Effects of Proteolysis on the Photochemical Activity and Polypeptide Composition of the Photosystem II Core Complex Isolated from Spinach Chloroplasts

The photosystem II core complex (TSF-IIa) is composed of polypeptides of molecular weight 54-, 47-, 42-, and 30 kilodaltons (kD) and cytochrome b-559. After treatment with trypsin or α-chymotrypsin for 20 hours, the TSF-IIa particles still retained their photochemical activity and the light-induced cytochrome b-559 signal, although all of the polypeptides of the complexes, except the 30 kD unit were extensively degraded. Proteolytic treatment decreased the apparent molecular weight of the complex from 250,000 to 100,000 daltons as determined by gel filtration, and also decreased the protein to chlorophyll ratio by 40%. Chlorophyll a appeared to be associated with the 47- and 42 kD polypeptides. Proteolysis of the complex produced a single chlorophyll a band with a slightly higher electrophoretic mobility. This band was not equivalent to the 30 kD polypeptide. Proteolysis also reduced the sensitivity of the TSF-IIa particles to 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea (DCMU), but did not completely abolish it.     

Isolation and Characterization of a Light-Harvesting Chlorophyll a/b Protein Complex Associated with Photosystem I

A chlorophyll a/b protein complex has been isolated from a resolved native photosystem I complex by mildly dissociating sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The chlorophyll a/b protein contains a single polypeptide of molecular weight 20 kilodaltons, and has a chlorophyll a/b ratio of 3.5 to 4.0. The visible absorbance spectrum of the chlorophyll a/b protein complex showed a maximum at 667 nanometers in the red region and a 77 K fluorescence emission maximum at 681 nanometers. Alternatively, by treatment of the native photosystem I complex with lithium dodecyl sulfate and Triton, the chlorophyll a/b protein complex could be isolated by chromatography on Sephadex G-75. Immunological assays using antibodies to the P₇₀₀-chlorophyll a-protein and the photosystem II light-harvesting chlorophyll a/b protein show no cross-reaction between the photosystem I chlorophyll a/b protein and the other two chlorophyll-containing protein complexes.     

Inactivation of Photosynthetic Oxygen Evolution and Concomitant Release of Three Polypeptides in the Photosystem II Particles of Spinach Chloroplasts

Photosystem II particles having an oxygen evolution activityas high as 300 µmol mg^–1 chlorophyll hr ^–1were prepared from spinach chloroplasts using Triton X-100.The oxygen evolution system in these particles was stable; 70%of the original activity remained after storage of the particlesat 0?C for 7 days. When the particles were treated at pH 9.3,the oxygen evolution was specifically inactivated and threepolypeptides having apparent molecular weights of 32,000. 24,000and 15,000 were simultaneously released. This observation suggeststhat these polypeptides are associated with the oxygen evolutionsystem of photosynthesis. ¹ Present address: Department of Chemistry, Faculty of Science,Toho University, Miyama 2-2-1, Funabashi, Chiba 274, Japan. (Received January 4, 1982; Accepted February 19, 1982)     

Light-induced Changes of the Electrical Potential in Chloroplasts Associated with the Activity of Photosystem I and Photosystem II

The electric potential changes induced by flashing and continuouslight were measured with microcapillary electrodes in isolatedwhole chloroplasts of Peperomia inetallica. In continuous lightthe chloroplast electrical potential rose in two phases. Theinitial rapid phase coincided in extent with the flash-inducedpotential and was insensitive to the electron transfer inhibitorDBMIB. The subsequent phase was relatively slow (20–30ms) and was inhibited by DBMIB. Electron acceptors of photosystemII (p-phenylendiamine, p-benzoquinone) added to DBMIB-treatedchloroplasts produced a suppression of the flash-induced responseand a considerable increase in the steady level of the potentialin the light. The electrical potential associated with the activityof photosystem II rose in continuous light much more slowlythan that associated with the activity of photosystem I aloneor the activities of both photosystems. Illumination of chloroplastswith successive flashes at a repetition rate 5 Hz in the presenceof oxaloacetate, a terminal acceptor of photosystem I, was accompaniedwith a gradual decline of the flash-induced potential. The specificrole of two photosystems in the light-induced H⁺ transport andthe electrogenesis across the chloroplast thylakoid membranesis discussed.     

