Thylakoid Membrane Protein Complexes Research Articles

Dark-adapted spinach thylakoid protein heterogeneity offers insights into the photosystem II repair cycle

In higher plants, thylakoid membrane protein complexes show lateral heterogeneity in their distribution: photosystem (PS) II complexes are mostly located in grana stacks, whereas PSI and adenosine triphosphate (ATP) synthase are mostly found in the stroma-exposed thylakoids. However, recent research has revealed strong dynamics in distribution of photosystems and their light harvesting antenna along the thylakoid membrane. Here, the dark-adapted spinach (Spinacia oleracea L.) thylakoid network was mechanically fragmented and the composition of distinct PSII-related proteins in various thylakoid subdomains was analyzed in order to get more insights into the composition and localization of various PSII subcomplexes and auxiliary proteins during the PSII repair cycle. Most of the PSII subunits followed rather equal distribution with roughly 70% of the proteins located collectively in the grana thylakoids and grana margins; however, the low molecular mass subunits PsbW and PsbX as well as the PsbS proteins were found to be more exclusively located in grana thylakoids. The auxiliary proteins assisting in repair cycle of PSII were mostly located in stroma-exposed thylakoids, with the exception of THYLAKOID LUMEN PROTEIN OF 18.3 (TLP18.3), which was more evenly distributed between the grana and stroma thylakoids. The TL29 protein was present exclusively in grana thylakoids. Intriguingly, PROTON GRADIENT REGULATION5 (PGR5) was found to be distributed quite evenly between grana and stroma thylakoids, whereas PGR5-LIKE PHOTOSYNTHETIC PHENOTYPE1 (PGRL1) was highly enriched in the stroma thylakoids and practically missing from the grana cores. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.

Organization and functionality of chlorophyll–protein complexes in thylakoid membranes isolated from Pb-treated Secale cereale

In this study, Secale cereale seedlings cultivated under 0 (control), 2 or 5mM Pb(NO3)2 concentrations were used to examine alterations in the organization and functionality of chlorophyll–protein complexes in thylakoid membranes under Pb ion stress. The studies were conducted on whole leaves of rye seedlings or thylakoid membranes isolated from Pb-treated and control plants. Using non-denaturing electrophoresis, it was assessed that increasing Pb concentrations resulted in an increase in the value of the ratio of the content of LHCII oligomers (mainly trimers) to the content of LHCII monomers. The parameters of chlorophyll fluorescence induction (qp and qn) indicated that the change in the LHCII supramolecular organization in the presence of Pb ions was connected with an increase in non-photochemical fluorescence quenching. Quantification of photosynthetic pigments showed that both Pb concentrations decreased the content of chlorophyll a, chlorophyll b, and carotenoids. The changes in the pigment content led to a significant reduction in light absorption by antenna complexes. However, the absorption spectra showed that red light was preferentially absorbed by antenna complexes in thylakoid membranes isolated from the Pb-treated plants. Examination of fluorescence emission spectra revealed that Pb ions decreased the fluorescence quantum yield of PSII. The emission spectra of thylakoids indicated a relative increase in the intensity of fluorescence emission from the trimeric and aggregated forms of the LHCII complexes in comparison to the intensity of fluorescence emission from PSI antenna complexes under excitation at 440nm. Simultaneously, under excitation at 470nm, we observed a rise in fluorescence intensity from the LHCII trimer after addition of 5mM Pb as well as a decrease in fluorescence intensity from the LHCII aggregates and PSI core and LHCI antenna complexes under both Pb concentrations. Pb treatments also reduced excitation energy absorbed by chlorophyll b and carotenoids within antenna complexes and transferred to chlorophyll a species emitting at 680nm.

