Photosynthetic vesicles with bound phycobilisomes from Anabaena variabilis

Photosynthetically active vesicles with attached phycobilisomes from Anabaena variabilis, were isolated and shown to transfer excitation energy from phycobiliproteins to F696 chlorophyll (Photosystem II). The best results were obtained when cells were disrupted in a sucrose/phosphate/citrate mixture (0.3 : 0.5 : 0.3 M, respectiely) containing 1.5% serum albumin. The vesicles showed a phycocyanin/chlorophyll ratio essentially identical to that of whole cells, and oxygen evolution rates of 250 μmol O₂/h per mg chlorophyll (with 4 mM ferricyanide added as oxidant), whereas whole cells had rates of up to 450. Excitation of the vesicles by 600 nm light produced fluorescence peaks (?196°C) at 644, 662, 685, 695, and 730 nm. On aging of the vesicles, or upon dilution, the fluorescence yield of the 695 nm emission peak gradually decreased with an accompanying increase and final predominant peak at 685 nm. This shift was accompanied by a decrease in the quantum efficiency of Photosystem II activity from an initial 0.05 to as low as 0.01 mol O₂/einstein (605 nm), with a lesser change in the V_max values. The decrease in the quantum efficiency is mainly attributed to excitation uncoupling between phycobilisomes and Photosystem II. It is concluded that the F685 nm emission peak, often exclusively attributed to Photosystem II chlorophyll, arises from more than one component with phycobilisome emission being a major contributor. Vesicles from which phycobilisomes had been removed, as verified by electron microscopy and spectroscopy, had an almost negligible emission at 685 nm.     

Specificity of energy transfer to photosystem II by in vitro reassociated homologous and heterologous membrane-bound phycobilisomes

In a previous publication we have reported the in vitro reassociation of phycobiliproteins with thylakoids of Fremyella diplosiphon to form homologous, functional, membrane-bound phycobilisomes (Kirilovsky, D., Kessel, M. and Ohad, I (1983) Biochim. Biophys. Acta 724, 416–426). In the present work, using the same experimental system, we demonstrate the in vitro formation of heterologous, membrane-bound phycobilisomes. Analysis of phycobiliprotein association and binding curves disclosed two types of binding sites: specific sites which allow energy transfer to Photosystem II and non-specific sites which become occupied only after saturation of the Photosystem II specific sites. Binding to non-specific sites does not result in energy transfer. Both types of sites are present on cyanophyte thylakoids. Thylakoids of eukaryotic chloroplasts such as those of Chlamydomonas reinhardtii or Euglena gracilis can bind phycobiliproteins which reassociate to form intact membrane-bound phycobilisomes. However, only non-specific binding occurs in such heterologous systems. Limited proteolysis of membrane-bound phycobilisomes results in a rapid loss of the 94–95 kDa polypeptide assumed to be required for binding and energy transfer (Redlinger, T. and Gantt, E. (1982) Proc. Natl. Acad. Sci. USA 79, 5542–5546). Phycobilisomes lacking this polypeptide cannot bind to either specific or non-specific sites. Based on these results, we conclude that the 94–95 kDa polypeptide is required for the association of the phycobilisomes to both homologous and heterologous membranes; however, additional factors within the Photosystem II unit of cyanophytes are also required for establishing energy transfer.     

Regulation of Photosystem I electron flow activity by phosphatidylglycerol in thylakoid membranes as revealed by phospholipase treatment