Organization and Stability of Polypeptides Associated with the Chlorophyll a-b Light-Harvesting Complex of Photosystem-II

In the oxygen-evolving photosystem-II (PSII) of higher plantchioroplasts and green algae, most of the light-harvesting functionis performed by the chlorophyll (Chl) a-b-protein complex (LHC-II).On the average, the LHC-II contains about 210 Chl (a+b) moleculesper PSII reaction center. The polypeptide composition, copynumber and organization of assembly in the LHC-II complex arenot fully understood at present. This work utilized the chlorinaf2 mutant of barley (lacking Chl b and having a LHC-II antennaof only 13 Chl a molecules) to determine the organization andstability of assembly of proteins in the LHC-II. High-resolutionSDS-PAGE and immunoblot analysis showed the presence of fourmain constitutive polypeptides in the wild-type LHC-II (termedhere subunits a, b, c and d) with molecular masses in the range30–25 kDa. Of those, only subunit d (a 25 kDa polypeptide)was found to occur at an equal copy number per PSII reactioncenter in both wild-type and in the Chl b-less chlorina f2 mutant.All other subunits were either absent or existed in much loweramounts in the mutant. Subunit d is a polypeptide constituentof the major Chl-protein subcomplex (CPII) of the LHC-II. Itis stably incorporated in the thylakoid membrane in the absenceof Chl b and probably binds the 13 Chl a molecules in the residualLHC-II antenna of the chlorina f2 mutant. We propose that, ofall LHC-II polypeptides, subunit d is most proximal to the PSIIcore and may serve as a linker in the process of excitationenergy transfer from the bulk LHC-II to the PSII reaction centerin chloroplasts. (Received February 25, 1992; Accepted May 12, 1992)     

Organization of Photosystem I Polypeptides (A Structural Interaction between the PsaD and PsaL Subunits)

The wild-type, PsaD-less, and PsaL-less strains of the cyanobacterium Synechocystis sp. PCC 6803 were used to study subunit interactions in photosystem I (PSI). When the membranes of a PsaD-less strain were solubilized with Triton X-100 and PSI was purified using ion-exchange chromatography and sucrose-gradient ultracentrifugation, the PsaL subunit was substantially removed from the core of PSI, whereas other subunits, such as PsaE and PsaF, were quantitatively retained during purification. When the wild-type PSI was exposed to increasing concentrations of NaI, the PsaE, PsaD, and PsaC subunits were gradually removed, whereas PsaF, PsaL, PsaK, and PsaJ resisted removal by up to 3 M NaI. The absence of PsaL enhanced the accessibility of PsaD to removal by NaI. Treatment of the wild-type PSI complexes with glutaraldehyde at 4[deg] C resulted in a 29-kD cross-linked product between PsaD and PsaL. The formation of such cross-linked species was independent of PSI concentrations, suggesting an intracomplex cross-linking between PsaD and PsaL. Taken together, these results demonstrate a structural interaction between PsaD and PsaL that plays a role in their association with the PSI core.     

A Fluorescence Detected Magnetic Resonance Investigation of the Carotenoid Triplet States Associated with Photosystem II of Isolated Spinach Thylakoid Membranes

The carotenoid triplet populations associated with the fluorescence emission chlorophyll forms of Photosystem II have been investigated in isolated spinach thylakoid membranes by means of fluorescence detected magnetic resonance in zero field (FDMR). The spectra collected in the 680–690 nm emission range, have been fitted by a global analysis procedure. At least five different carotenoid triplet states coupled to the terminal emitting chlorophyll forms of PS II, peaking at 682 nm, 687 nm and 692 nm, have been characterised. The triplets associated with the outer antenna emission forms, at 682 nm, have zero field splitting parameters |D| = 0.0385 cm⁻¹, |E| = 0.00367 cm⁻¹; |D| = 0.0404 cm⁻¹, |E| = 0.00379 cm⁻¹ and |D| = 0.0386 cm⁻¹, |E| = 0.00406 cm⁻¹ which are very similar to those previously reported for the xanthophylls of the isolated LHC II complex. Therefore the FDMR spectra recorded in this work provide insights into the organisation of the LHC II complex in the unperturbed environment represented by thylakoid membranes. The additional carotenoid triplet populations, detected by monitoring the chlorophyll emission at 687 and 692 nm, are assigned to carotenoids bound to inner antenna complexes and hence attributed to β-carotene molecules.     

