The role of chloride ion in Photosystem II I. Effects of chloride ion on Photosystem II electron transport and on hydroxylamine inhibition

1. Chloroplasts washed with Cl^?-free, low-salt media (pH 8) containing EDTA, show virtually no DCMU-insensitive silicomolybdate reduction. The activity is readily restored when 10 mM Cl^? is added to the reaction mixture. Very similar results were obtained with the other Photosystem II electron acceptor 2,5-dimethylquinone (with dibromothymoquinone), with the Photosystem I electron acceptor FMN, and also with ferricyanide which accepts electrons from both photosystems.2. Strong Cl^?-dependence of Hill activity was observed invariably at all pH values tested (5.5–8.3) and in chloroplasts from three different plants: spinach, tobacco and corn (mesophyll).3. In the absence of added Cl^? the functionally Cl^?-depleted chloroplasts are able to oxidize, through Photosystem II, artificial reductants such as catechol, diphenylcarbazide, ascorbate and H₂O₂ at rates which are 4–12 times faster than the rate of the residual Hill reaction.4. The Cl^?-concentration dependence of Hill activity with dimethylquinone as an electron acceptor is kinetically consistent with the typical enzyme activation mechanism: E(inactive) + Cl^?ag E · Cl^? (active), and the apparent activation constant (0.9 mM at pH 7.2) is unchanged by chloroplast fragmentation.5. The initial phase of the development of inhibition of water oxidation in Cl^?-depleted chloroplasts during the dark incubation with NH₂OH ($\text{1}\text{2}$ H₂SO₄) is 5 times slower when the incubation medium contains Cl^? than when the medium contains NH₂OH alone or NH₂OH plus acetate ion. (Acetate is shown to be ineffective in stimulating O₂ evolution.)6. We conclude that the Cl^?-requiring step is one which is specifically associated with the water-splitting reaction, and suggests that Cl^? probably acts as a cofactor (ligand) of the NH₂OH-sensitive, Mn-containing O₂-evolving enzyme.     

Photosystem II energy coupling in chloroplasts with H2O2 as electron donor.

NH2OH-treated, non-water-splitting chloroplasts can oxidize H2O2 to O2 through Photosystem II at substantial rates (100--250 muequiv . h-1 . mg-1 chlorophyll with 5 mM H2O2) using 2,5-dimethyl-p-benzoquinone as an electron acceptor in the presence of the plastoquinone antagonist dibromothymoquinone. This H2O2 leads to Photosystem II leads to dimethylquinone reaction supports phosphorylation with a P/e2 ratio of 0.25--0.35 and proton uptake with H+/e values of 0.67 (pH 8)--0.85 (pH 6). These are close to the P/e2 value of 0.3--0.38 and the H+/e values of 0.7--0.93 found in parallel experiments for the H2O leads to Photosystem II leads to dimethylquinone reaction in untreated chloroplasts. Semi-quantitative data are also presented which show that the donor leads to Photosystem II leads to dibromothymoquinone (leads to O2) reaction can support phosphorylation when the donor used is a proton-releasing reductant (benzidine, catechol) but not when it is a non-proton carrier (I-, ferrocyanide).     

Enhancement by chloride ions of photoactivation of oxygen evolution in manganese-depleted photosystem II membranes

The Mn cluster that catalyzes photosynthetic oxygen evolution was removed from the photosystem II (PSII) complex by treating PSII membranes with 1.0 mM NH2OH with concomitant inactivation of oxygen evolution. The cluster was reconstituted by incubating the treated membranes with 1.0 mM Mn2+, 20 mM Ca2+, 10 microM 2,6-dichlorophenolindophenol, and Cl- under illumination with continuous or flashing light to restore the oxygen-evolving capacity. This light-dependent activation (photoactivation) of oxygen evolution did not occur to a significant extent at 3 mM Cl-, but markedly accelerated at higher Cl- concentrations without showing a saturation phenomenon even at 1 M Cl-. At 10 mM Cl- only about 10% of the oxygen-evolving activity before NH2OH treatment was restored by 5-min illumination with continuous light, whereas at 600 mM Cl- about 60% of the original activity was recovered. This acceleration resulted from at least two different actions of Cl-: (1) stabilization of the intermediate state involved in the photoactivation process and (2) increase in the quantum yield of photoactivation. The stabilization of the intermediate was saturated at about 150 mM Cl-, whereas the increase in yield did not show saturation. The Cl(-)-induced increase in quantum yield did not involve any changes in the affinity of either Mn2+ binding or Ca2+ binding for photoactivation, but was rather ascribed to a protective effect of Cl- against inhibition of photoactivation by high concentrations of Mn2+. We also found that removal of the extrinsic 33-kDa protein from the PSII complex increased the Cl- requirement for photoactivation.     

