Isolation of Photosystem II enriched membrane vesicles from spinach chloroplasts by phase partition

Partition in an aqueous Dextran-polyethylene glycol two-phase system has been used for the separation of chloroplast membrane vesicles obtained by press treatment of a grana-enriched fraction after unstacking in a low salt buffer.

The fractions obtained were analysed with respect to chlorophyll, photochemical activities and ultrastructural characteristics. The results reveal that the material partitioning to the Dextran-rich bottom phase consisted of large membrane vesicles possessing mainly Photosystem II properties with very low contribution from Photosystem I. Measurements of the H₂O to phenyl-p-benzoquinone and ascorbate-Cl₂Ind to NADP⁺ electron transport rates indicate a ratio of around six between Photosystem II and I.

The total fractionation procedure could be completed within 2–3 h with high recovery of both the Photosystem II water-splitting activity and the Photosystem I reduction of NADP⁺.

These data demonstrate that press treatment of low-salt destabilized grana membranes yields a population of highly Photosystem-II enriched membrane vesicles which can be discriminated by the phase system. We suggest that such membrane vesicles originate from large regions in the native grana membrane which contain virtually only Photosystem II.     

Inhibitory effect of p-nitrothiophenol in the light on the Photosystem II activity of spinach chloroplasts

The treatment of spinach chloroplasts with p-nitrothiophenol in the light at acidic and neutral pH's caused specific inhibition of the Photosystem II activity, whereas the same treatment in the dark did not affect the activity at all. The photosystem I activity was not inhibited by p-nitrothiophenol both in the light and in the dark. The inhibition was accompanied by changes of fluorescence from chloroplasts. As observed at room temperature, the 685-nm band was lowered by the p-nitrothiophenol treatment in the light and, at liquid nitrogen temperature, the relative height of the 695-nm band to the 685-nm band increased and the 695-nm band shifted to longer wavelengths. The action spectra for these effects of p-nitrothiophenol on the activity and fluorescence showed a peak at 670 nm with a red drop at longer wavelengths. It was concluded that the light absorbed by Photosystem II is responsible for the chemical modification of chloroplasts with p-nitrothiophenol to causing the specific inhibition of Photosystem II.     

Chloroplast membranes of the green alga Acetabularia mediterranea. I. Isolation of the Photosystem II

1. In the presence of Triton X-100, chloroplast membranes of the green alga Acetabularia mediterranea were disrupted into two subchloroplast fragments which differed in buoyant density. Each of these fractions had distinct and unique complements of polypeptides, indicating an almost complete separation of the two fragments.

2. One of the two subchloroplast fractions was enriched in chlorophyll b. It exhibited Photosystem II activity, was highly fluorescent and was composed of particles of approx. 50 Å diameter.

3. The light-harvesting chlorophyll-protein complex of the Photosystem II-active fraction had a molecular weight of 67 000 and contained two different subunits of 23 000 and 21 500. The molecular ratio of these two subunits was 2:1.     

Studies on electron transport associated with Photosystem I. I. Functional site of plastocyanin: Inhibitory effects of HgCl2 on electron transport and plastocyanin in chloroplasts

1. Incubation of chloroplasts with HgCl₂ at a molar ratio of HgCl₂ to chlorophyll of about unity, induced a complete inhibition of the methyl viologen Hill reaction, as well as methyl viologen photoreduction with reduced 2,6-dichlorophenolindophenol (DCIP) as electron donor. Photooxidation of cytochrome ? was similarly sensitive towards HgCl₂, whereas photooxidation of P700 was resistant to the poison. Photoreduction of cytochrome ? and light-induced increase in fluorescence yield were enhanced by the HgCl₂ treatment of chloroplasts.     

The effect of magnesium ions on action spectra for reactions mediated by Photosystems I and II in spinach chloroplasts

Action spectra were measured for positive changes in variable fluorescence (emission > 665 nm) excited by a beam of 485 nm chopped at 75 Hz. The action of two further beams was compared, one being variable, the other (reference) constant with respect to wavelength and intensity. Comparison was achieved by alternating the reference and the variable wavelength beams at 0.3 Hz and adjusting the intensity of the latter such as to cancel out any 0.3 Hz component in the 75 Hz fluorescence signal. The relative action then was obtained as the reciprocal of the intensity of the variable wavelength beam. Similarly, action spectra were measured for O₂ evolution with ferricyanide/p-phenylenediamine as electron acceptor, and for O₂ uptake mediated by methyl viologen with ascorbate 3-(p-chlorophenyl)-1,1-dimethylurea as electron donor in the presence of 2,6-dichlorophenolindophenol.Addition of 5 mM MgCl₂ increases the relative action around 480 nm for the change in variable fluorescence and p-phenylenediamine-dependent O₂ evolution, and decreases it for methyl viologen-mediated O₂ uptake with 2,6-dichlorophenolindophenol/ascorbate as electron donor in the presence of 3-(p-chlorophenyl-1,1-dimethylurea. The change in variable fluorescence and O₂ evolution are stimulated by MgCl₂, whereas O₂ uptake is inhibited by it.The results are discussed in terms of a model assuming a tripartite organization. of the photosynthetic pigments (Thornber, J. P. and Highkin, H. R. (1974) Eur. J. Biochem. 41, 109–116; Butler, W. L. and Kitajima, M. (1975) Biochim. Biophys. Acta 396, 72–85). MgCl₂ is thought to promote energy transfer to Photosystem II from a light-harvesting pigment complex serving both photosystems.     