FA/FB Protein from the Spinach Photosystem I Complex: Isolation in a Native State and Some Properties of the Iron-Sulfur Clusters

The F_A/F_B protein of the photosystem I complex was isolatedfrom spinach leaves in a native state by use of anaerobic systems.The protein contained 8.5 non-heme iron atoms and 8.0 acid-labilesulfur atoms per molecule, consistent with the current conceptthat it has two [4Fe-4S] clusters. Its absorption spectrum wasvery similar to those of bacterial-type ferredoxins. The ratioof the absorbance at 390 nm to that at 280 nm was 0.6, and themolar extinction coefficient at 390 nm was 32,000 M^–.cm^–.Theoxidation-reduction properties of the iron-sulfur clusters wereexamined by redox potentiometry and EPR spectroscopy. The twoclusters were distinguishable in terms of their oxidation-reductionmidpoint potentials; their E_m values were determined to be about-470mV and-560 mV, respectively. (Received July 16, 1990; Accepted October 8, 1990)     

Synthesis and Assembly of the Polypeptides of Photosystem I and II in Isolated Etiochloroplasts of Wheat

Synthesis and assembly of photosystems (PS) I and II polypeptides in etiochloroplasts isolated from greening wheat (Triticum aestivum L. cv Norin 61) seedlings were studied. The isolated etiochloroplasts synthesized PSI polypeptides of 66 and 15 kilodaltons, PSII polypeptides of 46 and 42 kilodaltons, and atrazine-binding 34 to 32 kilodalton polypeptide. Their assembly processes in the thylakoid membrane were studied by pulse-chase labeling with [³⁵S]methionine, mild solubilization of the thylakoid membrane with Triton X-100, sucrose density gradient centrifugation, and polyacrylamide gel electrophoresis. The newly synthesized polypeptides of 66, 46, 42, 34, and 32 kilodaltons were first integrated into the complexes of 7.5, 5.9, 7.5, 6.3, and 7.5 Svedberg units, respectively, in 20 minutes. After the chase with excess amount of methionine for 100 min, they were found in complexes of 9.5, 9.1, 9.1, 9.1, and 9.1 Svedberg units, respectively. In this condition, stained polypeptides of PSI and PSII were found in the complexes of 11.1 and 10.3 Svedberg units, respectively. These results indicated that newly synthesized PSI or PSII polypeptides are integrated into intermediate complexes, but not complete complexes in the isolated etiochloroplasts. The relationship between the processing of the atrazine-binding 32 kilodalton polypeptide and its assembly into the PSII complex is also discussed.     

Quantitative Analysis of the Inactivation of Photosynthetic Oxygen Evolution and the Release of Polypeptides and Manganese in the Photosystem II Particles of Spinach Chloroplasts

Photosynthetic oxygen evolution by photosystem II particleswas inactivated by treatment with NaCl, NH₂OH or high pH. Whenthe degree of inactivation was compared with the degree of releasefrom the particles of Mn and three polypeptides having molecularmasses of 33, 24 and 18kdaltons, two types of inactivation werefound: one, brought about with 960 mM NaCl, was related to therelease of the 24 kdalton polypeptide, and the other, broughtabout with 1.5 mM NH₂OH or high pH, seemed to be related tothe release of Mn. ¹Present address: Department of Chemistry, Faculty of Science,Toho University, Miyama 2-2-1, Funabashi 274, Japan. (Received January 31, 1983; Accepted March 28, 1983)     

