The site of inhibition of photosynthetic electron transfer by amphotericin B.

The inhibitory effect of the polyene antibiotic, amphotericin B, on photosynthetic electron transfer has been investigated. Treatment of chloroplasts with the inhibitor results in the release of plastocyanin from its site in the chloroplast membrane. This release is accompanied by a shift in the pH curve for ferricyanide photoreduction from water, which is similar to that observed when chloroplasts are treated by sonication or passage through a French press. Delayed light emission from photosystem 2 is not destroyed by amphotericin B treatment, indicating that photosystem 2 is not damaged. Amphotericin B does not inhibit photoreduction of ferricyanide from water by chloroplast preparations which are deficient in plastocyanin, such as maize bundle-sheath chloroplast fragments, Euglena chloroplasts, or maize mesophyll chloroplasts passed through a French press. Chloroplasts treated with amphotericin B are not able to photooxidize plastocyanin. This result demonstrates that little structural damage occurs to the membrane during treatment with the antibiotic as a capacity to photooxidize plastocyanin is observed only in damaged chloroplast membranes.     

Mechanism of KCN inhibition of photosystem I.

Experiments with chloroplasts and purified spinach plastocyanin suggest a mechanism for KCN inhibition of Photosystem I. KCN inhibition can be bypassed by a detergent or reversed by replacement of the inactive plastocyanin. KCN bleaches and inactivates purified plastocyanin. KCN releases copper from chloroplast membranes and from purified plastocyanin. Cyanide does not bind to the apoprotein produced when plastocyanin is treated with KCN, and KCN-produced apoplastocyanin has a N-ethylmaleimide-reactive sulfhydryl group not found in holoplastocyanin. Apoplastocyanin is not active in restoring Photosystem I activity to plastocyanin-depleted membranes. Holoplastocyanin restores Photosystem I activities to plastocyanin-depleted membranes prepared from either control or KCN-treated chloroplasts to about the same extent. KCN-treated chloroplast membranes are found to have higher amounts of apoplastocyanin than do control chloroplast membranes. These results offer evidence that KCN removes the copper from plastocyanin in the chloroplast membrane, leaving the inactive apoplastocyanin which is unable to transfer electrons to Photosystem I.     

A combined procedure for preparation of plastocyanin, ferredoxin and CF1.

Homogeneous preparations of ferredoxin, plastocyanin, and chloroplast coupling factor (CF1) have been isolated from spinach by a combined procedure in which supernatants from preparation of chloroplasts are used for isolation of ferredoxin and the chloroplasts serve as the source of plastocyanin. The proteins were purified by DEAE-cellulose chromatography and gel filtration, after precipitation with acetone in the case of ferredoxin or release from membranes in the case of plastocyanin. The proteins obtained by this procedure are pure, as evidenced by absorption ratios (ferredoxin, A420/A276 = 0.47-0.48; plastocyanin, A278/A597 = 1.2) and by the fact that both proteins migrate as single bands on polyacrylamide gels in the presence of sodium dodecyl sulfate.     

Reversibility of the cyanide inhibition of electron transport in spinach chloroplast thylakoids

The prior treatment of thylakoids with cyanide (30 mM) was shown to inhibit plastocyanin-dependent electron transport reactions. We find that cyanide inhibition of electron flow from either water or diaminodurene to methyl viologen, but not from water to ferricyanide, is partially reversed when the thylakoids are collected by centrifugation and resuspended in a cyanide-free medium. However, methyl viologen reduction in thylakoids pretreated with cyanide is sensitive to cyanide (～1 mM) added to the reaction mixtures, whereas that in control thylakoids is unaffected. The cyanide must be added in the dark. Electron transport to methyl viologen in chloroplasts pretreated with cyanide is also sensitive to inhibition by EDTA and bathocuproine sulfonate. Thus, KCN inhibition of electron transport in thylakoids is partially reversible. Moreover, the accessibility of plastocyanin to various reagents is probably altered by the KCN treatment.     

Plastocyanin participation in chloroplast Photosystem I

French pressure cell disruption of spinach chloroplasts releases much of the plastocyanin from chloroplast membranes. Heavy particles obtained from French pressure cell disrupted chloroplasts lose most of their plastocyanin while light particles retain a high plastocyanin to chlorophyll ratio. Photosystem I activity is dependent on the presence of plastocyanin in our preparations.     