Effects of α-benzyl-α-bromo-malodinitrile on the primary electron acceptor of Photosystem II in spinach chloroplasts

Reactions at the reducing side of Photosystem II in spinach chloroplasts are modified by α-benzyl-α-bromo-malodinitrile (BBMD).On addition of 50 μM BBMD to chloroplasts the following phenomena can be observed: (1) electron flow to an acceptor like 2,6-dichlorophenolindophenol is partly deflected to electron flow to oxygen; (2) the electron flow to oxygen is carbonyl cyanide m-chlorophenylhydrazone sensitive but 3-(3,4-dichlorophenyl)-1,1-dimethylurea insensitive; (3) variable fluorescence is abolished but basal fluorescence is not altered; (4) a strong photobleaching of carotenoids is induced. BBMD seems a very efficient acceptor for electrons from the primary electron acceptor of Photosystem II, resulting in a BBMD-mediated electron transport from this primary acceptor to oxygen.On pretreatment of chloroplasts with 50 μM BBMD the effects are different; (1) electron flow to 2,6-dichlorophenolindophenol, ferricyanide, or NADP is almost completely inhibited and is not restored by addition of artificial electron donors: (2) no electron flow to oxygen is observable unless BBMD again is added to reaction media; (3) no variable fluorescence is observable but basal fluorescence is not affected; (4) there is no photobleaching of carotenoids unless BBMD again is added; (5) no reduction of C-550 can be recorded. Pretreatment of chloroplasts with BBMD seems to induce an intense cycling of electrons around Photosystem II and only anew added BBMD can interrupt this cycling.     

3-(3,4-Dichlorophenyl)-1, 1-dimethylurea-insensitive oxygen production in a cell-free preparation from Phormidium luridum that shows redox potential dependent coupling to one-electron oxidants

A cell-free preparation has been isolated from Phormidium luridum that evolves oxygen when coupled to one-electron oxidants, that is insensitive to 3-(3,4-dichlorophenyl)-1,1-dimethylurea, and that yields oxygen at a rate dependent on redox potential. In this preparation the Hill oxidant couples closer to the oxygen-producing apparatus than in any other cell-free system. Light saturation curve data for the cell-free preparation shows a stabilization, by the Hill oxidant, of intermediates in oxygen synthesis. In whole cells coupled to CO₂ or to K₃ Fe(CN)₆ no such stabilization occurs and a 2nd order light intensity dependence of the oxygen-production rate is observed.     

Primary reactions of Photosystem II at low pH. 2. Light-induced changes of absorbance and electron spin resonance in spinach chloroplasts

The effects of lowering the pH on Photosystem II have been studied by measuring changes in absorbance and electron spin resonance in spinach chloroplasts.At pH values around 4 a light-induced dark-reversible chlorophyll oxidation by Photosystem II was observed. This chlorophyll is presumably the primary electron donor of system II. At pH values between 5 and 4 steady state illumination induced an ESR signal, similar in shape and amplitude to signal II, which was rapidly reversed in the dark. This may reflect the accumulation of the oxidized secondary donor upon inhibition of oxygen evolution. Near pH 4 the rapidly reversible signal and the stable and slowly decaying components of signal II disappeared irreversibly concomitant with the release of bound manganese.The results are discussed in relation to the effects of low pH on prompt and delayed fluorescence reported earlier (van Gorkom, H. J., Pulles, M. P. J., Haveman, J. and den Haan, G. A. (1976) Biochim. Biophys. Acta 423, 217–226).     

C-550 in photosystem II subchloroplast particles

The photoreactions mediated by Photosystem II at low temperatures were examined in subchloroplast particles enriched in Photosystem II to determine if the Photosystem II activity was independent of C-550 in such particle preparations. Two types of Photosystem II particle preparations were tested, one enriched in chlorophyll b and the other purified further to eliminate the chlorophyll b. The Photosystem II-mediated photoreactions at low temperature were the same in both of the Photosystem II particle preparations as they were in normal chloroplasts. C-550 acted as if it were the primary electron acceptor of Photosystem II in all of the experiments performed.     