Changes of Photosynthetic Membrane Function and Protein Complexes in Flag Leaves of Liangyoupeijiu during Leaf Senescence

The purpose of this study was to explore the content variation of thylakoid membrane protein complexes and its relationship with light absorption,transformation and transfer in flag leaves of high-yield hybrid rice Liangyoupeijiu during senescence.A rice cultivar,Liangyoupeijiu was grown in the field.Through chlorophyll a fluorescence transient,physiological and biochemical techniques,we studied the function of photosynthetic membrane.The content changes of thylakoid membrane protein complexes were also investigated by Blue-native polyacrylamide gel-electrophoresis(BN-PAGE).The results showed that chlorophyll content,photosynthesis and thylakoid membrane protein content of flag leaves rose and peaked at heading stage,then began to decline.It still maintained a high level at flowering and filling stages,and the significant decline appeared untill the stage of milky and wax ripeness.The stability of protein complexes(i.e.inverse degradation rate)during leaf senescence showed an order of LHCII PSIIcore PSIcore ATPase Cyt b6/f LHCI.Protein complexes and electron transport activity of PSI declined later but more quickly than those of PSII.The decreased chlorophyll a/b ratio could be explained by the relative enrichment of light-harvesting antenna to reaction center with leaf senescence.The stable LHCII kept the high level of absorbing energy in the late of flag leaf growth,which may play an important role in adjusting the energy distribution and dissipation during leaf senescence.

Regulatory factors for the assembly of thylakoid membrane protein complexes

Major multi-protein photosynthetic complexes, located in thylakoid membranes, are responsible for the capture of light and its conversion into chemical energy in oxygenic photosynthetic organisms. Although the structures and functions of these photosynthetic complexes have been explored, the molecular mechanisms underlying their assembly remain elusive. In this review, we summarize current knowledge of the regulatory components involved in the assembly of thylakoid membrane protein complexes in photosynthetic organisms. Many of the known regulatory factors are conserved between prokaryotes and eukaryotes, whereas others appear to be newly evolved or to have expanded predominantly in eukaryotes. Their specific features and fundamental differences in cyanobacteria, green algae and land plants are discussed.

Optimized native gel systems for separation of thylakoid protein complexes: novel super- and mega-complexes

Gel-based analysis of thylakoid membrane protein complexes represents a valuable tool to monitor the dynamics of the photosynthetic machinery. Native-PAGE preserves the components and often also the conformation of the protein complexes, thus enabling the analysis of their subunit composition. Nevertheless, the literature and practical experimentation in the field sometimes raise confusion owing to a great variety of native-PAGE and thylakoid-solubilization systems. In the present paper, we describe optimized methods for separation of higher plant thylakoid membrane protein complexes by native-PAGE addressing particularly: (i) the use of detergent; (ii) the use of solubilization buffer; and (iii) the gel electrophoresis method. Special attention is paid to separation of high-molecular-mass thylakoid membrane super- and mega-complexes from Arabidopsis thaliana leaves. Several novel super- and mega-complexes including PS (photosystem) I, PSII and LHCs (light-harvesting complexes) in various combinations are reported.

A Small Zinc Finger Thylakoid Protein Plays a Role in Maintenance of Photosystem II in Arabidopsis thaliana

This work identifies LOW QUANTUM YIELD OF PHOTOSYSTEM II1 (LQY1), a Zn finger protein that shows disulfide isomerase activity, interacts with the photosystem II (PSII) core complex, and may act in repair of photodamaged PSII complexes. Two mutants of an unannotated small Zn finger containing a thylakoid membrane protein of Arabidopsis thaliana (At1g75690; LQY1) were found to have a lower quantum yield of PSII photochemistry and reduced PSII electron transport rate following high-light treatment. The mutants dissipate more excess excitation energy via nonphotochemical pathways than wild type, and they also display elevated accumulation of reactive oxygen species under high light. After high-light treatment, the mutants have less PSII-light-harvesting complex II supercomplex than wild-type plants. Analysis of thylakoid membrane protein complexes showed that wild-type LQY1 protein comigrates with the PSII core monomer and the CP43-less PSII monomer (a marker for ongoing PSII repair and reassembly). PSII repair and reassembly involve the breakage and formation of disulfide bonds among PSII proteins. Interestingly, the recombinant LQY1 protein demonstrates a protein disulfide isomerase activity. LQY1 is more abundant in stroma-exposed thylakoids, where key steps of PSII repair and reassembly take place. The absence of the LQY1 protein accelerates turnover and synthesis of PSII reaction center protein D1. These results suggest that the LQY1 protein may be involved in maintaining PSII activity under high light by regulating repair and reassembly of PSII complexes.

