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.     

Feedback controlling oxygen production in a cross-reaction between two photosystems in photosynthesis

The non-linear light-saturation curve for oxygen production in both Chlorella vulgaris and Phormidium luridium at low light intensities, under anaerobic conditions is shown to be caused by the reduction of a pool of electron carriers coupled to both an endogenous reducing agent R, and to oxygen. The light dependence of oxygen production in these algae was studied by a repetitive-flash method, which allows a direct analysis of the steady-state kinetics of pool reduction. We propose a kinetic model which quantitatively accounts for these kinetics and several transient phenomena. This model centers on a novel cross reaction at the pool of photo and dark electron input and output, allowing a delicate poising of oxygen production by the environment. This model shows a positive feedback of oxygen on oxygen production.     

The stoichiometry (ATP/2e− ratio) of non-cyclic photophosphorylation in isolated spinach chloroplasts

1. The stoichiometry of non-cyclic photophosphorylation and electron transport in isolated chloroplasts has been re-investigated. Variations in the isolation and assay techniques were studied in detail in order to obtain optimum conditions necessary for reproducibly higher ADP/O (equivalent to ATP/2e^?) and photosynthetic control ratios.2. Studies which we carried out on the possible contribution of cyclic phosphorylation to non-cyclic phosphorylation suggested that not more than 10% of the total phosphorylation found could be due to cyclic phosphorylation.3. Photosynthetic control, and the uncoupling of electron transport in the presence of NH₄Cl, were demonstrated using oxidised diaminodurene as the electron acceptor. A halving of the ADP/O ratio was found, suggesting that electrons were being accepted between two sites of energy conservation, one of which is associated with Photosystem I and the other associated with Photosystem II.4. ATP was shown to inhibit State 2 and State 3 of electron transport, but not State 4 electron transport or the overall ADP/O ratio, thus confirming its activity as an energy transfer inhibitor. It is suggested that part of the non-phosphorylating electron transport rate (State 2) which is not inhibited by ATP is incapable of being coupled to subsequent phosphorylation triggered by the addition of ADP (State 3). If the ATP-insensitive State 2 electron transport is deducted from the State 3 electron transport when calculating the ADP/O ratio, a value of 2.0 is obtained.5. The experiments reported demonstrate that there are two sites of energy conservation in the non-cyclic electron transfer pathway: one associated with Photosystem II and the other with Photosystem I. Thus, non-cyclic photophosphorylation can probably produce sufficient ATP and NADPH “in vivo” to allow CO₂ fixation to proceed.     

Trypsin inhibition of electron transport

1. 1. A relaxation spectrophotometer was employed to measure the effects of trypsin treatment on electron transport in both cyclic and non-cyclic chloroplast reactions. The parameters measured were electron flow rate through P700 (flux) and the time constant for dark reduction of P700.

2. 2. In the reduction of methyl viologen by the ascorbate-2,6-dichlorophenol-indophenol (DCIP) donor couple, there was no effect of trypsin on P700 flux or on the time constant for dark reduction of P700. In the phenazine methosulfate (PMS) cyclic system, trypsin had either a slightly stimulatory or slightly inhibitory effect on the P700 flux, depending on the presence or absence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU): either effect being marginal compared to trypsin effects on Photosystem II.With both ferricyanide and methyl viologen reduction from water, trypsin treament gave a first order decline in P700 flux: which matched the trypsin-induced decline in electron transport with the water to DCIP system, measured by dye reduction. This implies that Photosystem II is inhibited. The inhibition of Photosystem II was up to 90% with a 6–10-min trypsin treatment. This result is consistent with the concept of Photosystem I (P700) being in series with Photosystem II in the electron transfer sequence.

3. 3. Cyclic phosphorylation was severely inhibited (85%) by trypsin treatment which had a somewhat stimulatory effect on P700 flux, indicating uncoupling. Non-cyclic phosphorylation was uncoupled as well as electron flow being inhibited since the P/2e ratio decreased more rapidly as a function of trypsin incubation time than inhibition of electron flow. The two effects, uncoupling and non-cyclic electron flow inhibition, are separate actions of trypsin. It is probably that the uncoupling action of trypsin is due to attack on the coupling factor protein, known to be exposed on the outer surface of thylakoids.

4. 4. Trypsin treatment caused an increase in the rate constant, k_d, for the dark H⁺ efflux, resulting in a decreased steady state level of proton accumulation. The increased proton efflux and the inhibition of phosphorylation are consistent with an uncoupling effect on trypsin.

5. 5. Trypsin treatment did not reduce the manganese content of chloroplasts: as reported by others, Tris washing did remove about 30% of the chloroplast manganese.

6. 6. Electron micrographs of both negatively stained and thin-sectioned preparations showed that, under these conditions, trypsin does not cause a general breakdown of chloroplast lamellae. Inhibition by trypsin must therefore result from attacks on a few specific sites.