Localization of the reaction side of plastocyanin from immunological and kinetic studies with inside-out thylakoid vesicles

(1) The effect of four active antisera against plastocyanin on Photosystem I-driven electron transport and phosphorylation was investigated in spinach chloroplasts. Partial inhibition of electron transport and stimulation of plastocyanin-dependent phosphorylation were sometimes observed after adding amounts of antibodies which were in large excess and not related to the plastocyanin content of the chloroplasts. This indicates effects of the antibodies on the membrane. (2) The antibodies against plastocyanin neither directly nor indirectly agglutinated unbroken chloroplast membranes. (3) The plastocyanin content of right-side-out and inside-out thylakoid vesicles isolated by aqueous polymer two-phase partition from chloroplasts disrupted by Yeda press treatment was determined by quantitative rocket electroimmunodiffusion. Right-side-out vesicles retained about 25%, inside-out vesicles none of the original amount of plastocyanin. (4) The effect of externally added plastocyanin on the reduction of P-700 was studied by monitoring the absorbance changes at 703 nm after a long flash. In inside-out vesicles P-700 was reduced by the added plastocyanin but not in right-side-out vesicles and class II chloroplasts. These results provide strong evidence for a function of plastocyanin at the internal side of the thylakoid membrane.     

Chloroplast membrane sidedness. Location of plastocyanin determined by chemical modifiers

Intact grana and stroma membranes (outer membrane absent) and detergent or sonication disrupted thylakoid membranes were treated with the hydrophilic covalent chemical modifiers [³⁵S]diazonium benzene sulfonic acid ([³⁵S]DABS) and [¹⁴C]glycine ethylester plus 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide metho-p-toluenesulfonate (CDIS). Plastocyanin was purified using column chromatography followed by polyacrylamide gel electrophoresis and the incorporation of [³⁵S]DABS and [¹⁴C]glycine ethylester into plastocyanin was determined by slicing the gels and counting the radioactivity in the plastocyanin band. Plastocyanin isolated from thylakoids disrupted prior to chemical modification binds two to four times as much of either modifier than the plastocyanin isolated from intact chloroplasts. This ratio is five to ten times lower than the ratio expected for a component buried behind the permeability barrier of a membrane. The data suggest that plastocyanin is partially exposed at the external surface of the thylakoid membrane rather than being completely buried in, or behind, the lipo-protein membrane.     

Lateral distribution and diffusion of plastocyanin in chloroplast thylakoids

The lateral distribution of plastocyanin in the thylakoid lumen of spinach and pea chloroplasts was studied by combining immunocytochemical localization and kinetic measurements of P700+ reduction at high time resolution. In dark-adapted chloroplasts, the concentration of plastocyanin in the photosystem I containing stroma membranes exceeds that in photosystem II containing grana membranes by a factor of about two. Under these conditions, the reduction of P700+ with a halftime of 12 microseconds after a laser flash of saturating intensity indicates that to greater than 95% of total photosystem I a plastocyanin molecule is bound. An analysis of the labeling densities, the length of the different lumenal regions, and the total amounts of plastocyanin and P700 shows that most of the remaining presumable mobile plastocyanin is found in the granal lumen. This distribution of plastocyanin is consistent with a more negative surface charge density in the stromal than in the granal lumen. During illumination the concentration of plastocyanin in grana increases at the expense of that in stroma lamellae, indicating a light-driven diffusion from stroma to grana regions. Our observations provide evidence that a high concentration of plastocyanin in grana in the light favors the lateral electron transport from cytochrome b6/f complexes in appressed grana across the long distance to photosystem I in nonappressed stroma membranes.     

Kinetic analysis of P-700 photoconversion: Effect of secondary electron donation and plastocyanin inhibition

The kinetics of P-700 photoconversion under weak continuous actinic illumination were quantitatively analyzed to provide information on the relative absorption cross-section σ_PSI of the light-harvesting pigments associated with photosystem I and on the number of electrons stored between the two photosystems in dark-adapted chloroplasts. The theory of chemical kinetics for a system of monomolecular consecutive first-order reactions is reviewed briefly to provide support for the experimental approach taken. A complete inhibition of plastocyanin by cyanide eliminated all secondary electron donation to P-700⁺ and allowed the registration of the exponential (monomolecular) P-700 photoconversion at room temperature. The rate constant K_p-700 of the exponential kinetics was independent of the ionic (± Mg²⁺) and osmotic (± sucrose) strength of the chloroplast suspension medium, and of the oxidation-reduction state of photosystem II. The extent of plastocyanin inhibition in partially inhibited samples was greater under low ionic and low osmotic conditions. In dark-adapted chloroplast samples that were not cyanide treated, the number of electrons stored between the two photosystems was 3.9 ± 0.2 and independent of divalent cations. It is concluded that plastocyanin inhibition by cyanide is favored under low ionic and low osmotic conditions. The Mg²⁺ ion and redox state of photosystem II-independent photoconversion of P-700 does not support significant changes in the spillover of excitation from photosystem II to photosystem I in isolated chloroplasts.     