Comparative analysis of leaf‐type ferredoxin‐NADP+ oxidoreductase isoforms in Arabidopsis thaliana

Physiological roles of the two distinct chloroplast-targeted ferredoxin-NADP(+) oxidoreductase (FNR) isoforms in Arabidopsis thaliana were studied using T-DNA insertion line fnr1 and RNAi line fnr2. In fnr2 FNR1 was present both as a thylakoid membrane-bound form and as a soluble protein, whereas in fnr1 the FNR2 protein existed solely in soluble form in the stroma. The fnr2 plants resembled fnr1 in having downregulated photosynthetic properties, expressed as low chlorophyll content, low accumulation of photosynthetic thylakoid proteins and reduced carbon fixation rate when compared with wild type (WT). Under standard growth conditions the level of F(0)'rise' and the amplitude of the thermoluminescence afterglow (AG) band, shown to correlate with cyclic electron transfer (CET), were reduced in both fnr mutants. In contrast, when plants were grown under low temperatures, both fnr mutants showed an enhanced rate of CET when compared with the WT. These data exclude the possibility that distinct FNR isoforms feed electrons to specific CET pathways. Nevertheless, the fnr2 mutants had a distinct phenotype upon growth at low temperature. The fnr2 plants grown at low temperature were more tolerant against methyl viologen (MV)-induced cell death than fnr1 and WT. The unique tolerance of fnr2 plants grown at low temperature to oxidative stress correlated with an increased level of reduced ascorbate and reactive oxygen species (ROS) scavenging enzymes, as well as with a scarcity in the accumulation of thylakoid membrane protein complexes, as compared with fnr1 and WT. These results emphasize a critical role for FNR2 in the redistribution of electrons to various reducing pathways, upon conditions that modify the photosynthetic capacity of the plant.

Novel Nuclear-encoded Subunits of the Chloroplast NAD(P)H Dehydrogenase Complex

The NAD(P)H dehydrogenase (NDH) complex functions in photosystem I cyclic electron transfer in higher plant chloroplasts and is crucial for plant responses to environmental stress. Chloroplast NDH complex is a close relative to cyanobacterial NDH-1L complex, and all fifteen subunits so far identified in NDH-1L have homologs in the chloroplast NDH complex. Here we report on the identification of two nuclear-encoded proteins NDH48 and NDH45 in higher plant chloroplasts and show their intimate association with the NDH complex. These two membrane proteins are shown to interact with each other and with the NDH complex enriched in stroma thylakoids. Moreover, the deficiency of either the NDH45 protein or the NDH48 protein in respective mutant plants leads to severe defects in both the accumulation and the function of the NDH complex. The NDH48 and NDH45 proteins are not components of the hydrophilic connecting domain of the NDH complex but are strongly attached to the hydrophobic membrane domain. We conclude that NDH48 and NDH45 are novel nuclear-encoded subunits of the chloroplast NDH complex and crucial both for the stable structure and function of the NDH complex.

