes both the ATPase activity and oligomerization capacity of the protein. Interestingly, the acidic residues in helix α9a of CeFIGL-1-AAA are not conserved in other homologous AAA domains that have relatively low ATPase activities. These results demonstrate that the sequence of CeFIGL-1-AAA has adapted to establish an intersubunit charge interaction, which contributes to its strong oligomerization and ATPase activity. These unique properties of CeFIGL-1-AAA distinguish it from other homologous proteins, suggesting that CeFIGL-1 may have a distinct biological function.  相似文献   

3.
The AAA-type ATPase Vps4 functions with components of the ESCRT (endosomal sorting complex required for transport) machinery in membrane fission events that are essential for endosomal maturation, cytokinesis, and the formation of retroviruses. A key step in these events is the assembly of monomeric Vps4 into the active ATPase complex, which is aided in part by binding of Vps4 via its N-terminal MIT (microtubule interacting and trafficking) domain to its substrate ESCRT-III. We found that the 40-amino acid linker region between the MIT and the ATPase domain of Vps4 is not required for proper function but plays a role in regulating Vps4 assembly and ATPase activity. Deletion of the linker is expected to bring the MIT domains into close proximity to the central pore of the Vps4 complex. We propose that this localization of the MIT domain in linker-deleted Vps4 mimics a repositioning of the MIT domain normally caused by binding of Vps4 to ESCRT-III. This structure would allow the Vps4 complex to engage ESCRT-III subunits with both the pore and the MIT domain simultaneously, which might be essential for the ATP-driven disassembly of ESCRT-III.  相似文献   

4.
5.
Jaschkowitz K  Seidler A 《Biochemistry》2000,39(12):3416-3423
In Azotobacter vinelandii and Escherichia coli NifS or NifS-like proteins are involved in FeS protein assembly by mobilizing sulfur from free cysteine. This sulfur together with Fe(2+) is then incorporated into apo-FeS proteins to form an FeS center. A different activity termed C-DES [for cyst(e)ine desulfurylase] was recently isolated from the cyanobacterium Synechocystis PCC 6714 which also mobilized sulfur and which was able to incorporate the FeS center into apoferredoxin. In the genome of the cyanobacterium Synechocystis PCC 6803, there are three open reading frames (orfs) that are similar to NifS and one that is similar to C-DES, indicating that this bacterium might contain both activities, NifS and C-DES. One orf from Synechocystis PCC 6803 encoding a NifS-like protein, slr0387, was overexpressed in E. coli and purified. The molecular mass of the recombinant protein was determined to be about 82 kDa, indicating that it is a homodimer. The absorption spectrum was typical for PLP-containing proteins with an absorption maximum at 390 nm at pH 9.0 and at 425 nm at pH 6.5. The pH dependence of the absorption spectrum correlated with enzyme activity. Maximal activity measured as sulfide production was observed between pH 8.5 and 10. The activity decreased at lower pH values and was undetectable at pH 5.5. pH-dependent changes in the absorption spectrum and activity were attributed to protonation of the Schiff base formed by a lysine side chain and the PLP cofactor. Studies on substrate specificity demonstrated that cysteine derivatives other than cysteine methyl ester and cysteine-sulfinic acid could not serve as substrates for this enzyme. In particular, cystine was not a substrate for the Synechocystis NifS-like protein, whereas it is the best substrate for C-DES. In the presence of Fe(2+), cysteine, and a reductant, the NifS-like protein was able to produce holoferredoxin from apoferredoxin. The implications of two different activities for FeS center biosynthesis in Synechocystis are discussed.  相似文献   

