Spinach Thylakoid Membranes Research Articles

Inhibitory Effect of Crown Compound on Photoelectron Transport Activity of Beet Spinach Thylakoid Membranes

Abstract The effect of K-picrate-18-crown-6 (crown) on the photoelectron transport activity of beet spinach thylakoid membranes was investigated. Addition of micromolar concentration of crown to thylakoid preparation inhibited p-benzoquinone, chloride-indophenol, methyl viologen supported Hill activities maximally by 75 per cent in a concentration dependent manner. However, the photosystem I catalyzed reaction remained insensitive to crown suggesting that crown specifically inhibits photosystem II electron transport. Addition of exogenous electron donors like hydroxylamine or diphenylcarbazide failed to restore the crown induced inhibition of photosystem II electron transport and lowering of steady state chlorophyll a fluorescence yield. These observations suggest that crown also inhibits photosystem II catalyzed electron transport after the donation sites of these exogenous donors. Washing of the crown pre-treated thylakoids with isolation buffer, relieved the crown inhibited electron transport activity, indicating that this inhibition is reversible. Furthermore, in hydroxylamine washed thylakoids which are devoid of O2 evolution capacity, the hydroxylamine induced increase in chlorophyll a fluorescence of variable yield was quenched by the addition of crown. These observations suggest that crown affects the oxygen evolution and inhibits at a site close to photosystem II reaction centres.

The 55-kDa polypeptide released from spinach thylakoid membranes with 1 M LiCl is not the beta subunit of chloroplast F1.

It was reported by Frasch et al. (Frasch, W. D., Green, J., Caguiat, J., and Mejia, A. (1989) J. Biol. Chem. 264, 5064-5069) that washing spinach thylakoid membranes with 1 M LiCl caused the release of the beta subunit of chloroplast F1 (CF1) which, existing as 180-kDa complexes of beta 3, retained considerable ATPase activity. We repeated their procedures and confirmed that a CF1 beta-like 55-kDa polypeptide was a major constituent of the 1 M LiCl-washed extract. However, the extract contained another polypeptide of which the Mr was 14,000, and these two polypeptides comprised a complex with approximate Mr 550,000 that had the same mobility in native polyacrylamide gel electrophoresis as that of ribulose-1,5-bisphosphate carboxylase. Only very low ATPase activity, less than 1% of the reported value, was detected for the extract and the purified complex. Antibody against the beta subunit of F1 from a thermophilic bacterium PS3 showed a clear cross-reactivity with the CF1 beta subunit but not with the 55-kDa polypeptide. Analysis of the N-terminal amino acid sequences of the 55- and 14-kDa polypeptides and the whole complex revealed that the complex was ribulose-1,5-bisphosphate carboxylase and that the 55- and 14-kDa polypeptides were its large and small subunits, respectively.

Restoration of light induced photosystem II inhibition without de novo protein synthesis

Illumination of isolated spinach thylakoid membranes under anaerobic conditions gave rise to severe inhibition of photosystem II electron transport but did not result in D 1-protein degradation. When these photoinhibited thylakoids were incubated in total darkness the photosystem II activity could be fully restored in vitro in a process that required 1–2 h for completion.

Light saturation response of inactive photosystem II reaction centers in spinach

The effective absorption cross section of inactive photosystem II (PS II) centers, which is the product of the effective antenna size and the quantum yield for photochemistry, was investigated by comparing the light saturation curves of inactive PS II and active reaction centers in intact chloroplasts and thylakoid membranes of spinach (Spinacia oleracea). Inactive PS II centers are defined as the impaired PS II reaction centers that require greater than 50 ms for the reoxidation of QA (-) subsequent to a single turnover flash. Active reaction centers are defined as the rapidly turning over PS II centers (recovery time less than 50 ms) and all of the PS I centers. The electrochromic shift, measured by the flash-induced absorbance increase at 518 nm, was used to probe the activity of the reaction centers. Light saturation curves were generated for inactive PS II centers and active reaction centers by measuring the extent of the absorbance increase at 518 nm induced by red actinic flashes of variable energy. The light saturation curves show that inactive PS II centers required over twice as many photons as active reaction centers to achieve the same yield. The ratio of the flash energy required for 50% saturation for active reaction centers (PS II active + PS I) compared to inactive PS II centers was 0.45±0.04 in intact chloroplasts, and 0.54±0.11 in thylakoid membranes. Analysis of the light saturation curves using a Poisson statistical model in which the ratio of the antenna size of active PS II centers to that of PS I is considered to range from 1 to 1.5, indicates that the effective absorption cross section of inactive PS II centers was 0.54-0.37 times that of active PS II centers. If the quantum yield for photochemistry is assumed to be one, we estimate that the antenna system serving the inactive PS II centers contains approx. 110 chlorophyll molecules.