Intrinsically Unstructured Phosphoprotein TSP9 Regulates Light Harvesting in Arabidopsis thaliana

Thylakoid-soluble phosphoprotein of 9 kDa, TSP9, is an intrinsically unstructured plant-specific protein [Song, J., et al. (2006) Biochemistry 45, 15633-15643] with unknown function but established associations with light-harvesting proteins and peripheries of both photosystems [Hansson, M., et al. (2007) J. Biol. Chem. 282, 16214-16222]. To investigate the function of this protein, we used a combination of reverse genetics and biochemical and fluorescence measurement methods in Arabidopsis thaliana. Differential gene expression analysis of plants with a T-DNA insertion in the TSP9 gene using an array of 24000 Arabidopsis genes revealed disappearance of high light-dependent induction of a specific set of mostly signaling and unknown proteins. TSP9-deficient plants had reduced levels of in vivo phosphorylation of light-harvesting complex II polypeptides. Recombinant TSP9 was phosphorylated in light by thylakoid membranes isolated from the wild-type and mutant plants lacking STN8 protein kinase but not by the thylakoids deficient in STN7 kinase, essential for photosynthetic state transitions. TSP9-lacking mutant and RNAi plants with downregulation of TSP9 showed reduced ability to perform state transitions. The nonphotochemical quenching of chlorophyll fluorescence at high light intensities was also less efficient in the mutant compared to wild-type plants. Blue native electrophoresis of thylakoid membrane protein complexes revealed that TSP9 deficiency increased relative stability of photosystem II dimers and supercomplexes. It is concluded that TSP9 regulates plant light harvesting acting as a membrane-binding protein facilitating dissociation of light-harvesting proteins from photosystem II.

Spatial location of photosystem pigment–protein complexes in thylakoid membranes of chloroplasts of Pisum sativum studied by chlorophyll fluorescence

Ultrastructure of plant chloroplasts was studied by a single-molecule spectroscopy setup at a temperature of 77 K exploring spatial location of photosystems. Two chloroplast thylakoid membrane regions were visualized by fluorescence microscopy and detected at different wavelengths. The size of these regions and the spatial resolution of the microscope allowed us to measure their chlorophyll fluorescence emission spectra of these membrane domains. While the grana regions are characterized by a predominant presence of Photosystem II pigment–protein complexes emitting at 685 nm, Photosystem I complexes are localized in stroma regions and emit at 730 nm.

Kinetic model for electron and proton transport in chloroplasts with a nonuniform distribution of protein complexes in thylakoid membranes

A mathematical model for describing electron and proton transport in chloroplasts of higher plants with nonuniform distribution of photosystem 1 (PS1) and photosystem 2 (PS2) between granal and stromal thylakoids was proposed. Along with the noncyclic (linear) electron transport from PS2 to terminal acceptor PS1, we considered cyclic electron transport around PS1, ATP synthesis coupled to transmembrane proton transfer through ATP synthase, and ATP consumption in the Calvin cycle. Numerical simulations of the kinetics of the photoinduced redox transformations of the primary PS1 donor (P700) and the behavior of pH in the intrathylakoid space (pHi) and interthylakoid space (pH0), as well as the time evolution of the ATP concentration for various conditions of the functioning of the chloroplast were performed.

Mobilization of Photosystem II Induced by Intense Red Light in the Cyanobacterium Synechococcus sp PCC7942

We use confocal fluorescence microscopy and fluorescence recovery after photobleaching to show that a specific light signal controls the diffusion of a protein complex in thylakoid membranes of the cyanobacterium Synechococcus sp PCC7942 in vivo. In low light, photosystem II appears completely immobile in the membrane. However, exposure to intense red light triggers rapid diffusion of up to approximately 50% of photosystem II reaction centers. Particularly intense or prolonged red light exposure also leads to the redistribution of photosystem II to specific zones within the thylakoid membranes. The mobilization does not result from photodamage but is triggered by a specific red light signal. We show that mobilization of photosystem II is required for the rapid initiation of recovery from photoinhibition. Thus, intense red light triggers a switch from a static to a dynamic configuration of thylakoid membrane protein complexes, and this facilitates the rapid turnover and repair of the complexes. The localized concentrations of photosystem II seen after red light treatment may correspond to specific zones where the repair cycle is active.