Effect of metalloproteins on the photochemical activity of chloroplasts treated with polyene antibiotics]

The effects of various metall-containing proteins (plastocyanin, plantacyanin, azurine and cytochromes of the f type) on the activity of photosystem I of chloroplasts, treated with polyene antibiotics, were studied. The inhibiting effect of the polyenes, surgumycin and philipin, was completely removed by an addition of copper-containing protein plastocyanin. No similar effect was exerted by other Cu-containing proteins--azurine and plantacyanin. The cytochromes of the f type isolated from the green algae chlorella, blue-green algae spiruline and aphanezomenone, having different electrophoretic properties, restored the activity of photosystem I of chloroplasts incubated with antibiotics in a different degree. Acid cytochrome f of chlorella restored the activity by 80--100%; less acid cytochrome f from spiruline-only by 50%. The least restoring effect was exerted by aphanezomenone cytochrome, which possesses some basic properties. The chloroplasts treatment with surgumycin did not affect the isolation of the terminal enzyme of the chloroplast electron-transporting chain of ferredoxin--NADP--reductase. Possible environment of plastocyanin in the chloroplast membrane and the mechanism of photosystem I restoration are discussed.     

Studies on well coupled Photosystem I-enriched subchloroplast vesicles. Content and redox properties of electron-transfer components

Stable and well coupled Photosystem (PS) I-enriched vesicles, mainly derived from the chloroplast stroma lamellae, have been obtained by mild digitonin treatment of spinach chloroplasts. Optimal conditions for chloroplast solubilization are established at a digitonin/chlorophyll ratio of 1 ($\text{w}\text{w}$) and a chlorophyll concentration of 0.2 mM, resulting in little loss of native components. In particular, plastocyanin is easily released at higher digitonin/chlorophyll ratios. On the basis of chlorophyll content, the vesicles show a 2-fold enrichment in ATPase, chlorophyll-protein Complex I, P-700, plastocyanin and ribulose-1,5-bisphosphate carboxylase as compared to chloroplasts, in line with the increased activities of cyclic photophosphorylation and PS I-associated electron transfer as shown previously (Peters, A.L.J., Dokter, P., Kooij, T. and Kraayenhof, R. (1981) in Photosynthesis I (Akoyunoglou, G., ed.), pp. 691–700, Balaban International Science Services, Philadelphia). The vesicles have a low content of the light-harvesting chlorophyll-protein complex and show no PS II-associated electron transfer. Characterization of cytochromes in PS I-enriched vesicles and chloroplasts at 25°C and 77 K is performed using an analytical method combining potentiometric analysis and spectrum deconvolution. In PS I-enriched vesicles three cytochromes are distinguished: c-554 (E′₀ = 335 mV), b-559_LP (E′₀ = 32 mV) and b-563 (E′₀ = ? 123 mV); no b-559_HP is present (LP, low-potential; HP, high-potential). Comparative data from PS I vesicles and chloroplasts are consistent with an even distribution of the cytochrome b-563- cytochrome c-554 redox complex in the lateral plane of exposed and appressed thylakoid membranes, an exclusive location of plastocyanin in the exposed membranes and a dominant location of plastoquinone in the appressed membranes. The results are discussed in view of the lateral heterogeneity of redox components in chloroplast membranes.     

Requirement of galactolipids for Photosystem I activity in lyophilized spinach chloroplasts

1. The effect of monogalactosyl diacylglycerol and digalactosyl diacylglycerol on reconstitution of Photosystem I activity in heptane-extracted and galactolipase-treated spinach chloroplasts was investigated.2. Both galactolipids, in a molar ratio with chlorophyll of 2.5, partially restored Photosystem I activity in heptane-extracted chloroplasts. An addition o saturating amounts of plastocyanin caused complete reactivation of Photosystem I.3. Similarly, with galactolipase-treated chloroplasts, both galactolipids partially restored Photosystem I activity and additional amounts of plastocyanin were required for complete reactivation.4. The action of galactolipids on partial reconstitution of Photosystem I supports the suggestion of their structural role in the restoration of thylakoid membranes.     