Chlorophyll α fluorescence measurements of isolated spinach thylakoids obtained by using single‐laser‐based flow cytometry

Flow cytometry data of spinach thylakoid membrane preparations indicate the presence of a homogeneous thylakoid population. Fluorescence data from a flow cytometer and comparison with data from two other fluorometers show that chlorophyll a fluorescence detected with a flow cytometer has the character of maximum fluorescence (Fmax), not of the constant component (Fo). This conclusion is important since Fo measures fluorescence that is affected mostly by changes in excitation energy transfer and Fmax-Fo (the variable fluorescence) by changes in photochemistry. This was demonstrated by: 1) The light intensity as well as diffusion rate dependence of the quenching effect of various quinones (p-benzoquinone, phenyl-benzoquinone, and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, DBMIB) on fluorescence yield; quenching for the same concentration of these quinones was lower at the higher than at the lower light intensities. 2) Temperature dependence of the fluorescence yield; increasing the temperature from 20 to 70 degrees C did not show an increase in fluorescence yield using a flow cytometer in contrast to measurements with weak excitation light, but similar to those obtained for Fmax. 3) Addition of an inhibitor diuron up to 100 microM did not change the fluorescence intensity. A comparison of quenching of fluorescence by various quinones obtained by flow cytometry with those by other fluorometers suggests that the high intensity used in the cytometry produces unique results: the rate of reduction of quinones in much larger than the rate of equilibration with the bulk quinones.

Studies on the heterogeneity of the soluble chloroplast coupling factor 1: The formation of ε-deficient isozymes

Release of the chloroplast coupling factor 1 (CF1) from thylakoid membranes by chloroform yields a heterogeneous population of isozymes. Following solubilization, subtle changes in the isozyme distribution occur. In particular, the percentage of e-deficient isozymes increases. Five parameters were evaluated for their role in the distribution of isozymes and net enzyme yield, (i) Extraction of spinach thylakoid membranes yields a five-subunit holoenzyme and derivative isozymes whereas Chlamydomonas reinhardtii membranes yield isozymes lacking a δ-subunit. (ii) Supplementing extraction buffers with MgCl2 or CaCl2 dramatically suppresses the release of total CF1, but does not appear to affect the distribution of isozymes. (iii) Loss of CF 1by non-specific protein denaturation becomes detectable 24–48 h after enzyme solubilization. Analysis of CF1 samples stored at room temperature for extended periods (up to 2 months) shows that the loss of solubility is uniform for the isozyme complex, rather than selective for agiven subunit. (iv) Proteolysis does not significantly affect the distribution of the e-subunit either before or after enzyme solubilization. (v) Dithiothreitol greatly enhances the formation of e-deficient isozymes from e-containing isozymes in both species studied and appears to account for the subtle changes observed following CF1 solubilization.