Light-dependent reversal of dark-chilling induced changes in chloroplast structure and arrangement of chlorophyll–protein complexes in bean thylakoid membranes

Changes in chloroplast structure and rearrangement of chlorophyll–protein (CP) complexes were investigated in detached leaves of bean ( Phaseolus vulgaris L. cv. Eureka), a chilling-sensitive plant, during 5-day dark-chilling at 1 °C and subsequent 3-h photoactivation under white light (200 μmol photons m −2 s −1) at 22 °C. Although, no change in chlorophyll (Chl) content and Chl a/ b ratio in all samples was observed, overall fluorescence intensity of fluorescence emission and excitation spectra of thylakoid membranes isolated from dark-chilled leaves decreased to about 50%, and remained after photoactivation at 70% of that of the control sample. Concomitantly, the ratio between fluorescence intensities of PSI and PSII (F736/F681) at 120 K increased 1.5-fold upon chilling, and was fully reversed after photoactivation. Moreover, chilling stress seems to induce a decrease of the relative contribution of LHCII fluorescence to the thylakoid emission spectra at 120 K, and an increase of that from LHCI and PSI, correlated with a decrease of stability of LHCI–PSI and LHCII trimers, shown by mild-denaturing electrophoresis. These effects were reversed to a large extent after photoactivation, with the exception of LHCII, which remained partly in the aggregated form. In view of these data, it is likely that dark-chilling stress induces partial disassembly of CP complexes, not completely restorable upon photoactivation. These data are further supported by confocal laser scanning fluorescence microscopy, which showed that regular grana arrangement observed in chloroplasts isolated from control leaves was destroyed by dark-chilling stress, and was partially reconstructed after photoactivation. In line with this, Chl a fluorescence spectra of leaf discs demonstrated that dark-chilling caused a decrease of the quantum yield PSII photochemistry ( F v/ F m) by almost 40% in 5 days. Complete restoration of the photochemical activity of PSII required 9 h post-chilling photoactivation, while only 3 h were needed to reconstruct thylakoid membrane organization and chloroplast structure. The latter demonstrated that the long-term dark-chilled bean leaves started to suffer from photoinhibition after transfer to moderate irradiance and temperature conditions, delaying the recovery of PSII photochemistry, independently of photo-induced reconstruction of PSII complexes.

Dynamics of photosystem II: a proteomic approach to thylakoid protein complexes

Oxygenic photosynthesis produces various radicals and active oxygen species with harmful effects on photosystem II (PSII). Such photodamage occurs at all light intensities. Damaged PSII centres, however, do not usually accumulate in the thylakoid membrane due to a rapid and efficient repair mechanism. The excellent design of PSII gives protection to most of the protein components and the damage is most often targeted only to the reaction centre D1 protein. Repair of PSII via turnover of the damaged protein subunits is a complex process involving (i) highly regulated reversible phosphorylation of several PSII core subunits, (ii) monomerization and migration of the PSII core from the grana to the stroma lamellae, (iii) partial disassembly of the PSII core monomer, (iv) highly specific proteolysis of the damaged proteins, and finally (v) a multi-step replacement of the damaged proteins with de novo synthesized copies followed by (vi) the reassembly, dimerization, and photoactivation of the PSII complexes. These processes will shortly be reviewed paying particular attention to the damage, turnover, and assembly of the PSII complex in grana and stroma thylakoids during the photoinhibition-repair cycle of PSII. Moreover, a two-dimensional Blue-native gel map of thylakoid membrane protein complexes, and their modification in the grana and stroma lamellae during a high-light treatment, is presented.

Differential changes in the steady state levels of thylakoid membrane proteins during senescence in Cucumis sativus cotyledons.