6.
Sll1951 is the surface layer (S-layer) protein of the cyanobacterium Synechocystis sp. strain PCC 6803. This large, hemolysin-like protein was found in the supernatant of a strain that was deficient in S-layer attachment. An sll1951 deletion mutation was introduced into Synechocystis and was easily segregated to homozygosity under laboratory conditions. By thin-section and negative-stain transmission electron microscopy, a ∼30-nm-wide S-layer lattice covering the cell surface was readily visible in wild-type cells but was absent in the Δsll1951 strain. Instead, the Δsll1951 strain displayed a smooth lipopolysaccharide surface as its most peripheral layer. In the presence of chaotropic agents, the wild type released a large (>150-kDa) protein into the medium that was identified as Sll1951 by mass spectrometry of trypsin fragments; this protein was missing in the Δsll1951 strain. In addition, Sll1951 was prominent in crude extracts of the wild type, indicating that it is an abundant protein. The carotenoid composition of the cell wall fraction of the Δsll1951 strain was similar to that of the wild type, suggesting that the S-layer does not contribute to carotenoid binding. Although the photoautotrophic growth rate of the Δsll1951 strain was similar to that of the wild-type strain, the viability of the Δsll1951 strain was reduced upon exposure to lysozyme treatment and hypo-osmotic stress, indicating a contribution of the S-layer to the integrity of the Synechocystis cell wall. This work identifies the S-layer protein in Synechocystis and shows that, at least under laboratory conditions, this very abundant, large protein has a supportive but not a critical role in the function of the cyanobacterium.  相似文献   

7.
To advance our knowledge of the model cyanobacterium Synechocystis sp. PCC 6803 we investigated the three-dimensional organization of the cytoplasm using standard transmission electron microscopy and electron tomography. Electron tomography allows a resolution of ~5 nm in all three dimensions, superior to the resolution of most traditional electron microscopy, which is often limited in part by the thickness of the section (70 nm). The thylakoid membrane pairs formed layered sheets that followed the periphery of the cell and converged at various sites near the cytoplasmic membrane. At some of these sites, the margins of thylakoid membranes associated closely along the external surface of rod-like structures termed thylakoid centers, which sometimes traversed nearly the entire periphery of the cell. The thylakoid membranes surrounded the central cytoplasm that contained inclusions such as ribosomes and carboxysomes. Lipid bodies were dispersed throughout the peripheral cytoplasm and often juxtaposed with cytoplasmic and thylakoid membranes suggesting involvement in thylakoid maintenance or biogenesis. Ribosomes were numerous and mainly located throughout the central cytoplasm with some associated with thylakoid and cytoplasmic membranes. Some ribosomes were attached along internal unit-membrane-like sheets located in the central cytoplasm and appeared to be continuous with existing thylakoid membranes. These results present a detailed analysis of the structure of Synechocystis sp. PCC 6803 using high-resolution bioimaging techniques and will allow future evaluation and comparison with gene-deletion mutants.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.  相似文献   

8.
Folea IM  Zhang P  Aro EM  Boekema EJ 《FEBS letters》2008,582(12):1749-1754
The supramolecular organization of photosystem II (PSII) complexes in the photosynthetic membrane of the cyanobacterium Synechocystis 6803 was studied by electron microscopy. After mild detergent solubilization, crystalline PSII arrays were extracted in which dimeric PSII particles associate in multiple rows. Image processing of the arrays shows that the PSII dimers are tightly packed at distances of 12.2 and 16.7 nm. The domains are considered to be an important type of association for preventing either spill-over energy from PSII towards photosystem I (PSI) or direct energy flow from phycobilisomes to PSI, because the latter can only be at periphery of the arrays.  相似文献   

9.
A naturally occurring split intein from the dnaE gene of Synechocystis sp. PCC6803 (Ssp DnaE intein) has been shown to mediate efficient in vivo and in vitro trans-splicing in a foreign protein context. A cis-splicing Ssp DnaE intein construct displayed splicing activity similar to the trans-splicing form, which suggests that the N- and C-terminal intein fragments have a high affinity interaction. An in vitro trans-splicing system was developed that used a bacterially expressed N-terminal fragment of the Ssp DnaE intein and either a bacterially expressed or chemically synthesized intein C-terminal fragment. Unlike artificially split inteins, the Ssp DnaE intein fragments could be reconstituted in vitro under native conditions to mediate splicing as well as peptide bond cleavage. This property allowed the development of an on-column trans-splicing system that permitted the facile separation of reactants and products. Furthermore, the trans-splicing activity of the Ssp DnaE intein was successfully applied to the cyclization of proteins in vivo. Also, the isolation of the unspliced precursor on chitin resin allowed the cyclization reaction to proceed in vitro. The Ssp DnaE intein thus represents a potentially important protein for in vivo and in vitro protein manipulation.  相似文献   