Diversity of Bacterial Communities Associated with the Indian Ocean Sponge Tsitsikamma favus That Contains the Bioactive Pyrroloiminoquinones, Tsitsikammamine A and B

Tsitsikamma favus is a latrunculid sponge endemic to the coast of South Africa that produces unique pyrroloiminoquinones known as tsitsikammamines. Wakayin and makaluvamine A are structurally similar to the tsitsikammamines and are the only pyrroloiminoquinones isolated from a source other than Porifera (namely a Fijian ascidian Clavelina sp. and a laboratory culture of the myxomycete Didymium bahiense, respectively). The source of the tsitsikammamines is hypothesised to be microbial, which could provide a means of overcoming the current supply problem. This study focuses on characterising the microbial diversity associated with T. favus. We have used denaturing gradient gel electrophoresis together with clonal and deep sequencing of microbial 16S rRNA gene amplicons to show that specimens of this sponge species contain a distinct and conserved microbial population, which is stable over time and is dominated by a unique Betaproteobacterium species.     

Variation and Estimation of the Differential Absorption Coefficient of P-700 in Spinach Photosystem I Preparations

Treatment of spinach Photosystem I particles with detergentsinduced an apparent blue shift of chlorophyll at 690 nm inthe difference spectrum of P-700. As a consequence of the bandshift, the differential absorption coefficient of P-700 wasincreased from 63 to 87 mM^–1 .cm^–1. A curve waspresented, with which changes in the apparent differential absorptioncoefficient of P-700 can be estimated by measuring magnitudesof light-induced absorption decreases at 680 nm and 700 nm.The curve was compared with that for cyanobacterial preparationsand the mechanism of the detergent-induced band shift was discussed. (Received July 11, 1990; Accepted August 23, 1990)     

Polypeptide Composition of Influenza B Viruses and Enzymes Associated with the Purified Virus Particles

Influenza B/LEE/40, B/Rome/1/67, B/Hong Kong/8/73, and B/Victoria/98926/70 viruses have a similar polypeptide composition as analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. These viruses are composed of six or seven polypeptides, depending on whether one or two high-molecular-weight polypeptides are resolved, ranging in molecular weights from 27,000 to 90,400. Three of these polypeptides, namely the heavy and light hemagglutinin chains and the neuraminidase, have attached carbohydrate. Highly purified influenza B/LEE/40 and B/Rome/1/67 virus preparations have RNA-dependent RNA polymerase activity equivalent to the incorporation of 100 and 30 pmol, respectively, of (3)H-UMP per mg of virus protein per h at 37 C, which is demonstrated only in detergent-treated virus suspensions. However, no RNA-dependent DNA polymerase enzyme activity was detected in the two viruses although virus suspensions were "activated" by heat, alpha-chymotrypsin, and detergents. Other enzymatic activities were associated with purified preparations of influenza B virus and were attributed to minor contamination of virus with host cell enzymes. Thus, nucleoside and deoxynucleoside phosphohydrolase enzymes were active in the absence of detergents and catalyzed the release of 1,200 and 1,800 nmol of P(i) per mg of virus protein in 30 min at 37 C from ATP and dATP substrates. Thin-layer chromatography indicated that the products of the phosphohydrolase enzymes of influenza B/LEE/40 were mainly nucleoside diphosphate and monophosphate. The latter enzymes were tightly bound to influenza B/LEE/40 virus and could not be removed completely by repeated centrifugation, including centrifugation of the virus to equilibrium in density gradients of 25 to 40% (wt/vol) cesium chloride. A low degree of RNase (approximately 0.01 mug% contamination) and phosphatase (10-30 nmol of P(i) released per mg of virus protein per 30 min) activity was detected in some, but not all, influenza B/LEE/40 virus preparations.     