Plastocyanin stimulation of whole chain and photosystem I electron transport in inside-out thylakoid vesicles

Inside-out and right-side-out thylakoid vesicles were isolated from spinach chloroplasts by aqueous-polymer two-phase (dextran/polyethylene glycol) partitioning. Externally added plastocyanin stimulated the whole-chain and PSI electron transport rates in the inside-out thylakoid vesicles by about 500 and 350%, respectively, compared to about 50% stimulation for both assays in the fraction enriched in right-side-out vesicles. No apparent stimulation by plastocyanin was observed in unbroken Class II thylakoids. The electron transport between PSII and PSI in inside-out thylakoid vesicles appears to be interrupted due to plastocyanin release from the thylakoids by the Yeda press treatment, but it was restored by externally added plastocyanin. The P700 content of the inside-out membrane preparations, measured by chemical and photochemical methods, was 1 P700 per 1100 to 1500 chlorophylls while it was about 1 P700 per 500 chlorophylls for the right-side-out vesicles. The data presented support the concept of lateral heterogeneity of PS I and II in thylakoid membranes, but does not support a virtual or total absence of PSI in the appressed grana partitions. Further, the heterogeneity does not appear to be as extreme as suggested earlier. Although PSI is somewhat depleted in the appressed grana membrane region, there is adequate photochemically active P700, when sufficient plastocyanin is available, to effectively couple PSI electron transfer with the preponderant PSII in linear electron transport.     

The action of lipases on chloroplast membranes I. The release of plastocyanin from galactolipase-treated thylakoid membranes

Bean chloroplasts treated with galactolipase (lipolytic acyl hydrolase) isolated from bean leaves showed an inhibition of photosystem I activity as measured by methyl viologen-mediated oxygen uptake and NADP⁺ photoreduction. This inhibition was partially reversed by exogenous plastocyanin added to galactolipase-treated thylakoid membranes. Galactolipase released substantial amounts of endogenous plastocyanin (about 40%) from bean chloroplasts. The results are discussed with regard to the localization of plastocyanin in thylakoid membranes.Abbreviations chlf chlorophyll - DCMU 3-(2,4-dichlorophenyl)-1,1-dimethylurea - DGDG digalactosyldiacylglycerol - MGDG monogalactosyldiacylglycerol - MV methyl viologen - NADP⁺ nicotinamide dinucleotide phosphate - PC phosphatidylcholine - PG phosphatidylglycerol - PE phosphatidylethanolamine - PI phosphatidylinositol - SQDG sulphoquinovosyldiacylglycerol - SDS sodium dodecyl sulphate - TMPD N,N,N,N-tetramethyl-p-phenylenediamine - Tricine N-Tris-(hydroxymethyl)-methylglycine - Tris Tris-(hydroxymethyl)-aminomethane     

Requirement of galactolipids for photosystem I activity in lyophilized spinach chloroplasts.

1. The effect of monogalactosyl diacylglycerol and digalactosyl diacylglycerol on reconstitution of Photosystem I activity in heptane-extracted and galactolipase-treated spinach chloroplasts was investigated. 2. Both galactolipids, in a molar ratio with chlorophyll of 2.5, partially restored Photosystem I activity in heptane-extracted chloroplasts. An addition of saturating amounts of plastocyanin caused complete reactivation of Photosystem I. 3. Similarly, with galactolipase-treated chloroplasts, both galactolipids partially restored Phostosystem I activity and additional amounts of plastocyanin were required for complete reactivation. 4. The action of galactolipids on partial reconstitution of Photosystem I supports the suggestion of their structural role in the restoration of thylakoid membranes.     

Cytochrome f from spinach and cyanobacteria. Purification and characterization

Cytochrome f has been purified from spinach chloroplasts and from the photosynthetic membranes of the cyanobacterium Spirulina maxima. The spinach protein has an isoelectric point of 5.2 and gives a single band on isoelectric focusing gels. The S. maxima cytochrome shows a major band with a pI of 4.01 and a minor band with a pI of 3.97. S. maxima cytochrome f has a molecular weight approximately 38,000 and is monomeric, while the spinach protein is slightly smaller, approximately 36,000 daltons, and aggregates to form an octamer. S. maxima cytochrome f has an E'0 of +339 mV which is close to that of cytochromes f from higher plants. The NH2-terminal amino acid sequences of the cytochromes show striking similarities. Spinach cytochrome f shows a clear preference for oxidation by spinach plastocyanin and S. maxima cytochrome f is more readily oxidized by its in vivo reaction partner, cytochrome c553.     