10.
11.
Ivleva  N. B.  Sidoruk  K. V.  Pakrasi  H. B.  Shestakov  S. V. 《Microbiology》2002,71(4):433-437
To understand the functional role of CtpB and CtpC proteins, which are similar to the C-terminal processing CtpA peptidase, the effect of the insertional inactivation of the ctpB and ctpCgenes on the phenotypic characteristics of Synechocystis sp. PCC 6803 was studied. The inactivation of the ctpC gene was found to be lethal to the cyanobacterium, which indicates a vital role of the CtpC protein. The mutant with the inactivated ctpB gene had the same photosynthetic characteristics as the wild-type strain. The double mutant ctpActpB with the two deleted genes was identical, in the phenotypic characteristics, to the mutant with a knock-out mutation in the ctpAgene, which was unable to grow photoautotrophically. The data obtained suggest that, in spite of the high similarity of the Ctp proteins, they serve different functions in Synechocystis sp. PCC 6803 cells and cannot compensate for each other.  相似文献   

12.
The cph1 gene from the unicellular cyanobacterium Synechoycstis sp. PCC 6803 encodes a protein with the characteristics of plant phytochromes and histidine kinases of two-component phospho-relay systems. Spectral and biochemical properties of Cph1 have been intensely studied in vitro using protein from recombinant systems, but virtually nothing is known about the situation in the natural host. In the present study, His6-tagged Cph1 was isolated from Synechocystis cells. The cph1-his gene was expressed either under the control of the natural cph1 promoter or over-expressed using the strong promoter of the psbA2 gene. Upon purification with nickel affinity chromatography, the presence of Cph1 in extracts was confirmed by immunoblotting and Zn2+-induced fluorescence. The Cph1 extracts exhibited a red/far-red photoactivity characteristic of phytochromes. Difference spectra were identical with those of the phycocyanobilin adduct of recombinant Cph1, implying that phycocyanobilin is the chromophore of Cph1 in Synechocystis.  相似文献   

13.
《Process Biochemistry》2014,49(12):2071-2077
Lactate is an important industrial material with numerous potential applications, and its production from carbon dioxide is very attractive. d-Lactate is an essential monomer for production of thermostable polylactide. The photoautotrophic prokaryote cyanobacterium Synechocystis sp. PCC 6803 represents a promising host for biosynthesis of d-lactate from CO2 as it only contains d-lactate dehydrogenase. The production of d-lactate from CO2 by an engineered strain of Synechocystis sp. PCC 6803 with overexpressing d-lactate dehydrogenase and a soluble transhydrogenase has been reported recently. Here, we report an alternative engineering strategy to produce d-lactate from CO2. This strategy involves blocking two competitive pathways, the native poly-3-hydroxybutyrate and acetate pathways from the acetyl-CoA node, and introducing a more efficient d-lactate dehydrogenase into Synechocystis sp. PCC 6803. The engineered strain of Synechocystis sp. PCC 6803 was capable of producing 1.06 g/L of d-lactate from CO2. This alternative strategy for the production of optically pure d-lactate could also be used to produce other acetyl-CoA-derived chemicals from CO2 by using engineered cyanobacteria.  相似文献   

14.
Periplasmic proteins were obtained from control cells and salt-adapted cells of the cyanobacterium Synechocystis sp. PCC 6803 using the method of cold osmotic shock. Two of these proteins (PP 1, apparent mol. mass 27.6 kDa, and PP 3, apparent mol. mass 39.9 kDa) were accumulated in high amounts in the periplasm of salt-adapted cells, while the major periplasmic protein (PP 2, apparent mol. mass 36.0 kDa) was accumulated independently from salt. After isolation from gels and partial sequencing, the proteins could be assigned to proteins deduced from the complete genome sequence of Synechocystis. Neither salt-induced periplasmic proteins (PP 1, Slr0924 and PP 3, Slr1485) exhibited sequence similarity to proteins of known function from databases. The major protein (PP 2-Slr0513) showed significant sequence similarities to iron-binding proteins. All proteins included typical leader sequences at their N-terminus. Received: 21 September 1998 / Accepted: 17 December 1998  相似文献   