Thylakoid membranes were treated by potato lipolytic acyl hydrolase, phospholipases A₂ from pancreas and snake venom, and by phospholipase C from Bacillus cereus under various conditions. The changes in the uncoupled rates of electron transport through Photosystem I (PS I) and in lipid composition were followed during these treatments. Pancreatic phospholipase A₂ which destroyed all phospholipids in thylakoid membranes stimulated the NADP⁺ reduction supported by reduced 2,6-dichlorophenolindophenol. This stimulation concerned only the dark but not the light reactions of this pathway. The main site of action of pancreatic phospholipase A₂ may be located on the donor side of PS I; the hydrolysis of phospholipids at this site caused an increased ability of reduced 2,6-dichlorophenolindophenol and ascorbate alone to feed electrons into PS I. A second site may be located on the acceptor side of PS I, probably between the primary acceptor and the ferredoxin system. When thylakoid membranes were first preincubated with or without lipolytic acyl hydrolase at 30°C (pH 8), the NADP⁺ photoreduction was inhibited whilst the methyl viologen-mediated O₂ uptake was stimulated. A subsequent addition of pancreatic phospholipase A₂ (which had the same hydrolysis rates for phosphatidylglycerol but not for phosphatidylcholine) further stimulated the O₂ uptake and restored NADP⁺ photoreduction. The extent of this stimulation, which depended on the presence of lipolytic acyl hydrolase, was ascribed partly to the hydrolysis of the phospholipids and partly to the generation of their lyso derivatives but not to the release of free fatty acids. On the contrary, phospholipase C which destroyed only phosphatidylcholine failed to restore this activity. It is suggested that phosphatidylglycerol is the only phospholipid associated with thylakoid membrane structures supporting PS I activities and that this lipid may play a physiological role in the regulation of these activities.     

Organization of chlorophyll-protein complexes of Photosystem I in Chlamydomonas reinhardii

The polypeptide pattern, chlorophyll-protein complexes, fluorescence emission spectra and light intensity required for saturation of electron flow via Photosystem (PS) II and PS I in a pale-green photoautotrophic mutant, y-lp, were compared to those of the parent strain, Chlamydomonas reinhardii y-1 cells. The mutant exhibits a 686 nm fluorescence yield at 25°C and 77 K 2–6-fold higher than that of the parent strain cells, and is deficient in thylakoid polypeptides 14, 17.2, 18 and 22 according to the nomenclature of Chua (Chua, N.-H. (1980) Methods Enzymol. 60C, 434–446). All chlorophyll-protein complexes ascribed to PS II and the CP I complex were present in both type of cells. However, a chlorophyll-protein complex CP Ia containing — in the parent strain — the 66–68 kDa polypeptides of CP I and the four above-mentioned polypeptides, was absent in the mutant. It was previously reported that a chlorophyll-protein complex, CP O, obtained from C. reinhardii contains five polypeptides, namely, 14, 15, 17.2, 18 and 22 (Wollman, F.A. and Bennoun, P. (1982) Biochim. Biophys. Acta 680, 352–360). A CP O-like complex was present also in the mutant y-lp cells but it contains only one polypeptide, 15. Energy transfer from PS II to PS I was not impaired in the mutant, although a 4-fold higher light intensity was required for the saturation of PS I electron flow in the y-lp cells as compared with the parent strain. No difference was found in the light saturation curves for PS II activity between the mutant and parent strain cells. Based on these and additional data (Gershoni, J.M., Shochat, S., Malkin, S. and Ohad, I. (1982) Plant Physiol. 70, 637–644), it is concluded that the chlorophyll-protein complexes of PS I in Chlamydomonas comprise a reaction center-core antenna complex containing the 66–68 kDa polypeptides (CP I), a connecting antenna consisting of four polypeptides (14, 17.2, 18 and 22), and a light-harvesting antenna containing one polypeptide, 15. These appear to be organized as a complex, CP Ia. The interconnecting antenna is deficient in the y-lp mutant and thus the CP Ia complex is unstable and energy is not transferred from CP O to CP I. The effective cross-section of PS I antenna is thus reduced and a high fluorescence is emitted at 686 nm.     