The oxygen-evolving complex requires chloride to prevent hydrogen peroxide formation.

Illumination of PSII core preparations can cause the production of H2O2 at rates which approach 60 mumol of H2O2 (mg of Chl.h)-1. The rate of peroxide production is maximal at pH 7.2 at low sucrose concentrations and at concentrations of Cl- (1.5-3.0 mM) that limit the rate of the oxidation of water to O2. The rate of H2O2 production increased with pH from pH 6.8 to 7.2 and was inversely proportional to the oxidation of water to O2 from pH 6.8 to 7.5. While EDTA does not inhibit H2O2 production, this reaction is abolished by 5 mM NH2OH and inhibited by the same concentrations of NH3 that affect water oxidation which indicates that the oxygen-evolving complex is responsible for the production of peroxide generated upon illumination of PSII core preparations. These results support a mechanism in which bound Cl- in the S2 state is displaced by OH- ions which are then oxidized by the OEC to form H2O2. Thus, the OEC requires Cl- to prevent access to the active site of the OEC until four oxidizing equivalents can be generated to allow the oxidation of water to O2.     

Surface charge asymmetry and a specific calcium ion effect in chloroplast photosystem II

We have used the decay kinetics of Signal IIf in Tris-washed chloroplasts as a direct probe to reactions on the oxidizing side of Photosystem II. A study of the salt concentration dependence of the rate of reduction of Z . + by the ascorbate monoanion has been interpreted by using the Gouy-Chapman diffuse double layer model and allows the calculation of an inner membrane surface charge density of -3.4 +/- 0.3 microC . cm-2 at pH = 8.0 in the vicinity of Photosystem II. We have also measured the outer membrane surface charge density at this pH in Tris- and sucrose-washed chloroplasts by monitoring the rate of potassium ferricyanide oxidation of Q-, and arrive at values of -2.2 +/- 0.3 microC . cm-2 and -2.1 microC . cm-2, respectively. From these experiments we conclude that in dark-adapted chloroplasts at pH 8.0 there exists a transmembrane electric field in the vicinity of Photosystem II which arises from this surface charge asymmetry. In the presence of 10 mM monovalent salts, the transmembrane potential difference is of the order of 20 mV, corresponding to a field of 4 . 10(4) V . cm-1 (negative inside) for a 50A membrane. It is both smaller in magnitude and in the opposite direction compared to the photoinduced transmembrane field which gives rise to the 515 nm absorption change. We have also found non-double layer Ca2+ effects on the decay kinetics of Signal IIf with both charged (ascorbate monoanion) and neutral (diphenylcarbazide) donors. These results suggest a change in the environment of Z from lipophilic to hydrophilic upon specific binding of Ca2+.     

Sulfide inhibition of photosystem II in cyanobacteria (blue-green algae) and tobacco chloroplasts.

The present study shows that in the presence of 600 nm light, sulfide acts as a specific inhibitor of photosynthetic electron transport between water and Photosystem II in the cyanobacteria Aphanothece halophytica and Synechococcus 6311 as well as in tobacco chloroplasts. In the presence of 600 nm light sulfied affects the fast fluorescence transients as does a low concentration (10 mM) of hydroxylamine; the fluorescence yield decreases in the presence of either chemical and can be restored by the addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea. In chloroplasts, however, NH2OH, an electron donor at high concentrations (40 mM), relieves the sulfide effect. In the dark, sulfide affects the cyanobacterial fluorescence transients through decrease of oxygen tension. The fluorescence yield increases in a similar pattern to that observed under nitrogen flushing. Upon omission of sulfide in A. halophytica, the characteristic aerobic fluorescence transients return, consistent with the ease of alternation between oxygenic and sulfide-dependent anoxygenic photosynthesis in many cyanobacteria.     