Ammonia binds to the catalytic manganese of the oxygen-evolving complex of photosystem II. Evidence by electron spin-echo envelope modulation spectroscopy

The mechanism of ammonia inhibition of photosynthetic oxygen evolution has been examined by the pulsed EPR technique of electron spin-echo envelop modulation (ESEEM), revealing the direct coordination of an ammonia-derived ligand to the catalytic Mn complex during the S{sub 1} {yields}S{sub 2} transition of the oxygen evolution S-state cycle. ESEEM experiments were performed on the multiline Mn EPR signal observed in photosystem II enriched spinach thylakoid membranes which were treated with either {sup 14}NH{sub 4}Cl or {sup 15}NH{sub 4}Cl (100 mM, pH 7.5). {sup 15}NH{sub 4}Cl treatment produced modulation of the electron spin-echo signal which arises from an I = 1/2 {sup 15}N nucleus with an isotropic hyperfine coupling A({sup 15}N) = 3.22 MHz. {sup 14}NH{sub 4}Cl treatment produced a different ESEEM pattern resulting from an I = 1 {sup 14}N nucleus with A({sup 14}N) = 2.29 MHz, and with electric quadrupole parameters e{sup 2}qQ = 1.61 MHz and {eta} = 0.59. The {sup 14}n electric quadrupole parameters are interpreted with respect to possible chemical structures for the ligand. An amido (NH{sub 2}) bridge between metal ions is proposed as the molecular identity of the ammonia-derived ligand to the catalytic Mn of photosystem II.

Comparative study of the photochemistry of chloroplast membranes and photosystem II particles

A comparative study of the photoreducing potentials of spinach thylakoid membranes and spinach photosystem II particles has been made. Hexachloroplatinate ions have been used as electron acceptors in a Hill-like assay for oxygen evolution measurements with both thylakoid membranes and photosystem II particles. However, unlike other Hill acceptors, such as ferricyanide, hexachloroplatinate can be fully reduced to metallic platinum that is catalytically active for hydrogen evolution. This is experimentally confirmed in the ability of chloroplast membranes to photoprecipitate platinum and photoproduce molecular hydrogen. Although similar experiments with photosystem II particles resulted in hexachloroplatinate-supported oxygen evolution, hydrogen evolution was not observed. Moreover, photosystem II particles coupled to ferredoxin and hydrogenase resulted in neither hydrogen nor oxygen evolution—a distinct contrast to the results obtained with chloroplast membranes.

Ferredoxin Cross-Links to a 22 kD Subunit of Photosystem I

We have used a cross-linking approach to study the interaction of ferredoxin (Fd) with photosystem I (PSI). The cross-linking reagent N-ethyl-3-(3-dimethylaminopropyl) carbodiimide was found to cross-link spinach Fd to a 22 kilodalton subunit of PSI in both isolated spinach (Spinacia oleracea) PSI complexes and spinach thylakoid membranes. The product had an apparent molecular weight of 38 kilodaltons on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was identified as a cross-linked product using specific antibodies to Fd and the 22 kilodalton subunit. In both a native PSI complex (200 Chl/P700) and a PSI core complex (100 Chl/P700), a second cross-linked product at 36 kilodaltons was seen. The latter cross-reacted with an antibody to Fd but did not cross-react with antibodies directed against the 24.3, 22, 19, 17.3 or 8.5 kilodalton, or psaC subunits of PSI. Its composition remains to be determined. In thylakoids only the 38 kilodalton product was observed along with a cross-linked complex of Fd and Fd:NADP(+) reductase.

Further enzymatic characteristics of a thylakoid protein kinase.

The enzymatic characteristics of a protein kinase purified from thylakoids are further described. ATP (KM approximately 30 microM) and Mg2+ ion (greater than 1.0 mM) were required for activity, while ADP was a competitive inhibitor (Ki = 100 microM). Activity was 55% inhibited by the sulfhydryl inhibitor p-chloromercuribenzoate (1 mM) and was less sensitive to substituted maleimides. Lysine-rich histones (H1) were utilized as an exogenous phosphorylation substrate both by thylakoid-bound kinase and by isolated enzyme; threonine was predominantly phosphorylated by the in situ enzyme, whereas the isolated enzyme phosphorylated closely related serine residues as determined by peptide mapping. Detergents that proved useful in extracting the kinase from thylakoids markedly inhibited activity of the isolated enzyme, whereas Triton X-100 and 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonic acid had little effect. The enzyme could be freed from detergent and behaved as an active monomer on size-exclusion chromatography. The phosphate contents of the light-harvesting chlorophyll a/b protein complex of photosystem II isolated from maximally phosphorylated thylakoid membranes of spinach and pea were equivalent to approximately 6% and approximately 19% phosphorylation, respectively. Corresponding values for nonphosphorylated membranes were approximately 3% and approximately 14.5%.