Electron spin resonance studies of the bound iron-sulfur centers in Photosystem I. Photoreduction of center A occurs in the absence of center B

The Photosystem I acceptor system of a subchloroplast particle from spinach was investigated by optical and electron spin resonance (ESR) spectroscopy following graduated inactivation of the bound iron-sulfur proteins by urea/ferricyanide solution. The chemical analysis of iron and sulfur and the ESR properties of centers A, B and X are consistent with the participation of three iron-sulfur centers in Photosystem I. A differential decrease in centers A, B and X is observed under conditions that induce S^2? →S⁰ conversion in the bound iron-sulfur proteins. Center B is shown to be the most susceptible, while center ‘X’ is the least susceptible component to oxidative denaturation. Stepwise inactivation experiments suggest that electron transport in Photosystem I does not occur sequentially from X→B→A, since there is quantitative photoreduction of center A in the absence of center B. We propose that center A is directly reduced by X; thus, X may serve as a branch point for parallel electron flow through centers A and B.     

Purification of cytochrome b-559 from oxygen-evolving Photosystem II preparations of spinach and maize

A rapid and simple procedure is presented for the purification of chloroplast cytochrome b-559. The method is based on the protocol devised by Garewal and Wasserman (Garewal, H.S. and Wasserman, A.R. (1974) Biochemistry 13, 4063–4071), which we have modified to eliminate the requirement for a lengthy electrophoretic step. Novel features of our method include: the use of oxygen-evolving Photosystem II preparations (Kuwabara, T. and Murata, N. (1982) Plant Cell Physiol. 23, 533–539) as the starting material; isocratic elution of cytochrome b-559 from a DEAE-cellulose column (yielding the protein in a pure state); and a simple column procedure for removal of excess Triton X-100. The procedure has been applied to both spinach and maize (Zea mays L.). Purified cytochromes b-559 from these species have similar optical spectra and mobility during gel electrophoresis under native conditions. Lithium dodecyl sulfate polyacrylamide gel electrophoresis of cytochrome b-559 from both spinach and maize reveals a major polypeptide band (apparent molecular mass = 9 kDa), and two minor bands (apparent molecular masses = 10 kDa and 6 kDa).     

Partial reconstitution of the photosynthetic oxygen evolution system by rebinding of the 33-kDa polypeptide

Treatment with 2.6 M urea of the Photosystem II particles depleted of two polypeptides of 24 kDa and 18 kDa completely released a polypeptide of 33 kDa and eliminated the oxygen-evolution activity. The 33-kDa polypeptide rebound to the urea-treated particles and partially reactivated the oxygen evolution. A quantitative analysis of the rebinding suggests tha there is a specific binding site for the 33-kDa polypeptide on the membrane surface.     

Manganese proteins isolated from spinach thylakoid membranes and their role in O2 evolution: II. A binuclear manganese-containing 34 kilodalton protein,a probable component of the water dehydrogenase enzyme