The rapid component of electron paramagnetic resonance signal II: A candidate for the physiological donor to Photosystem II in spinach chloroplasts

Rapid light-induced transients in EPR Signal IIf (F^?+) are observed in 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)-treated, Tris-washed chloroplasts until the state F P680 Q^? is reached. In the absence of exogenous redox mediators several flashes are required to saturate this photoinactive state. However, the Signal IIf transient is observed on only the first flash following DCMU addition if an efficient donor to Signal IIf, phenylenediamine or hydroquinone, is present. Complementary polarographic measurements show that under these conditions oxidized phenylenediamine is produced only on the first flash of a series. The DCMU inhibition of Signal IIf can be completely relieved by oxidative titration of a one-electron reductant with E′_08.0 = +480 mV. At high reduction potentials the decay time of Signal IIf is constant at about 300 ms, whereas in the absence of DCMU the decay time is longer and increases with increasing reduction potential.A model is proposed in which Q^?, the reduced Photosystem II primary acceptor, and D, a one-electron 480 mV donor endogenous to the chloroplast suspension, compete in the reduction of Signal IIf (F^?+). At high potentials D is oxidized in the dark, and the (Q^? + F^?+) back reaction regenerates the photoactive F P680 Q state. The electrochemical and kinetic evidence is consistent with the hypothesis that the Signal IIf species, F, is identical with Z, the physiological donor to P680.     

Kinetics of reduction of the oxidized primary electron donor of Photosystem II in spinach chloroplasts and in Chlorella cells in the microsecond and nanosecond time ranges following flash excitation

Absorption changes (ΔA) at 820 nm, following laser flash excitation of spinach chloroplasts and Chlorella cells, were studied in order to obtain information on the reduction time of the photooxidized primary donor of Photosystem II at physiological temperatures.In the microsecond time range the difference spectrum of ΔA between 750 and 900 nm represents a peak at 820 nm, attributable to a radical-cation of chlorophyll a. In untreated dark-adapted material the signal can be attributed solely to P⁺?700; it decays in a polyphasic manner with half-times of 17 μs, 210 μs and over 1 ms. The oxidized primary donor of Photosystem II (P⁺_II) is not detected with a time resolution of 3 μs. After treatment with 3–10 mM hydroxylamine, which inhibits the donor side of Photosystem II, P⁺_II is observed and decays biphasically (a major phase with $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 20–40 μ}\text{s}$, and a minor phase with $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{? 200 μ}\text{s}$), probably by reduction by an accessory electron donor.In the nanosecond range, which was made accessible by a new fast-response flash photometer operating at 820 nm, it was found the P⁺_II is reduced with a half-time of 25–45 ns in untreated dark-adapted chloroplasts. It is assumed that the normal secondary electron donor is responsible for this fast reduction.     

Iodination of chloroplasts. I. Properties of iodinated chloroplasts

Spinach chloroplasts exposed to iodide can be washed free of the bulk of the iodide. In the presence of lactoperoxidase and H₂O₂, iodide can be introduced into chloroplasts in high amounts and in non diffusible forms. The resultant particles, which have been named iodochloroplasts, extrude their iodide upon stimulation by light. The form and the amount of extruded iodide bears a definite relationship to the amount of incident light. A flash of marginally effective light is additive to the next such flash even after a lapse of 10 min of darkness. These and other properties of iodochloroplasts may make them of great use in the study of intermediate reactions of photosynthesis.     

Primary and secondary electron donors in Photosystem II of chloroplasts. Rates of electron transfer and location in the membrane

Absorption changes at 820 or 515 nm after a short laser flash were studied comparatively in untreated chloroplasts and in chloroplasts in which oxygen evolution is inhibited.In chloroplasts pre-treated with Tris, the primary donor of Photosystem II (P-680) is oxidized by the flash, as observed by an absorption increase at 820 nm. After the first flash it is re-reduced in a biphasic manner with half-times of 6 μs (major phase) and 22 μs. After the second flash, the 6 μs phase is nearly absent and P-680⁺ decays with half-times of 130 μs (major phase) and 22 μs. Exogenous electron donors (MnCl₂ or reduced phenylenediamine) have no direct influence on the kinetics of P-680⁺.In untreated chloroplasts the 6 and 22 μs phases are of very small amplitude, either at the 1st, 2nd or 3rd flash given after dark-adaptation. They are observed, however, after incubation with 10 mM hydroxylamine.These results are interpreted in terms of multiple pathways for the reduction of P-680⁺: a rapid reduction (<1 μs) by the physiological donor D₁; a slower reduction (6 and 22 μs) by donor D′₁, operative when O₂ evolution is inhibited; a back-reaction (130 μs) when D′₁ is oxidized by the pre-illumination in inhibited chloroplasts. In Tris-treated chloroplasts the donor system to P-680⁺ has the capacity to deliver only one electron.The absorption change at 515 nm (electrochromic absorption shift) has been measured in parallel. It is shown that the change linked to Photosystem II activity has nearly the same magnitude in untreated chloroplasts or in chloroplasts treated with hydroxylamine or with Tris (first and subsequent flashes). Thus we conclude that all the donors (P-680, D₁, D′₁) are located at the internal side of the thylakoid membrane.     