Catechols stimulate ferricyanide reduction in chloroplast photosystem II.

In isolated chloroplasts (Spinacia olearacea), where electron transport to Photosystem I is blocked by the plastoquinone antagonist, dibromothymoquinone, lipophilic catechols in concentrations of 50--150 microM stimulate ferricyanide reduction in Photosystem II and associated O2 evolution. Non-permeating catechols, such as Tiron, are unable to stimulate this reaction. Those quinones, such as 2,5-dimethylbenzoquinone, which act as class III electron acceptors, do not lead to stimulation of ferricyanide reduction in Photosystem II or stimulation fo associatied O2 evolution, when electron transport to Photosystem I is blocked by dibromoquinone. Stimulation of ferricyanide reduction is not observed in Tris-treated chloroplasts, implying that electron donation to Photosystem II by catechols is not responsible for the stimulation. Various mechanisms for this stimulation in class II chloroplasts are discussed.     

Flash-induced absorption changes of the primary donor of photosystem II at 820 nm in chloroplasts inhibited by low pH or tris-treatment.

A comparative study is made, at 15 degrees C, of flash-induced absorption changes around 820 nm (attributed to the primary donors of Photosystems I and II) and 705 nm (Photosystem I only), in normal chloroplasts and in chloroplasts where O2 evolution was inhibited by low pH or by Tris-treatment. At pH 7.5, with untreated chloroplasts, the absorption changes around 820 nm are shown to be due to P-700 alone. Any contribution of the primary donor of Photosystem II should be in times shorter than 60 mus. When chloroplasts are inhibited at the donor side of Photosystem II by low pH, an additional absorption change at 820 nm appears with an amplitude which, at pH 4.0, is slightly higher than the signal due to oxidized P-700. This additional signal is attributed to the primary donor of Photosystem II. It decays (t 1/2 about 180 mus) mainly by back reaction with the primary acceptor and partly by reduction by another electron donor. Acid-washed chloroplasts resuspended at pH 7.5 still present the signal due to Photosystem II (t 1/2 about 120 mus). This shows that the acid inhibition of the first secondary donor of Photosystem II is irreversible. In Tris-treated chloroplasts, absorption changes at 820 nm due to the primary donor of Photosystem II are also observed, but to a lesser extent and only after some charge accumulation at the donor side. They decay with a half-time of 120 mus.     

Inhibition of Photosystem II by uncouplers at alkaline pH and its reversal by artificial electron donors

When chloroplasts are aged for 5 min at pH 9.6, or are exposed to uncouplers at pH 8.5–9.0, electron flow from water to Hill acceptors is inhibited. Both treatments induce rapid millisecond dark decay of delayed light emission. 3-(3,4-Dichlorophenyl)-1,1-dimethylurea-sensitive electron transport through Photosystem II can be regenerated in both types of inhibited chloroplasts by the artificial electron donor, 1,5-diphenylcarbohydrazide. Neither treatment inhibits electron flow through Photosystem I. Uncouplers at alkaline pH, when added in the light, are less effective in producing the inhibition than when added in the dark. These results are interpreted as indicating inhibition of the oxygen-evolving apparatus by alkaline intrathylakoid pH.     