Extraction conditions have been found which result in the retention of managanese to the 33–34 kDa protein, first isolated as an apoprotein by Kuwabara and Murata (Kuwabara, T. and Murata, N. (1979) Biochim. Biophys Acta 581, 228–236). By maintaining an oxidizing-solution potential, with hydrophilic and lipophilic redox buffers during protein extraction of spinach grana-thylakoid membranes, the 33–34 kDa protein is observed to bind a maximum of 2 Mn/protein which are not released by extended dialysis versus buffer. This manganese is a part of the pool of 4 Mn/Photosystem II normally associated with the oxygen-evolving complex. The mechanism for retention of Mn to the protein during isolation appears to be by suppression of chemical reduction of natively bound, high-valent Mn to the labile Mn(II) oxidation state. This protein is also present in stoichiometric levels in highly active, O₂-evolving, detergent-extracted PS-II particles which contain 4–5 Mn/PS II. Conditions which result in the loss of Mn and O₂ evolution activity from functional membranes, such as incubation in 1.5 mM NH₂OH or in ascorbate plus dithionite, also release Mn from the protein. The protein exists as a monomer of 33 kDa by gel filtration and 34 kDa by gel electrophoresis, with an isoelectric point of 5.1 ± 0.1. The protein exhibits an EPR spectrum only below 12 K which extends over at least 2000 G centered at g = 2 consisting of non-uniformly separated hyperfine transitions with average splitting of 45–55 G. The magnitude of this splitting is nominally one-half the splitting observed in monomeric manganese complexes having O or N donor ligands. This is apparently due to electronic coupling of the two ⁵⁵Mn nuclei in a presumed binuclear site. Either a ferromagnetically coupled binuclear Mn₂(III,III) site or an antiferromagnetically coupled mixed-valence Mn₂(II,III) site are considered as possible oxidation states to account for the EPR spectrum. Qualitatively similar hyperfine structure splittings are observed in ferromagnetically coupled binuclear Mn complexes having even-spin ground states. The extreme temperature dependence suggests the population of low-lying excited spin states such as are present in weakly coupled dimers and higher clusters of Mn ions, or, possibly, from efficient spin relaxation such as occurs in the Mn(III) oxidation state. Either 1.5 mM NH₂OH or incubation with reducing agents abolishes the low temperature EPR signal and releases two Mn(II) ions to solution. This is consistent with the presence of Mn(III) in the isolated protein. The intrinsically unstable Mn₂(II,III) oxidation state observed in model compounds favors the assignment of the stable protein oxidation state to the Mn₂(III,III) formulation. This protein exhibits characteristics consistent with an identification with the long-sought Mn site for photosynthetic O₂ evolution. An EPR spectrum having qualitatively similar features is observable in dark-adapted intact, photosynthetic membranes (Dismukes, G.C., Abramowicz, D.A., Ferris, F.K., Mathur, P., Upadrashta, B. and Watnick, P. (1983) in The Oxygen-Evolving System of Plant Photosynthesis (Inoue, Y., ed.), pp. 145–158, Academic Press, Tokyo) and in detergent-extracted, O₂-evolving Photosystem-II particles (Abramowicz, D.A., Raab, T.K. and Dismukes, G.C. (1984) Proceedings of the Sixth International Congress on Photosynthesis (Sybesma, C., ed.), Vol. I, pp. 349–354, Martinus Nijhoff/Dr. W. Junk Publishers, The Hague, The Netherlands), thus establishing a direct link with the O₂ evolving complex.     

Energy transfer and quantum yield in Photosystem II

The photoreduction and dark reoxidation of Q_α and Q_β, the primary electron acceptors of Photosystems (PS) IIα and IIβ, respectively, in the presence of 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea (DCMU) were studied in tobacco chloroplasts by means of fluorescence and absorbance measurements. The magnitude of a correction for an absorbance change by the oxidizing side of PS II needed in our previous study of the quantum yield of Q reduction (Biochim. Biophys. Acta 635 (1981), 111–120) has been determined. The absorbance change occurs in PS IIα mainly. The maximum fluorescence yield was found to be the same as in the mutant Su/su, which has a 3-fold higher reaction center concentration and a lower PS IIα to PS IIβ ratio. The kinetics of the light-induced fluorescence increase were measured after various pretreatments and the corresponding kinetics of the integrated fluorescence deficit were analyzed into their α and β components. From the results the contribution to the minimum fluorescence level, the degree of energy transfer between units, and the quantum efficiency of Q reduction were calculated for both types of PS II. This led to the following conclusions. The absence of energy between PS IIβ antennae is confirmed. Fluorescence quenching in PS IIα was adequately described by the matrix model, except for a decrease in the energy transfer between units during photoreduction of Q_α, possibly due to the formation of ‘islets’ of closed centers. PS II reaction centers in which Q is reduced do not significantly quench fluorescence. The ratio of variable to maximum fluorescence, 0.77 in PS IIα and 0.92 in PS IIβ, multiplied by the fraction of Q remaining in the reduced state after one saturating flash, 0.88 in PS IIα and greater than 0.95 in PS IIβ, leads to a net quantum efficiency of Q reduction in the presence of DCMU and NH₂OH of 0.68 in PS IIα and about 0.90 in PS IIβ. These values are in good agreement with the measured overall quantum efficiency of Q reduction.     