15.
The biosynthesis of chlorophyll, an essential cofactor for photosynthesis, requires the ATP-dependent insertion of Mg2+ into protoporphyrin IX catalyzed by the multisubunit enzyme magnesium chelatase. This enzyme complex consists of the I subunit, an ATPase that forms a complex with the D subunit, and an H subunit that binds both the protoporphyrin substrate and the magnesium protoporphyrin product. In this study we used electron microscopy and small-angle x-ray scattering to investigate the structure of the magnesium chelatase H subunit, ChlH, from the thermophilic cyanobacterium Thermosynechococcus elongatus. Single particle reconstruction of negatively stained apo-ChlH and Chl-porphyrin proteins was used to reconstitute three-dimensional structures to a resolution of ∼30 Å. ChlH is a large, 148-kDa protein of 1326 residues, forming a cage-like assembly comprising the majority of the structure, attached to a globular N-terminal domain of ∼16 kDa by a narrow linker region. This N-terminal domain is adjacent to a 5 nm-diameter opening in the structure that allows access to a cavity. Small-angle x-ray scattering analysis of ChlH, performed on soluble, catalytically active ChlH, verifies the presence of two domains and their relative sizes. Our results provide a basis for the multiple regulatory and catalytic functions of ChlH of oxygenic photosynthetic organisms and for a chaperoning function that sequesters the enzyme-bound magnesium protoporphyrin product prior to its delivery to the next enzyme in the chlorophyll biosynthetic pathway, magnesium protoporphyrin methyltransferase.  相似文献   

16.
The γ and ε subunits of F(0)F(1)-ATP synthase from photosynthetic organisms display unique properties not found in other organisms. Although the γ subunit of both chloroplast and cyanobacterial F(0)F(1) contains an extra amino acid segment whose deletion results in a high ATP hydrolysis activity (Sunamura, E., Konno, H., Imashimizu-Kobayashi, M., Sugano, Y., and Hisabori, T. (2010) Plant Cell Physiol. 51, 855-865), its ε subunit strongly inhibits ATP hydrolysis activity. To understand the physiological significance of these phenomena, we studied mutant strains with (i) a C-terminally truncated ε (ε(ΔC)), (ii) γ lacking the inserted sequence (γ(Δ198-222)), and (iii) a double mutation of (i) and (ii) in Synechocystis sp. PCC 6803. Although thylakoid membranes from the ε(ΔC) strain showed higher ATP hydrolysis and lower ATP synthesis activities than those of the wild type, no significant difference was observed in growth rate and in intracellular ATP level both under light conditions and during light-dark cycles. However, both the ε(ΔC) and γ(Δ198-222) and the double mutant strains showed a lower intracellular ATP level and lower cell viability under prolonged dark incubation compared with the wild type. These data suggest that internal inhibition of ATP hydrolysis activity is very important for cyanobacteria that are exposed to prolonged dark adaptation and, in general, for the survival of photosynthetic organisms in an ever-changing environment.  相似文献   

17.
18.
In the unicellular cyanobacterium Synechocystis sp. PCC 6803, the mrgA gene is part of the PerR regulon that is upregulated during peroxide stress. We determined that an Δ mrgA mutant was highly sensitive to low peroxide levels and that the mutant upregulated a gene cluster ( sll1722-26 ) that encoded enzymes involved with exopolymeric substance (EPS) production. We made mutants in this EPS cluster in both a wild type and Δ mrgA background and studied the responses to oxidative stress by measuring cell damage with LIVE/DEAD stain. We show that Synechocystis sp. PCC 6803 becomes highly sensitive to oxidative stress when either mrgA or the sll1722-26 EPS components are deleted. The results suggest that the deletion of the EPS cluster makes a cell highly susceptible to cell damage, under moderate oxidative stress conditions. Mutations in either mrgA or the EPS cluster also result in cells that are more light and peroxide sensitive, and produce significantly less EPS material than in wild type. In this study, we show that in the absence of MrgA, which is known to be involved in the storage or mobilization of iron, cells can be more easily damaged by exogenous oxidative and light stress.  相似文献   

19.
F Rousseau  B Lagoutte 《FEBS letters》1990,260(2):245-248
We describe here the complete amino acid sequence of photosystem I subunit IV from Synechocystis 6803. The molecular mass of 8.0 kDa is lower than in higher plants and Chlamydomonas, due to the lack of a characteristic, proline-rich, N-terminal sequence. The remaining sequence exhibits a good conservation, with a hydrophilic and strongly basic N-tenninal head followed by two hydrophobic domains. There is no possibility of classical membrane-spanning alpha helices. This component is likely to be one of the most stroma accessible subunits of photosystem I.  相似文献   