Effect of imidazole on the activity of photosystem II in spinach chloroplasts

The activity of the Hill reaction was accelerated about 2–3times in the presence of 1–5 mM imidazole. However, inchloroplasts uncoupled by carbonyl cyanide m-chlorophenylhydrazone,the acceleration of the Hill activity was not observed in thesame imidazole concentration range, but a moderate inhibitionof the activity was observed. These results suggest that inthe coupled chloroplasts, the enhanced Hill activity by imidazoleis due to uncoupling of phosphorylation. Imidazole-washing of chloroplasts at neutral pH caused a moderateinhibition of the Hill reaction to about 60% of the control,while a complete loss of the Hill activity was observed afterwashing at pH 10.5. In chloroplasts washed with imidazole atpH 10.5, the variable fluorescence yield was also diminished.These activities were restored by adding an artificial electrondonor to photosystem II. (Received October 14, 1978; )     

Mechanism of bicarbonate action on photosynthetic electron transport in broken chloroplasts

In CO2-depleted chloroplasts electron transport between the Photosystem II electron acceptor Q and plastoquinone is largely suppressed. In the presence of a high concentration of sodium formate (greater than 10 mM), which probably binds to the bicarbonate site, addition of bicarbonate restores the ferricyanide Hill reaction only after incubation in the dark. With lower formate concentrations bicarbonate is able to restore electron transport in the light. The Hill reaction rate in CO2-depleted chloroplasts after bicarbonate addition, divided by the rate in CO2-depleted chloroplasts before bicarbonate addition, shows a sharp optimum at pH 6.5. Furthermore, the rate-limiting step in bicarbonate action is probably diffusion. The results are explained in terms of a hypothetical model: the bicarbonate-binding site is located at the outer side of the thylakoid membrane, but not directly accessible from the "bulk". To reach the site from the bulk, the molecule has to pass a channel with negatively charge groups on its side walls. In the light these groups are more negatively charged than in the dark. Therefore, the formate ion cannot exchange for bicarbonate in the light, and a dark period is necessary to enable exchange of formate for bicarbonate.     

Chlorophyll radical cation in photosystem II of chloroplasts. Millisecond decay at low temperature.

We compare the absorption changes, in the near infrared and in the green part of the spectrum, induced in spinach chloroplasts suspensions, at -- 170 degrees C, by continuous light and by flashes. (1) Following flash excitation, an absorption increase peaking at 825 nm which reverses rapidly (t 1/2 = 3.0 ms) is not affected by ferricyanide; it is suppressed when chloroplasts are preilluminated in the presence of 3-(3',4'-dichlorophenyl)-1,1'-dimethylurea (DCMU) and hydroxylamine. The reversion of that signal is simultaneous with a partial back reoxidation of C-550 (fully reduced by the flash) and with partial (about 25%) oxidation of cytochrome b559. The magnitude of the signal peaking at 825 nm (that we attribute to the radical cation of the trap chlorophyll of Photosystem II, acting as a primary electron donor) decreases progressively within a series of successive flashes. (2) An absorption increase (40% of which is slowly reversible) with a broad peak around 810 nm is induced by continuous light or by a flash. It is suppressed by pretreatment with ferricyanide, but it is little affected by the treatment with 3-(3',4'-dichlorophenyl)-1,1'-dimethylurea and hydroxylamine. We attribute it to oxidized P700. (3) With chloroplasts pretreated with 10 mM ferricyanide, an absorption increase, whose magnitude is nearly independent of wavelength between 790 and 870 nm, can be induced by continuous light. One saturating flash produces only 20% of the signal. This absorption change (20% of which is reversible in 30 s) might be due to a secondary donor of Photosystem II.     

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 it is re-reduced in a biphasic manner with half-times of 6 microseconds (major phase) and 22 microseconds. After the second flash, the 6 microseconds phase is nearly absent and P-680+ decays with half-times of 130 microseconds (major phase) and 22 microseconds. Exogenous electron donors (MnCl2 or reduced phenylenediamine) have no direct influence on the kinetics of P-680+. In untreated chloroplasts the 6 and 22 microseconds 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 (less than 1 microseconds) by the physiological donor D1; a slower reduction (6 and 22 microseconds) by donor D'1, operative when O2 evolution is inhibited; a back-reaction (130 microseconds) 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.     

A rapid vectorial back reaction at the reaction centers of photosystem II in tris-washed chloroplasts induced by repetitive flash excitation.