Quantitative separation of spinach thylakoids into Photosystem II-enriched inside-out vesicles and Photosystem I-enriched right-side-out vesicles

Yeda press disruption of thylakoids in the presence of magnesium followed by aqueous polymer two-phase partitioning fractionated the total thylakoid membrane material into two distinctly different fractions. One fraction comprised approx. 60% of the material on a chlorophyll basis and contained inside-out vesicles while the other fraction (40%) contained right-side-out vesicles. The sidedness of the vesicles was determined from the direction of their light-induced proton translocation. The inside-out vesicles showed a pronounced Photosystem (PS) II enrichment as judged by their high PS II and low PS I activities. Moreover, they showed a high ratio between the PS II reaction centre chlorophyll-protein complex and the PS I reaction centre chlorophyll-protein complex (CP I). The chlorophyll $\text{a}\text{b}$ ratio was as low as 2.3 compared to 3.2 for the starting material. In contrast, the right-side-out vesicles showed a pronounced PS I enrichment. Their chlorophyll $\text{a}\text{b}$ ratio was 4.3–4.9. The tight stacking induced by Mg²⁺ allows a quantitative formation of inside-out vesicles from the appressed thylakoid regions while mainly non-appressed thylakoids turn right-side-out. The possibility of fractionating all of the thylakoid material into two sub-populations with markedly different composition with respect to PS I and PS II argues against a close physical association between the two photosystems and in favour of their spatial separation in the plane of the membrane. This fractionation procedure, which can be completed within 1 h and gives high yields of both PS II inside-out thylakoids and PS I right-side-out thylakoids, should be very useful for facilitating and improving studies on both the transverse and lateral organization of the thylakoid membrane.     

Malonyl CoA inhibition of carnitine palmityltransferase in rat heart mitochondria

The isolated beta 2-dimer of Escherichia coli tryptophan synthase exhibits reversible high-pressure deactivation and hybridization with an equilibrium transition at 690 and 870 bar for the apoenzyme and holoenzyme, respectively. To investigate the hypothetical dissociation mechanism ultracentrifugal analysis has been applied. In a conventional swing-out rotor (r(max) = 16 cm, fill-height 9 cm) a pressure gradient of 1 less than p less than 1840 bar is formed at maximum speed (40 000 rpm). Using a sucrose gradient to stabilize the particle distribution, pressure-dependent alterations of the state of association of oligomeric systems may be determined. In the present experiments ovalbumin (with a molecular mass close to the beta-monomer) has been used as a reference. The radial sedimentation velocity of the beta 2-dimer (in 5-20% sucrose, 10 degrees C) is found to decrease significantly at p approximately equal to 850 bar. From the slopes in an r-r(degrees) vs t plot the limiting values for the particle weight at the meniscus and the bottom of the tube are found to be the beta 2-dimer (M(r) = 85 800) and the beta-monomer (M(r) = 42 900), thus proving pressure-dependent dissociation. Since sucrose stabilizes the native quaternary structure, the beta 2 leads to 2 beta transition is shifted towards higher pressures compared to the dissociation in standard buffer. Conventional quench experiments in high-pressure cells in the presence of 13% (w/v) sucrose confirm the result of the sucrose gradient centrifugation with respect to the critical pressure where deactivation (and dissociation) occur.     