Chloroplast structure and function is known to alter during foliar senescence. Besides, the alterations in the structural organisation of thylakoid membranes changes in the steady state levels of thylakoid membrane proteins occur due to leaf ageing. We monitored temporal changes in some of the specific proteins of thylakoid membrane protein complexes by western blotting in the Cucumis sativus cotyledons as a function of the cotyledon age. We observed that the levels of D1 and D2 proteins of photosystem II started declining at the early stages of senescence of Cucumis cotyledons and continued to decline with the progress of cotyledon age. Similarly the level of Cyt f of Cyt b6/f complex declined rapidly with progress of senescence in these cotyledons. The reaction centre proteins of photosystem I were relatively found to be more stable than that of photosystem II reaction centre proteins reflecting possibly the disorganisation of photosystem II prior to photosystem I. The 33 kDa extrinsic protein (MSP) of oxygen evolving complex, the LHCII apoprotein and the beta-subunit of ATPsynthase showed the declined levels with the progress of cotyledon age. However, the extents of loss of these proteins were not as high as the reaction centre proteins of photosystem II and the Cyt f. These results provide that during senescence, proteins of thylakoid membranes degrade in a specific temporal sequence and thereby affect the temporal photochemical functions in Cucumis sativus cotyledons.

Requirement of phosphatidylglycerol for photosynthetic function in thylakoid membranes.

To investigate the role of phosphatidylglycerol (PG) in photosynthesis, we constructed a mutant defective in the CDP-diacylglycerol synthase gene from a cyanobacterium, Synechocystis sp. PCC6803. The mutant, designated as SNC1, required PG supplementation for growth. Growth was repressed in PG-free medium concomitantly with the decrease in cellular content of PG. These results indicate that PG is essential, and that SNC1 is defective in PG synthesis. Decrease in PG content was accompanied by a reduction in the cellular content of chlorophyll, but with little effect on the contents of phycobilisome pigments, which showed that levels of chlorophyll-protein complexes decreased without alteration of those of phycobilisomes. Regardless of the decrease in the PG content, CO(2)-dependent photosynthesis by SNC1 was similar to that by the wild type on a chlorophyll basis, but consequently became lower on a cell basis. Simultaneously, the ratio of oxygen evolution of photosystem II (PSII) measured with p-benzoquinone to that of CO(2)-dependent photosynthesis, which ranged between 1.3 and 1.7 in the wild type. However, it was decreased in SNC1 from 1.3 to 0.4 during the early growth phase where chlorophyll content and CO(2)-dependent photosynthesis were little affected, and then finally to 0.1, suggesting that PSII first lost its ability to reduce p-benzoquinone and then decreased in its level and actual activity. These results indicate that PG contributes to the accumulation of chlorophyll-protein complexes in thylakoid membranes, and also to normal functioning of PSII.

Physiological effects of sublethal atrazine on barley chloroplast thylakoid membranes

This study was conducted to more clearly define the physiological effects of PS II herbicides on chloroplast thylakoid membrane activity and composition. Barley (Hordeum vulgare L. cv Boone) was grown in hydroponic culture at 20°C in a growth chamber with a light intensity of 500 μmole photons m(-2) s(-1). Atrazine (2-chloro-4-ethylamino-6-isopropylamino-s-triazine), a Photosystem II herbicide, was supplied continuously via the roots to 7-day-old plants. Atrazine concentrations greater than 0.07 ppm (0.32 μM) were associated with decreased leaf chlorophyll (chl), lowered chl a/b ratio, inhibition of chloroplast electron transport, and plant death within 1 to 2 weeks. Atrazine at 0.07 ppm was defined as sublethal because no toxic effects were observed. Sublethal atrazine induced a decrease in chl a/b ratio with no effect on leaf chl content. Photosynthetic electron transport was either unaffected in fully expanded leaves or slightly stimulated in expanding leaves by treatment of intact plants with 0.07 ppm atrazine. The major effect of sublethal atrazine was on the chl-protein complex composition. Sublethal atrazine increased the level of the Photosystem II light-harvesting complex (LHC-II) and lowered the level of the CP1a Photosystem I complex relative to controls. The numbers of Photosystem II and Photosystem I reaction centers and cytochrome b 6/f complexes per unit chl were not affected by sublethal atrazine. The overall result was an atrazine-induced redistribution of light-harvesting chl from Photosystem I to Photosystem II with no effect on the number of thylakoid membrane-protein complexes associated with electron transport.