In Tris-washed chloroplasts, completely lacking the oxygen-evolving capacity, absorption changes in the range of 420--560 nm induced by repetitive flash excitation have been measured in the presence and absence of electron donors. It was found: (1) At 520 nm flash-induced absorption changes are observed, which predominantly decay via a 100--200-mus exponential kinetics corresponding to that of the back reaction between the primary electron donor and acceptor of Photosystem II (Haveman, J. and Mathis, P. (1976) Biochim. Biophys. Acta 440, 346--355; Renger, G. and Wolff, Ch. (1976) Biochim. Biophys. Acta 423, 610--614). In the presence of hydroquinone/ascorbate as donor couple the amplitude is nearly doubled and the decay becomes significantly slowed down. (2) The difference spectrum of the absorption changes obtained in the presence of hydroquinone/ascorbate, which are sensitive to ionophores, is nearly identical with that of normal chloroplasts in the range of 460--560 nm (Emrich, H.M., Junge, W. and Witt, H.T. (1969) Z. Naturforsch. 24b, 114--1146). In the absence of hydroquinone/ascorbate the difference spectrum of the absorption changes, characterized by a 100--200-mus decay kinetics, differs in the range of 460--500 nm and by a hump in the range of 530--560 nm. The hump is shown to be attributable to the socalled C550 absorption change, which reflects the turnover of the primary acceptor of Photosystem II (van Gorkom, H.J.(1976) Thesis, Leiden), while the deviations in the range of 460--500 nm are understandable as to be due to the overlapping absorption changes of chlorphyll alpha II+. The problems arising with the latter explanation are discussed. (3) The electron transfer due to the rapid turnover at Photosystem II, which can be induced by flash groups with a short dark time between the flashes, is not able to energize the ATPase and to drive photophosphorylation. On the basis of the present results it is inferred, that in Tris-washed chloroplasts under repetitive flash excitation a rapid transmembrane vectorial electron shuttle takes place between the primary acceptor (X320) and donor (Chl alpha II) of Photosystem II, which is not able to energize the photophosphorylation. Furthermore, the data are shown to confirm the localization of X320 and Chl alpha II within the thylakoid membrane at the outer and inner side, respectively.     

Flash-induced absorption changes of the primary donor of photosystem II at 820 nm in chloroplasts inhibited by low pH or tris-treatment

A comparative study is made, at 15 °C, of flash-induced absorption changes around 820 nm (attributed to the primary donors of Photosystems I and II) and 705 nm (Photosystem I only), in normal chloroplasts and in chloroplasts where O₂ evolution was inhibited by low pH or by Tris-treatment.At pH 7.5, with untreated chloroplasts, the absorption changes around 820 nm are shown to be due to P-700 alone. Any contribution of the primary donor of Photosystem II should be in times shorter than 60 μs.When chloroplasts are inhibited at the donor side of Photosystem II by low pH, an additional absorption change at 820 nm appears with an amplitude which, at pH 4.0, is slightly higher than the signal due to oxidized P-700. This additional signal is attributed to the primary donor of Photosystem II. It decays ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ about 180 μs) mainly by back reaction with the primary acceptor and partly by reduction by another electron donor. Acid-washed chloroplasts resuspended at pH 7.5 still present the signal due to Photosystem II ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ about 120 μs). This shows that the acid inhibition of the first secondary donor of Photosystem II is irreversible.In Tris-treated chloroplasts, absorption changes at 820 nm due to the primary donor of Photosystem II are also observed, but to a lesser extent and only after some charge accumulation at the donor side. They decay with a half-time of 120 μs.     