Protein composition and architecture of the photosynthetic membranes from the cyanobacterium, Anacystis nidulans R2

The protein composition of the photosynthetic membrane from the cyanobacterium, Anacystis nidulans R2, was analyzed by acrylamide gel electrophoresis following solubilization with lithium dodecyl sulfate. Autoradiograms of ³⁵S-labelled membranes revealed over 90 bands by this procedure. The effect of solubilization conditions on protein resolution was analyzed by modifying temperature and sulfhydryl concentrations. Labelling cells with ⁵⁹Fe yielded nine iron-containing bands on these gels. Three of these bands, at 33, 19, and 14 kDa, were also heme proteins as determined by tetramethylbenzidine staining, and represent cytochromes f, b₆ and c-552, respectively. The remaining iron proteins are highly sensitive to solubilization conditions, especially the presence of 2-mercaptoethanol, and we suggest that these bands may be Fe-S proteins. Lactoperoxidase-catalyzed iodination of the membranes indicated that at least 41 proteins have surface-exposed domains. Some of the known proteins with external surfaces include cytochrome c-552 and the chlorophyll-binding proteins of Photosystems I and II. Neither cytochrome f nor b₆ appear to be accessible to external labelling. When this structural information was combined with the isolation of functional submembrane complexes, we constructed a topological model of the membrane. Using this model we have discussed the protein architecture of the cyanobacterial membrane.     

Fluorescence polarization and spin-label studies of the fluidity of stromal and granal chloroplast membranes

The lipid fluidity of thylakoid membrane regions separated by Yeda press and sonication methods has been investigated using diphenylhexatriene fluorescence polarization measurements and rotational correlation times derived from the ESR spectra of the spin-labels 5-doxyldecane and 12-doxylstearate. According to both techniques, stromal lamellae vesicles with essentially only Photosystem I activity were more fluid than the granal membranes. The differences in lipid fluidity between the two fractions were interpreted in terms of the ratio of the amounts of protein compared to lipid in the membranes. Stromal lamellae fractions contained lower protein/lipid ratios compared with the granal membranes.     

Evidence for a new early acceptor in Photosystem I of plants. An ESR investigation of reaction center triplet yield and of the reduced intermediary acceptors

The yield of the triplet state of the primary electron donor of Photosystem I of photosynthesis (P^T-700) and the characteristic parameters (g value, line shape, saturation behavior) of the ESR signal of the photoaccumulated intermediary acceptor A have been measured for two types of Photosystem I subchloroplast particles: Triton particles (TSF 1, about 100 chlorophyll molecules per P-700) that contain the iron-sulfur acceptors F_X, F_B and F_A, and lithium dodecyl sulfate (LDS) particles (about 40 chlorophyll molecules per P-700) that lack these iron-sulfur acceptors. The results are: (i) In Triton particles the yield of P^T-700 upon illumination is independent of the redox state of A and of F_X,B,A and is maximally about 5% of the active reaction centers at 5 K. The molecular sublevel decay rates are k_x = 1100 s^?1 ± 10%, k_y = 1300 s^?1 ± 10% and k_z = 83 s^?1 ± 20%. In LDS particles the triplet yield decreases linearly with concentration of reduced intermediary acceptors, the maximal yield being about 4% at 5 K assuming full P-700 activity. (ii) In Triton particles the acceptor complex A consists of two acceptors A₀ and A₁, with A₀ preceding A₁. In LDS particles at temperatures below ?30°C only A₀ is photoactive. (iii) The spin-polarized ESR signal found in the time-resolved ESR experiments with Triton particles is attributed to a polarized P-700-A^?₁ spectrum. The decay kinetics are complex and are influenced by transient nutation effects, even at low microwave power. It is concluded that the lifetime at 5 K of P-700A₀A^?₁ must exceed 5 ms. We conclude that P^T-700 originates from charge recombination of P-700A^?₀, and that in Triton particles A₀ and A₁ are both photoaccumulated upon cooling at low redox potential in the light. Since the state P-700AF^?_X does not give rise to triplet formation the 5% triplet yield in Triton particles is probably due to centers with damaged electron transport.     