Molecular cloning of a maize gene involved in photosynthetic membrane organization that is regulated by Robertson's Mutator.

The maize photosynthetic mutant hcf106 has a distinctive and unusual thylakoid membrane organization, and fails to accumulate three of the five thylakoid membrane protein complexes. This mutant arose in a Robertson's Mutator background, and shows somatic instability typical of a transposon-induced allele. In addition, hcf106 is suppressed when Mu1 elements are inactive and modified in their terminal inverted repeats. Thus plants homozygous for the mutant allele adopt a mutant phenotype only when Mu1 elements are active and unmodified. DNA from the mutant allele has been cloned by 'transposon-tagging' using the transposon Mu1, and the identity of the clone confirmed by observing somatic excision of the transposon in a revertant sector. A 1.2 kb transcript homologous to the cloned DNA is found in wild-type and suppressed seedlings, but is not found in mutant seedlings, suggesting that suppression is mediated at the level of transcript accumulation.

86] Isolation and characterization of genes for cytochrome [formula omitted] complex

This chapter describes procedures used for the isolation and preliminary characterization of genes encoding the cytochrome b 6 /f complex from the filamentous cyanobacterium Nostoc PCC 7906. The proton-translocating thylakoid membrane cytochrome b 6 /f complex catalyzes electron transfer between the two photosystems (PS) and is required for cyclic electron flow around photosystem I (PSI). In cyanobacteria as in plant chloroplasts, the complex consists of four essential polypeptides: cytochromes f and b 6 , the Rieske Fe-S protein, and a subunit IV. In the cyanobacterium Anabaena these have apparent molecular weights of 31,000, 22,500, 22,000 and 16,000, respectively. Cloned genes can now be introduced by transformation or conjugation into several strains of cyanobacteria enabling, among other applications, a molecular genetic analysis of the structure and function of thylakoid membrane protein complexes. An additional requirement for this approach is the availability of cloned genes for these proteins and the determination of their nucleotide and derived amino acid sequences. Heterologous hybridization (the use of a gene from one source as a probe for isolating a gene from another source) was the approach first used to isolate cyanobacterial genes and perhaps remains the most widely used one. Its success ultimately depends on the level of nucleotide sequence similarity between the probe and the target DNA.

Characterization of electron transfer from water to plastocyanin catalyzed by resolved electron transfer complexes from chloroplasts

The photoreduction of soluble plastocyanin in a homologous reconstituted system requires a resolved Photosystem II preparation and the cytochrome b 6- f complex from spinach chloroplasts. Oxygen evolution is linked quantitatively to plastocyanin reduction in the reconstituted system, indicating that water is the electron donor for the photoreduction. The photoreduction is sensitive to inhibitors which specifically interact with the Photosystem II preparation [DCMU: 3-(3,4-dichlorophenyl)-1,1-dimethylurea] as well as those which interact with the cytochrome complex (DNP-INT: 2-iodo-6-isopropyl-3-methyl-2′,4,4′-trinitrodiphenyl ether; and DBMIB: 2,5-dibromo-3-methyl-6-isopropyl- p-benzoquinone). The plastocyanin requirement in the reconstituted system indicated a K m of 4.6 μ m and a V max of 4.2 μmol plastocyanin reduced per nmol cytochrome f per h. Inhibition curves for DNP-INT and DBMIB show that both are effective inhibitors (50% inhibition at a ratio of inhibitor to ctyochrome f of 1.0). The rate of reduction of plastocyanin was found to be stimulated by cations. This dependency was localized in the transfer of electrons from the Photosystem II preparation to the cytochrome complex. These results are considered in terms of organizational aspects of integral protein complexes in thylakoid membranes.