Modifications of the functioning of Photosystem II induced in the dark by alkaline pH in the presence of cations

Submission of chloroplasts to alkaline pH, in the range pH 7.5–9.5, leads to changes in their oxygen-evolving capacities. These changes are enhanced by the addition of divalent cations and also monovalent cations at high concentrations. (1) Dark incubation of chloroplasts at pH ? 9 gives rise to a time-dependent inactivation of electron transport from water to 2,6-dichlorophenolindophenol measured at neutral pH. The rate of inactivation is increased by adding cations. (2) The variable fluorescence is decreased with a dependence on incubation time and concentration of cations similar to that of the Hill reaction. Addition of the electron donor NH₂OH removes most of the fluorescence quenching, (3) EPR measurements indicate that the inactivations are accompanied by loss of Mn²⁺ and the appearance of signal II fast. (4) At lower pH (7.5) the oscillations of oxygen evolved per flash during a sequence of flashes show an increase in damping when 20 mM MgCl₂ is present instead of 100 mM KCI. These changes are not seen at pH 6. (5) None of these Mg²⁺-induced modifications are prevented by glutaraldehyde fixation. We conclude that the effects of alkaline pH and MgCl₂ do not involve major protein structural changes, and that both act on the manganese-containing protein of the oxygen-evolving site.     

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.     

Primary reactions of photosystem II at low pH. I. Prompt and delayed fluorescence.

Prompt and delayed chlorophyll fluorescence have been studied in broken spinach chloroplasts at pH values down to 2.6. No direct effect of low pH on the primary charge separation in Photosystem II was observed. The irreversible inactivation of a secondary electron donor in a narrow pH range around pH 4.5 was demonstrated. At lower pH values the photooxidized form of a more primary electron donor, revealed by its efficient fluorescence quenching, was reduced with a half time of about 200 mus, 25% by another electron donor and 75% by back reaction with the reduced acceptor. The electron donation had a half time of 800 mus and was practically irreversible. The back reaction had a pH dependent half time: about 270 mus at pH 4 and increasing towards lower pH. The competition of both reactions resulted in a net efficiency of the charge separation at pH 4 of 25%, increasing towards lower pH.     

Increase effected by calcium ion in the rate of oxygen evolution from preparations of Phormidium luridum.

The presence of Ca2+ causes a twentyfold or greater increase in the rate of oxygen evolution by cell-free preparations of Phormidium luridum. The requirement for Ca2+ is specific; other divalent cations are much less effective or are inhibitory. The rate of the Hill reaction is maximal at 30 mM CaCl2 in both detergent-free and Brij 35 preparations. The 3-(3,4-dichlorophenyl)-1,1-dimethylurea-insensitive component of oxygen-evolving activity in each preparation also shows the requirement for added Ca2+. This indicates that Ca2+ is acting close to the oxygen-evolving reaction center of Photosystem II. Defatted bovine serum albumin increases the rate of oxygen evolution in the detergent-free preparation, but does not compete with Ca2+, discounting fatty acid mediation of the effects of Ca2+. Neither excess Hill acceptor nor uncouplers of photophosphorylation diminish the stimulatory effects of Ca2+.     

Photophosphorylation and related properties of reaggregated vesicles from spinach photosystem I particles.

The small Photosystem I particles prepared from spinach chloroplasts by the action of Triton X-100 (TSF 1 particles) reaggregate into membrane structures when they are incubated with soybean phospholipids and cholate and then subjected to a slow dialysis. The membranes so formed are vesicular in nature and show the capability of catalyzing phenazine methosulfate-mediated cyclic photophosphorylation at rates which are usually about 20% of those observed with chloroplasts, but higher rates have been obtained. When coupling factor is removed from the chloroplasts by treatment with EDTA, a requirement for coupling factor can be shown for the subsequent ATP formation. The uncouplers carbonylcyanide 3-chlorophenyl-hydrazone, valinomycin, Triton X-100 and NH+4 are effective with the reformed vesicles, which do not show the typical light-induced pH gradient observed with chloroplasts. Incubation of the TSF 1 particles with phospholipids alone allows for the formation of membrane vesicles, but such vesicles are only slightly active in ATP formation. In most properties investigated, the reformed membrane vesicles resemble the original chloroplast membrane so far as phenazine methosulfate-mediated cyclic photophosphorylation is concerned, which indicates a high degree of selectivity in the reaggregation process. The major difference between chloroplasts and the reformed vesicles is the failure of the latter to show a light-induced pH gradient.