Action of cyanide on the photosynthetic water-splitting process

KCN inhibits O₂ evolution from inside-out chloroplast thylakoid vesicles suspended in a low chloride-containing medium. Evidence is presented to suggest that the inhibition is due to CN^? and that this anion blocks electron flow close to the water-splitting process by interacting with a photo-oxidised species.     

Identity of the Photosystem II reaction center polypeptide

A Photosystem-II (PS-II)-enriched chloroplast submembrane fraction has been subjected to non-denaturing gel-electrophoresis. Two chlorophyll a (Chl a)-binding proteins associated with the core complex were isolated and spectrally characterized. The Chl protein with apparent apoprotein mass of 47 kDa (CP47) displayed a 695 nm fluorescence emission maximum (77 K) and light-induced absorption characteristics indicating the presence of the reaction center Chl, P-680, and its primary electron acceptor, pheophytin. A Chl protein of apparent apoprotein mass of 43 kDa (CP43) displayed a fluorescence emission maximum at 685 nm. We conclude that CP43 serves as an antenna Chl protein and the PS II reaction center is located in CP47.     

Effects of physical and chemical treatments on chloroplast manganese. NMR and ESR studies

Measurements were made of the water proton relaxation rate (T^?1₂ = R₂), electron spin resonance (ESR) six-line signal of ‘free’ Mn²⁺, and O₂-evolution activity in thylakoid membranes from pea leaves. The main results are: (1) Aging of thylakoids at 35°C causes a parallel decrease in O₂-evolution activity, in R₂ and in the content of bound Mn, suggesting that R₂ may be related to the loosely bound Mn involved in O₂ evolution. (2) Treatment of thylakoids with tetraphenylboron (TPB) at [TPB] > 2 mM produces a 2-fold increase in R₂, without release of Mn²⁺. The titration curve exhibits three sharp end points. The first end point occurs at a $\text{[}\text{TPB}\text{]}\text{[}\text{chlorophyll}\text{]}$ of 1.25, at which the O₂ evolution is completely inhibited. (3) Treatment of thylakoids with NH₂OH also increases R₂ by nearly 2-fold, either by the reduction of the higher oxidation states of Mn to Mn²⁺ and / or by exposing the Mn to solvent protons. Also, progressive release of bound Mn occurs at [NH₂OH] ≥ 1 mM as shown by an increase increase in the Mn²⁺ ESR signal and a decrease in R₂. (4) Addition of H₂O₂ (0.1–1.0%) to thylakoids causes an enhancement of R₂ similar to that by NH₂OH, but without the release of Mn²⁺. (5) Heat treatment of thylakoids at 40–50°C releases Mn²⁺ and increases R₂. Conversely, pH values of 7 to 4 release Mn²⁺ without changing R₂ while pH values of 7–9 increase R₂ without releasing Mn²⁺. Thus, both high and low pH values as well as the heat treatment cause structural changes enhancing the relaxivity of the bound Mn or of other paramagnetic species.     

Partial disintegration and reconstitution of the photosynthetic oxygen evolution system. Binding of 24 kilodalton and 18 kilodalton polypeptides

Treatment with 1 M NaCl almost totally removed two polypeptides of 24 and 18 kDa from the Photosystem II particles of spinach chloroplasts and reduced the oxygen-evolution activity by about half. Both polypeptides were able to rebind to the NaCl-treated particles in a low-salt medium. The rebinding of the 24 kDa polypeptide showed a saturation curve whose maximum level was close to that naturally occurring in the untreated particles. In parallel with the amount of rebound 24 kDa polypeptide, the oxygen-evolution activity was recovered. The 18 kDa polypeptide bound to the NaCl-treated particles without saturation. When the 18 kDa polypeptide was added to the particles previously treated with NaCl and then supplemented with a saturating amount of 24 kDa polypeptide, there appeared, in addition to the binding without saturation, another binding of the 18 kDa polypeptide with saturation to a maximum level close to that naturally occurring in the untreated particles. The 18 kDa polypeptide did not restore the oxygen-evolution activity. These findings suggest that there are specific binding sites; one for the 24 kDa polypeptide located on the Photosystem II particles, and the other for the 18 kDa polypeptide on the 24 kDa polypeptide.     