Inhibition of photosynthetic electron transport by amphotericin B

By assaying partial reactions of the photosynthetic electron transport system using thylakoids from spinach as well as from the algae Bumilleriopsis, Dunaliella , and Anabaena , it was demonstrated that the polyene antibiotic amphotericin B has no specific effect on plastocyanin. Pretreating spinach and algal thylakoids with this antibiotic decreased photosystem-II as well as photosystem-I activity regardless of whether the membranes contained plastocyanin or cytochrome c-553. Different sensitivity of cell-free electron transport activity against this antibiotic was observed due to the species used. With Dunaliella , the photosystem-II region was inhibited more strongly than photosystem-I, while Bumilleriopsis chloroplasts – although not containing plastocyanin – exhibited a stronger inhibition of the photosystem-I region. Apparently, amphotericin B mainly solubilizes redox compounds that form connecting pools in the photosynthetic electron transport chain.     

Full-length plastocyanin precursor is translocated across isolated thylakoid membranes

In higher plants, the chloroplastic protein plastocyanin is synthesized as a transit peptide-containing precursor by cytosolic ribosomes and posttranslationally transported to the thylakoid lumen. En route to the lumen, a plastocyanin precursor is first imported into chloroplasts and then further directed across the thylakoid membrane by a second distinct transport event. A partially processed form of plastocyanin is observed in the stroma during import experiments using intact chloroplasts and has been proposed to be the translocation substrate for the second step (Smeekens, S., Bauerle, C., Hageman, J., Keegstra, K., and Weisbeek, P. (1986) Cell 46, 365-375). To further characterize this second step, we have reconstituted thylakoid transport in a system containing in vitro-synthesized precursor proteins and isolated thylakoid membranes. This system was specific for lumenal proteins since stromal proteins lacking the appropriate targeting information did not accumulate in the thylakoid lumen. Plastocyanin precursor was taken up by isolated thylakoids, proteolytically processed to mature size, and converted to holo form. Translocation was temperature-dependent and was stimulated by millimolar levels of ATP but did not strictly require the addition of stromal factors. We have examined the substrate requirements of thylakoid translocation by testing the ability of different processed forms of plastocyanin to transport in the in vitro system. Interestingly, only the full-length plastocyanin precursor, not the partially processed intermediate form, was competent for transport in this in vitro system.     

Internal ATP is the only energy requirement for the translocation of precursor proteins across chloroplastic membranes

The energy requirements for the import of nuclear-encoded proteins into isolated chloroplasts have been reinvestigated. We have shown that, in contrast to protein import into mitochondria, the translocation of the precursors to ferredoxin, plastocyanin (prPC) and the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (prSS) across all chloroplastic membranes is independent of a protonmotive force and requires only ATP. This extends previous works in which investigations were limited to prSS and demonstrates that our results are probably general to all chloroplastic protein precursors. Our results are particularly interesting for the import of prPC, since in addition to the two envelope membranes, this protein must traverse the energy-transducing thylakoid membranes en route to its proper location in the thylakoid lumen. This lack of involvement of a protonmotive force, specifically of a transmembrane electric potential, demonstrates that separate mechanisms operate during the import of proteins into chloroplasts and mitochondria. We also examined the question of whether ATP is utilized inside or outside of chloroplasts during protein import. Previous attempts to resolve this question have resulted in conflicting answers. We found, by two independent approaches, that ATP for protein import is utilized inside chloroplasts. The implications of these results on the possible mechanisms of protein import into chloroplasts are discussed.     

Effect of surface potential on P-700 reduction in chloroplasts

The effect of salt addition on the rate of reduction of P-700 oxidized by flash illumination was analyzed. In broken chloroplasts, the rate of P-700 reduction was accelerated by salts of mono-, di- and trivalent cations, with the increasing effectiveness in this order, in the presence of various artificial electron donors or acceptors. The rate was not dependent on the concentration and the valence of anions. On the other hand, in Photosystem I-enriched subchloroplast particles, added KCl did not induce the acceleration of direct reduction of P-700 by reduced DCIP.At low KCl concentrations (below 10 mM), the rate of P-700 reduction was also accelerated by added KCl in sonicated chloroplasts to which purified plastocyanin was added. The curves of dependence of the reduction rate on plastocyanin concentration were not of the Michaelis-Menten type, but sigmoidal. The maximal of P-700 reduction was higher at higher salt concentrations and the half-maximal plastocyanin concentration for P-700 reduction became lower with increasing NaCl concentrations.In broken chloroplasts treated with 50 mM glutaraldehyde, the rate of P-700 reduction was not accelerated by added KCl.The Debye-Hückel theory and the Gouy-Chapman theory were applied to our data to analyze the electrostatic interaction between electron tranfer components on thylakoid membranes. It is suggested that the major factor determining the rate of P-700 reduction is the donation of electrons from plastocyanin to P-700. Most of the observed effect is probably due to the increase in the local concentration or accessibility of plastocyanin to the site of P-700 reduction which is expected when the negative surface potential rises when salt is added.