Electron transfer through the quinone acceptor complex of Photosystem II after one or two actinic flashes in bicarbonate-depleted spinach thylakoid membranes

We report here the pH dependence of the kinetics of the decay of variable chlorophyll a fluorescence after one or two actinic flashes in the absence or the presence of DCMU (3-(3,4-dichlorophenyl)-1,1-dimethyl-urea) in HCO 2 −-depleted or anion-inhibited spinach thylakoid membranes. All the reported effects of HCO 3 − removal are reversed by the addition of 5 mM HCO 3 −. The initial first-order component for the oxidation of Q A − (the reduced primary plastoquinone acceptor of Photosystem II (PS II) by Q B (the secondary plastoquinone acceptor) was reversibly inhibited in a pH-dependent manner in HCO 3 −-depleted membranes. After a single actinic flash, the half-time of Q A − decay was 630 μs (amplitude, 29%) at pH 6.5 which changed to a value of 320 μs (amplitude, 66%) at pH 7.75. The rate and amplitude at pH 7.75 were approximately the same as found in the restored and control membranes which were pH independent over the same pH range. A similar observation was made after the second actinic flash. Thus, at alkaline pH HCO 3 −-depleted membranes behave as control membranes with respect to electron flow from Q A to Q B or to Q B −. The time ( t 50) at which the [Q A −] is 50% of the maximum [Q A −] during the back reaction between Q A − and the S 2 state of the oxygen-evolving complex, in the presence of 5 μM DCMU, was increased from 1.3 s in control and restored samples to 5.3 s in HCO 3 −-depleted samples below pH 7.0, but was unaffected above pH 7.5 (2.3–2.9 s in all cases). Furthermore, a new pathway of Q A − with a half-time of less than 100 μs was present at pH 8.0 in the presence of DCMU, in approx. one-third of the PS II centers in HCO 3 −-depleted membranes. The apparent equilibrium for the sharing of an electron between Q A and Q B is estimated to decrease by a factor of 4 at pH 6.0 in treated membranes ( K app ≈ 16) as compared to the restored or control membranes ( K app ≈ 62); there was no difference in K app at pH 7.75. Estimates of the operating redox potential for the Q B/Q B − couple from the results presented here indicated that the pH dependence of this parameter is greatly reduced in treated membranes (−60 mV at pH 6.0 to −72 mV at pH 7.75) as compared to restored or control membranes (−25 mV at pH 6.0 to −72 mV at pH 7.75). We iscuss our results in the context of a model that envisages HCO 3 − to act as a proton donor to the protein dissociable group believed to participate in the protonation of Q B −. Finally, the possibility of HCO 3 − being a ligand to Fe 2+ in the Q A-Fe-Q B complex of the PS II reaction center is also discussed.

Electron transfer through the quinone acceptor complex of Photosystem II in bicarbonate-depleted spinach thylakoid membranes as a function of actinic flash number and frequency

In this paper four major points with respect to HCO 3 −-reversible inhibition in spinach thylakoid membranes, depleted of HCO 3 − in the presence of inhibitory anions, are established. (1) The oxidation of Q A − (Q A is the primary quinone acceptor of Photosystem II) by Q B (Q B is the secondary quinone acceptor of Photosystem II) or Q B −, following one or two actinic flashes, respectively, exhibits a smaller t 50 time at which [Q A −] is 50% of maximum [Q A −]) after a flash at pH 7.5 than at pH 6.5 (2) The characteristic oscillations, due to differential rates of Q A − oxidation by Q B or Q B −, observed in the fluorescence flash pattern, generated by assaying the chlorophyll a fluorescence at specific times after an actinic flash and plotting these data as a function of flash number, are lost (i.e., the turnover of two electron gate is hampered). (3) At 1 Hz, the slowest oxidation of Q A −, as indicated by f 50 values, depends on both the pH and flash number: at pH 6.5, t 50 for Q A − decay reaches a maximum value after only three flashes (one turnover of the two-electron gate), whereas at pH 7.5, the t 50 is increased further until after five flashes (two turnovers of the two-electron gate). (4) The t 50 values of Q A − oxidation also depend on the actinic flash frequency: at 5 Hz, [Q A −] reaches its maximum after flash 5 even at pH 6.5. The increase observed in the t 50 values of Q A − oxidation in treated membranes is accompanied by the presence of slow components of Q A − oxidation in the 0.1–10 s range which can achieve an amplitude of more than 70%. These components are suggested to be related to protonation steps involved in the quinone acceptor complex of Photosystem II and support the conclusion that the rate-limiting step in electron transfer in HCO 3 −-depleted thylakoids may be the protonation of Q B − and possibly Q B 2−. A working hypothesis is presented that explains the flash, frequency and pH dependence of Q A − decay observed in this paper.