Cation-mediated regulation of excitation energy distribution in chloroplasts lacking organized Photosystem II complexes

Despite the total loss of Photosystem II activity, thylakoids isolated from the green nuclear maize mutant hcf1-3 contain normal amounts of the light-harvesting chlorophyll $\text{a}\text{b}$ pigment-protein complex (LHC). We interpret the spectroscopic and ultrastructural characteristics of these thylakoids to indicate that the LHC present in these membranes is not associated with Photosystem II reaction centers and thus exists in a ‘free’ state within the thylakoid membrane. In contrast, the LHC found in wild-type maize thylakoids shows the usual functional association with Photosystem II reaction centers. Several lines of evidence suggest that the free LHC found in thylakoids isolated from hcf1-3 is able to mediate cation-dependent changes in both thylakoid appression and energy distribution between the photosystems: (1) Thylakoids isolated from hcf1-3 and wild-type seedlings exhibit a similar Mg²⁺-dependent increase in the short/long wavelength fluorescence emission peak ratio at 77 K. This Mg²⁺ effect is lost following incubation of thylakoids isolated from either source with low concentrations of trypsin. Such treatment results in the partial proteolysis of the LHC in both membrane types. (2) Thylakoids isolated from both hcf1-3 and wild-type seedlings show a similar Mg²⁺ dependence for the enhancement of the maximal yield of room temperature fluorescence and light scattering; both Mg²⁺ effects are abolished by brief incubation of the thylakoids with low concentrations of trypsin (3) Mg²⁺ acts to reduce the relative quantum efficiency of Photosystem I-dependent electron transport at limiting 650 nm light in thylakoids isolated from hcf1-3. (4) The pattern of digitonin fractionation of thylakoid membranes, which is dependent upon structural membrane interactions and upon LHC in the thylakoids, is similar in thylakoids isolated from both hcf1-3 and wild-type seedlings. We conclude that the surface-exposed segment of the LHC, but not the LHC-Photosystem II core association, is necessary for the cation-dependent changes in both thylakoid appression and energy distribution between the two photosystems, and that the LHC itself is able to transfer excitation energy directly to Photosystem I in a Mg²⁺-dependent fashion in the absence of Photosystem II reaction centers. The latter phenomenon is equivalent to a cation-induced change in the absorptive cross-section of Photosystem I.     

Properties of thylakoids and thylakoid particles derived from structurally different chloroplasts

Structurally and functionally different tobacco chloroplasts were subjected to digitonin treatment and subsequent fractional centrifugation. The light-harvesting $\text{chlorophyll}\text{a}\text{chlorophyll}\text{b-}\text{protein}$ complex was found to be enriched in the most dense fraction regardless of the presence of grana in the original preparation. It is suggested that isolated thylakoid membranes and fragments thereof which contain sufficient light-harvesting protein may, under appropriate ionic conditions, form aggregates even when they originate from unstacked thylakoid systems. Comparative studies of fluorescence properties and polypeptide composition of the thylakoids suggest that the light-harvesting protein does not contribute significantly to the fluorescence spectrum of isolated chloroplasts as long as this protein is intimately associated with the Photosystem II (PS II) pigment-protein complex responsible for the 685 nm emission. While the PS II-deficient mutant chloroplasts of the variegated tobacco variety NC 95 lacked both the 685 nm fluorescence component and two or three PS II proteins, one of these proteins was found to be very prominent in our chlorophyll b-deficient mutant thylakoids which also displayed an intense 685 nm fluorescence peak. This correlation supports the contention that a 45 kdalton polypeptide is an apoprotein of pigments associated with the PS II reaction center.