20.
A method is presented for rapid extraction of the total plastoquinone (PQ) pool from Synechocystis sp. strain PCC 6803 cells that preserves the in vivo plastoquinol (PQH2) to -PQ ratio. Cells were rapidly transferred into ice-cold organic solvent for instantaneous extraction of the cellular PQ plus PQH2 content. After high-performance liquid chromatography fractionation of the organic phase extract, the PQH2 content was quantitatively determined via its fluorescence emission at 330 nm. The in-cell PQH2-PQ ratio then followed from comparison of the PQH2 signal in samples as collected and in an identical sample after complete reduction with sodium borohydride. Prior to PQH2 extraction, cells from steady-state chemostat cultures were exposed to a wide range of physiological conditions, including high/low availability of inorganic carbon, and various actinic illumination conditions. Well-characterized electron-transfer inhibitors were used to generate a reduced or an oxidized PQ pool for reference. The in vivo redox state of the PQ pool was correlated with the results of pulse-amplitude modulation-based chlorophyll a fluorescence emission measurements, oxygen exchange rates, and 77 K fluorescence emission spectra. Our results show that the redox state of the PQ pool of Synechocystis sp. strain PCC 6803 is subject to strict homeostatic control (i.e. regulated between narrow limits), in contrast to the more dynamic chlorophyll a fluorescence signal.The photosynthetic apparatus of oxygenic phototrophs consists of two types of photosynthetic reaction centers: PSII and PSI. Both photosystems are connected in series, with electrons flowing from PSII toward PSI through an intermediate electron transfer chain, which comprises the so-called plastoquinone (PQ) pool, plastocyanin and/or cytochrome c553, and the cytochrome b6f complex. The redox potential of the PQ pool is clamped by the relative rates of electron release into and uptake from this pool. Within the PSII complex, electrons are extracted from water at the lumenal side of the thylakoid membrane and transferred to the primary accepting quinone (QA) at the stromal side. The electron is subsequently transferred to a PQ molecule in the secondary accepting quinone (QB) of PSII. The intermediate QB semiquinone, which is formed accordingly, is stable in the QB site for several seconds (Diner et al., 1991; Mitchell, 1993) and subsequently can be reduced to plastoquinol (PQH2). The midpoint potential of QA reduction is approximately −100 mV (Krieger-Liszkay and Rutherford, 1998; Allakhverdiev et al., 2011), whereas the corresponding midpoint potential of the QB semiquinone is close to zero (Nicholls and Ferguson, 2013). PQH2 equilibrates with the PQ pool in the thylakoid membranes, which has a size that is approximately 1 order of magnitude larger than the number of PSII reaction centers (Melis and Brown, 1980; Aoki and Katoh, 1983).PQ is a lipophilic, membrane-bound electron carrier, with a midpoint potential of +80 mV (Okayama, 1976), that can accept two electrons and two protons to form PQH2 (Rich and Bendall, 1980). PQH2 can donate both electrons to the cytochrome b6f complex, one to low-potential cytochrome b6, by which reduced high-potential cytochrome b6 is formed, and one to the cytochrome f moiety on the lumenal side of the thylakoid membrane, where the two protons are released. High-potential cytochrome b6 then donates an electron back to PQ on the stromal side of the membrane, rendering a semiquinone in the PQ-binding pocket on the cytoplasmic face of the b6f complex ready as an acceptor of another electron from PSII, and reduced cytochrome f feeds an electron to a water-soluble electron carrier (i.e. either plastocyanin or cytochrome c553) for subsequent transfer to the reaction center of PSI or to cytochrome c oxidase, respectively (Rich et al., 1991; Geerts et al., 1994; Schubert et al., 1995; Paumann et al., 2004; Mulkidjanian, 2010).Electron transfer through the cytochrome b6f complex proceeds according to the Q-cycle mechanism (Rich et al., 1991). As a result, maximally two protons from the stroma are released into the lumen per electron transferred. This electrochemical proton gradient can be used for the synthesis of ATP by the ATP synthase complex (Walker, 1998). In PSI, another transthylakoid membrane charge separation process is energized by light. Electron transfer within the PSI complex involves iron-sulfur clusters and quinones and leads to the reduction of ferredoxin, the reduced form of which serves as the electron donor for NADPH by the ferredoxin:NADP+ oxidoreductase enzyme (van Thor et al., 1999). The ATP and NADPH generated this way are used for CO2 fixation in a mutual