Photosystem II disorganization and manganese release after photoinhibition of isolated spinach thylakoid membranes

The activity, the protein content and the manganese properties of photosystem II have been compared after photoinhibition of isolated thylakoid membranes. The results show a concomitant disappearance of the oxygen evolving activity and the ability to form the S 2-state multiline EPR signal. The D 1-protein is degraded in a subsequent event which closely correlates to release of manganese from the thylakoid membranes.

Nucleotide sequence of cDNA clones encoding the complete precursor for subunit delta of thylakoid-located ATP synthase from spinach

The nucleotide sequence of the entire nuclear-encoded precursor for subunit delta of the ATP synthase from spinach thylakoid membranes was determined by cDNA sequencing. Appropriate recombinant DNAs were selected from pBR322 and lambda gt11 libraries made from polyadenylated RNA of greening spinach seedlings. The mature protein consists of 187 amino acid residues corresponding to a molecular weight of 20468. The precursor protein (257 amino acid residues; M r=27676) is probably processed between a Met-Val bond. The predicted secondary structure of the transit sequence (70 residues; 7.2 kDa) resembles that of the Rieske Fe/S polypeptide, but shows little similarity with those of stromal or luminal proteins. The comparison of the chloroplast delta amino acid sequence with the published delta sequences from respiratory ATP synthases of bacterial and mitochondrial sources and from the thylakoid ATP synthase of the cyanobacterium Synechococcus suggests substantial divergence at the genic level although structural elements appear to be remarkably conserved.

ALL‐trans β‐CAROTENE‐5,6‐EPOXIDE IN THYLAKOID MEMBRANES

AbstractAll‐transβ‐carotene‐5,6‐epoxide has been found in the thylakoid membranes of spinach and of the cyanobacterium Synechococcus vulcanus Copeland. The epoxide was extracted from the thylakoid membranes with acetone, and was isolated by high‐performance liquid chromatography (HPLC). The structure of the epoxide was identified by means of mass, Raman, and electronic absorption spectroscopy. Changes in the amount of the epoxide, as a result of epoxidation and (apparent) de‐epoxidation reactions in the membranes, were traced by analysis of extracts on HPLC. In isolated thylakoid membranes, only the epoxidation reaction took place. The reaction was caused by irradiation or by the addition of ferricyanide, suggesting that electron transport reactions in the membranes are involved in the epoxidation. In intact spinach leaves, however, both epoxidation and de‐epoxidation took place; the extent of epoxidation correlated with the intensity of light incident on the leaves. The epoxidation and de‐epoxidation of all‐transβ‐carotene are contrasted with those of xanthophylls (in the violaxanthin cycle).

Expression of the psbA Gene in E. coli

A psbA gene fragment has been cloned into an expression vector in frame with the 3′end of the beta-galactosidase gene. Expression of this construct in E. coli results in an overproduction of a hybrid protein consisting of part of the herbicide binding protein and beta-galactosidase. An antiserum raised against this fusion protein specifically detects a 34 kDa polypeptide within the complex mixture of spinach thylakoid membrane proteins.