The detection,isolation and characterization of a light-harvesting complex which is specifically associated with Photosystem I

10% of the chlorophyll associated with a ‘native’ Photosystem (PS) I complex (110 chlorophylls/P-700) is chlorophyll (Chl) b. The Chl b is associated with a specific PS I antenna complex which we designate as LHC-I (i.e., a light-harvesting complex serving PS I). When the native PS I complex is degraded to the core complex by LHC-I extraction, there is a parallel loss of Chl b, fluorescence at 735 nm, together with 647 and 686 nm circular dichroism spectral properties, as well as a group of polypeptides of 24-19 kDa. In this paper we present a method by which the LHC-I complex can be dissociated from the native PS I. The isolated LHC-I contains significant amounts of Chl b (Chl $\text{a}\text{b ? 3.7}$). The long-wavelength fluorescence at 730 nm and circular dichroism signal at 686 nm observed in native PS I are maintained in this isolated complex. This isolated fraction also contains the low molecular weight polypeptides lost in the preparation of PS I core complex. We conclude that we have isolated the PS I antenna in an intact state and discuss its in vivo function.     

Effect of temperature on the photoreduction of centres A and B in Photosystem I,and the kinetics of recombination

When spinach Photosystem I particles, frozen in the dark with ascorbate, are illuminated at low temperatures, one electron is transferred from P-700 to either iron-sulphur centre A or B. It was found that the proportion of centre A or B reduced depended on the temperature of illumination. At 25 K, reduction of centre A, as detected by ESR spectroscopy, was strongly preferred. At higher temperatures, at about 150K, there was an increased proportion of reduced centre B. Reduction of B was more strongly preferred in particles frozen in 50% glycerol. The kinetics of dark reoxidation of A^? and B^? at various temperatures were followed by observing the radical signal of P-700⁺, and also by periodically cooling to 25 K to measure the ESR spectra of the iron-sulphur centres. The recombination of A^? and P-700⁺ occurred at lower temperatures than that at of B^?; at 150–200 K, centre B was the more stable electron trap. Dark reoxidation of both centres was more rapid in samples that were illuminated at 25 K than in samples illuminated at 150–215 K. In no case was net electron transfer between centres A and B observed. Differences in g values of the ESR spectra in particles illuminated at 25 and 200 K indicate that the iron-sulphur centres are in altered conformational states. It is concluded firstly that, in the frozen state, the rates of dark electron transfer decrease in the sequence A^? → P-700⁺ > B^? → P-700⁺ > B^? → A; secondly, that when centres A or B are photoreduced, a temperature-dependent conformational change takes place which slows down the rate of recombination with P-700⁺.     

The role of the membrane-bound iron-sulphur centres A and B in the Photosystem I reaction centre of spinach chloroplasts

Photosystem I particles prepared from spinach chloroplast using Triton X-100 were frozen in the dark with the bound iron-sulphur Centre A reduced. Illumination at cryogenic temperatures of such samples demonstrated the photoreduction of the second bound iron-sulphur Centre B. Due to electron spin-electron spin interaction between these two bound iron-sulphur centres, it was not possible to quantify amounts of Centre B relative to the other components of the Photosystem I reaction centre by simulating the line-shape of its EPR spectrum. However, by deleting the free radical signal I from the EPR spectra of reduced Centre A alone or both Centres A plus B reduced, it was possible to double integrate these spectra to demonstrate that Centre B is present in the Photosystem I reaction centre in amounts comparable to those of Centre A and thus also signal I ($\text{P-700}$) and X.Oxidation-reduction potential titrations confirmed that Centre A had $\text{E}{}_{\mathrm{<mtext>m</mtext>}}\text{? ?550}\text{mV}$, Centre B had $\text{E}{}_{\mathrm{<mtext>m</mtext>}}\text{? ?585}\text{mV}$. These results, and those presented for the photoreduction of Centre B, place Centre B before Centre A in the sequence of electron transport in Photosystem I particles at cryogenic temperatures. When both A and B are reduced, $\text{P-700}$ photooxidation is reversible at low temperature and coupled to the reduction of the component X. The change from irreversible to reversible $\text{P-700}$ photooxidation and the photoreduction of X showed the same potential dependence as the reduction of Centre B with $\text{E}{}_{\mathrm{<mtext>m</mtext>}}\text{? ?585}$ mV, substantiating the identification of X as the primary electron acceptor of Photosystem I.