Light-induced changes of absorbance and electron spin resonance in small Photosystem II particles

Photosystem II reaction center components have been studied in small system II particles prepared with digitonin. Upon illumination the reduction of the primary acceptor was indicated by absorbance changes due to the reduction of a plastoquinone to the semiquinone anion and by a small blue shift of absorption bands near 545 nm (C550) and 685 nm. The semiquinone to chlorophyll ratio was between 1/20 and 1/70 in various preparations. The terminal electron donor in this reaction did not cause large absorbance changes but its oxidized form was revealed by a hitherto unknown electron spin resonance (ESR) signal, which had some properties of the well-known signal II but a linewidth and g-value much nearer to those of signal I. Upon darkening absorbance and ESR changes decayed together in a cyclic or back reaction which was stimulated by 3-(3,4 dichlorophenyl)-1,1-dimethylurea. The donor could be oxidized by ferricyanide in the dark.

Illumination in the presence of ferricyanide induced absorbance and ESR changes, rapidly reversed upon darkening, which may be ascribed to the oxidation of a chlorophyll a dimer, possibly the primary electron donor of photosystem II. In addition an ESR signal with 15 to 20 gauss linewidth and a slower dark decay was observed, which may have been caused by a secondary donor.     

Light-induced changes of fluorescence and absorbance in spinach chloroplasts at −40 °C

1. 1. Spinach chloroplasts were stored in the dark for at least 1 h, rapidly cooled to −40 °C, and illuminated with continuous light or short saturating flashes. In agreement with the measurements of Joliot and Joliot, chloroplasts that had been preilluminated with one or two flashes just before cooling showed a less efficient increase in the yield of chlorophyll a fluorescence upon illumination at −40 °C than dark-adapted chloroplasts. The effect disappeared below −150 °C, but reappeared again upon warming to −40 °C. Little effect was seen at room temperature in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), added after the preillumination.

2. 2. Light-induced absorbance difference spectra at −40 °C in the region 500–560 nm indicated the participation of two components, the socalled 518-nm change (P518) and C-550. After preillumination with two flashes the absorbance change at 518 nm was smaller, and almost no C-550 was observed. After four flashes, the bands of C-550 were clearly visible again.

3. 3. The fluorescence increase and the absorbance change at 518 nm showed the same type of flash pattern with a minimum after the second and a maximum at the fourth flash. In the presence of 100 μM hydroxylamine, the fluorescence response was low after the fourth and high again after the sixth flash, which confirmed the hypothesis that the flash effect was related to the so-called S-state of the electron transport pathway from water to Photosystem 2.

4. 4. The kinetics of the light-induced absorbance changes were the same at each wavelength, and, apart from the size of the deflection, they were independent of preillumination. Flash experiments indicated that the absorbance changes were a one-quantum reaction. This was also true for the fluorescence increase in dark-adapted chloroplasts, but with preilluminated chloroplasts several flashes were needed to approximately saturate the fluorescence yield.

5. 5. The results are discussed in terms of a mechanism involving two electron donors and two electron acceptors for System 2 of photosynthesis.

Photosynthetic electron transport and phosphorylation reactions in thylakoid membranes from the blue-green alga Anacystis nidulans

Thylakoid membranes were prepared from the blue-green alga, Anacystis nidulans with lysozyme treatment and a short period of sonic oscillation. The thylakoid membrane preparation was highly active in the electron transport reactions such as the Hill reactions with ferricyanide and with 2,6-dichlorophenolindophenol, the Mehler reaction mediated by methyl viologen and the system 1 reaction with methyl viologen as an electron acceptor and 2,6-dichlorophenolindophenol and ascorbate as an electron donor system. The Hill reaction with ferricyanide and the system 1 reaction was stimulated by the phosphorylating conditions. The cyclic and non-cyclic phosphorylation was also active.These findings suggest that the preparation of thylakoid membranes retained the electron transport system from H₂O to reaction center 1, and that the phosphorylation reaction was coupled to the Hill reaction and the system 1 reaction.