Reassembly of solubilized chlorophyll-protein complexes in proteolipid particles — Comparison of monogalactosyldiacylglycerol and two phospholipids

Formation and properties of chlorophyll-proteolipid particles assembled from Triton-X-100-solubilized spinach thylakoid membrane proteins and monogalactosyldiacylglycerol (MG), or phosphatidylcholine (PC), or phosphatidylethanolamine (PE), were compared. MG particles differed from phospholipid particles in three respects. (1) Chlorophyll proteins were more readily embedded into MG particles than into PE or PC particles. (2) MG particles contained chlorophyll proteins and cytochromes in their in vivo ratios, whereas PE or PC particles were enriched in the light-harvesting chlorophyll a b- protein complex (LHC). (3) As judged by 77 K fluorescence emission spectra and P-700 oxidation kinetics, energy transfer from LHC to reaction center I which was lost in solubilized thylakoids, was restored almost completely in MG particles. In contrast, energy transfer was restored only partially or not at all in proteolipid particles formed with PE or PC, respectively. These unique properties of MG might be linked to its role as major lipid in thylakoid membranes.

Absolute absorption and relative fluorescence excitation spectra of the five major chlorophyll-protein complexes from spinach thylakoid membranes

The absolute absorption and relative fluorescence excitation spectra have been obtained for the five major chlorophyll-protein complexes from spinach chloroplasts, resolved by mild SDS-polyacrylamide gel electrophoresis. Electro-elution of the chlorophyll-protein complexes from the gels enabled their absolute absorption spectra to be determined. The mean molar extinction coefficient from 400 to 720 nm was independent of the type of protein complex and the chlorophyll a b ratio, averaging 2230 ± 40 m 2 per mol chlorophyll. The mean molar extinction coefficient was used to normalize the absorption spectra of the protein complexes while in the gel slices. The distribution of chlorophyll between the pigment complexes can be determined at 662 nm rather than from the mean of two scans at 650 nm and 675 nm. Recombining the absorption spectra from the pigment-protein complexes gave reasonable absolute agreement with the spectrum of spinach thylakoids. However, the solubilization led to (i) a shift in the red peak to shorter wavelengths by 4.5 nm, and (ii) a loss of carotenoids which also shifted the blue shoulder to shorter wavelengths. The absorption spectra for the two photosystems were predicted with phosphorylated (State 2) and nonphosphorylated (State 1) light-harvesting complexes. The 77 K fluorescence excitation spectra of the chlorophyll-protein complexes were measured in an attempt to assess the excitation spectra of PS I and PS II in terms of their chlorophyll-protein complexes. Due to the relative nature of fluorescence detection, the ratio of fluorescence excitation at 470 relative to that at 400 nm was used to compare the spectra. The discrepancies between the absorption and fluorescence spectra for the Photosystem I complexes meant that fluorescence emission was an insensitive method for assessing the degree of association of the light-harvesting chlorophyll a b- complex with each photosystem.

Expression of the psbA Gene in E. coli

A psbA gene fragment has been cloned into an expression vector in frame with the 3′ end of the beta-galactosidase gene. Expression of this construct in E. coli results in an overproduction of a hybrid protein consisting of part of the herbicide binding protein and beta-galactosidase. An antiserum raised against this fusion protein specifically detects a 34 kDa polypeptide within the complex mixture of spinach thylakoid membrane proteins.

Photoinhibition: Impairment of the primary charge separation between P-680 and pheophytin in photosystem II of chloroplasts

The question of the primary site of photoinhibition was investigated using the light-induced absorbance change of pheophytin a (at 685 nm) in spinach chloroplasts and Chlamydomonas reinhardtii cells. Photoinhibition of spinach thylakoid membranes resulted in a parallel decrease in the amplitude of the pheophytin a (Δ A 685) and Q A (Δ A 320) photoreduction signals. In intact Chlamydomonas reinhardtii cells the pheophytin photoreduction and oxygen evolution activity exhibited a similar decrease during photoinhibition. A complete recovery of both activities was attained within 60 min incubation in normal growth conditions. It is concluded that the primary site of photoinhibition involves the components (P-680 and/or pheophytin) of the primary charge separation.