stoichiometry that is close to the stoichiometry at which these two energy-rich compounds are formed at the thylakoid membrane. Normally, this ratio is ATP:NADPH = 3:2 (Behrenfeld et al., 2008).Photosynthetic and respiratory electron transport in cyanobacteria share a single PQ pool (Aoki and Katoh, 1983; Aoki et al., 1983; Matthijs et al., 1984; Scherer, 1990). Respiratory electron transfer provides cells the ability to form ATP in the dark, but this ability is not limited to those conditions. Transfer of electrons into the PQ pool is the result of the joint activity of PSII, respiratory dehydrogenases [in particular those specific for NAD(P)H and succinate], and cyclic electron transport around PSI (Mi et al., 1995; Cooley et al., 2000; Howitt et al., 2001;Yeremenko et al., 2005), whereas oxidation of PQH2 is catalyzed by the PQH2 oxidase, the cytochrome b6f complex, the respiratory cytochrome c oxidase (Nicholls et al., 1992; Pils and Schmetterer, 2001; Berry et al., 2002), and possibly plasma terminal oxidase (Peltier et al., 2010). Multiples of these partial reactions can proceed simultaneously, including respiratory electron transfer during illumination (Schubert et al., 1995), which includes oxygen uptake through a Mehler-like reaction (Helman et al., 2005; Allahverdiyeva et al., 2013).Because of its central location between the two photosystems, the redox state of the PQ pool has been identified as an important parameter that can signal photosynthetic imbalances (Mullineaux and Allen, 1990; Allen, 1995; Ma et al., 2010; Allen et al., 2011). Yet, an accurate estimation of the in vivo redox state of this pool has not been reported in cyanobacteria so far. Instead, the redox state of the PQ pool is widely assumed to be reflected in, or related to, the intensity of the chlorophyll a fluorescence emissions (Prasil et al., 1996; Yang et al., 2001; Gotoh et al., 2010; Houyoux et al., 2011). Imbalance in electron transport through the two photosystems may lead to a loss of excitation energy and, hence, to a loss of chlorophyll a fluorescence emission (Schreiber et al., 1986). Therefore, patterns of chlorophyll a fluorescence (pulse-amplitude modulated [PAM] fluorimetry; Baker, 2008) have widely been adopted for the analysis of (un)balanced photosynthetic electron transfer and, by inference, for indirect recording of the redox state of the PQ pool. However, the multitude of electron transfer pathways in the thylakoid membranes of cyanobacteria (see above) makes it much more complex to explain PAM signals in these organisms than in chloroplasts (Campbell et al., 1998). Additional regulatory mechanisms of nonphotochemical quenching, via the xanthophyll cycle in chloroplasts (Demmig-Adams et al., 2012) and the orange carotenoid protein (Kirilovsky and Kerfeld, 2012) in cyanobacteria, and energy redistribution via state transitions (Allen, 1995; Van Thor et al., 1998) complicate such comparisons even further.Several years ago, an HPLC-based technique was developed for the detection of the redox state of PQH2 in isolated thylakoids (Kruk and Karpinski, 2006), but these results have neither been related to physiological conditions nor to the results of chlorophyll a fluorescence measurements. In this report, we describe an adaptation of this method with elements of a method for estimation of the redox state of the ubiquinone pool in Escherichia coli (Bekker et al., 2007). This modified method allows for reliable measurements of the redox state of the PQ pool of Synechocystis sp. strain PCC 6803 under physiologically relevant conditions. The method uses rapid cell lysis in an organic solvent to arrest all physiological processes, followed by extraction and identification of PQH2 by HPLC separation with fluorescence detection. Next, we manipulated the redox state of the PQ pool with various redox-active agents, with inhibitors of photosynthetic electron flow, and by illumination with light specific for either PSII or PSI. The measured redox state of the PQ pool was then related to the chlorophyll a fluorescence signal and 77 K fluorescence emission spectra of cell samples taken in parallel and to oxygen-exchange rates measured separately. These experiments reveal that, despite highly fluctuating conditions of photosynthetic and respiratory electron flow, a remarkably stable redox state of the PQ pool is maintained. This homeostatically regulated redox state correlates poorly in many of the conditions tested with the more dynamic signal of chlorophyll a fluorescence emission, as measured with PAM fluorimetry. The latter signal only reflects the redox state of QA and not that of the PQ pool.  相似文献   

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