7. 7. Both System II inhibition and uncoupling occur rapidly when trypsin treatment is carried out in dilute buffer, a condition which leads to thylakoid unstacking, but both are prevented by the presence of 0.3 M sucrose and 0.1 M KCl, a condition that helps maintain stacked thylakoids. Evidently vulnerability to trypsin requires separation of thylakoids.

8. 8. Since trypsin does not appear to disrupt thylakoids nor prevent their normal aggregation in high sucrose-salt medium and since the trypsin molecule is probably impermeable, it is probable that the site(s) of trypsin attack in System II are exposed on the outer thylakoid surface.

Stabilization by glutaraldehyde of high-rate electron transport in isolated chloroplasts

Treatment of isolated chloroplasts with glutaraldehyde affects their ability to photoreduce artificial electron acceptors. The remaining rate of O₂ evolution approaches zero with methyl viologen, is low with ferricyanide, but nearly normal with lipophilic Photosystem II acceptors, like oxidized p-phenylenediamine and oxidized diaminodurene. Since Photosystem I donor reactions are also affected, a specific site of inhibition of electron transport to Photosystem I is indicated. At the same time, glutaraldehyde prolongs the longevity of the chloroplasts stored in dark. In control samples the half-life of Photosystem II activity varied between 5 days at 4 °C and 1 day at 25 °C. Glutaraldehyde treatment increased these half times approx. 3-fold. The glutaraldehyde doses required to induce inhibition and stabilization were very similar.     

Chlorophyll fluorescence induction in anaerobic Scenedesmus obliquus

Fluorescence time curves (Kautsky effect) were studied in anaerobic Scenedesmus obliquus, with an apparatus capable of simultaneous recording of O₂ exchange, and far-red actinic illumination. Results, as interpreted in terms of electron transport reactions, suggest: In the course of becoming anaerobic, fluorescence induction undergoes a series of changes, indicating at least three different effects of the absence of O₂ on electron transport. (1) Immediately on removal of O₂, once the pool of intermediates between the two photo-systems is reduced by light reaction II, electron flow stops, resulting in high fluorescence yield and a cessation of O₂ evolution. O₂ appears to regulate linear electron flow and cyclic feedback of electrons to the intermediate pool. (2) An endogenous reductant formed anaerobically reduces the System II acceptors in the dark. The time course of this reduction is at least biphasic, indicative of inhomogeneity of the primary acceptor pool. Prolonged dark anaerobic treatment induces maximal initial fluorescence which decays rapidly in light and with a System I action spectrum. (3) Anaerobic treatment eventually results in deactivation of the oxidizing side of System II, limiting System II even when the acceptors are oxidized by System I pre-illumination.     

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).     

Flash spectroscopic studies of cyclic electron flow in intact chloroplasts

The kinetic behaviours of cytochrome $\text{b}$-563 and cytochrome $\text{f}$ are shown to be consistent with their participation in coupled cyclic electron flow in intact chloroplasts. Electron transfer between cytochromes $\text{b}$-563 and cytochrome $\text{f}$ is antimycin sensitive. Fluorescence induction studies indicate that plastoquinone may function in a coupled step between the cytochromes.     

Restoration by silicotungstic acid of DCMU-inhibited photoreactions in spinach chloroplasts

The restoration by silicotungstic acid of the reversible light-induced pH rise mediated by pyocyanine in EDTA-treated chloroplasts corresponds to an irreversible fixation of the acid. The proton uptake is linearly related to the amount of fixed acid (4 protons per molecule of acid) as long as the amount of silicotungstic acid does not exceed 200 nmoles/mg of chlorophyll.In the same conditions silicotungstic acid partly restores ferricyanide reduction and O₂ evolution in chloroplasts suspensions supplemented with DCMU. These photoreactions are observed only with chloroplasts and these chloroplasts must have an unimpaired water-splitting mechanism.Silicotungstic acid does not impair DCMU fixation on the specific sites. More likely in its presence the properties of the membrane change and ferricyanide can accept electrons from a part of the electron transport chain, between the Photosystem II reaction center and the block of the electron flow by DCMU.     

The effects of carbon dioxide concentration on oxygen evolution and fluorescence transients in synchronous cultures of Chlorella pyrenoidosa

1. 1. The steady-state fluorescence yield of Chlorella pyrenoidosa is strongly affected by CO₂ concentration: the yield is approximately 2-fold higher in the presence than in the absence of CO₂. During induction, in the presence of saturating CO₂, accelerating oxygen evolution is paralleled by rising fluorescence (M₂-P₃ transient); in the absence of CO₂, fluorescence yield remains at the low M₂ level.

2. 2. Both illumination and CO₂ content are important in determining the steady-state fluorescence yield: at lower illuminations, lower concentrations of CO₂ are required to obtain a maximum fluorescence yield.

3. 3. The slow fluorescence transients are not affected directly by pH but only indirectly through the CO₂ concentration.

4. 4. The CO₂-dependent fluorescence rise (M₂-P₃ transient) is most readily observed in cells harvested early in the light period of a synchronous culture, but it can also be elicited in cells harvested during the dark period.

5. 5. Addition of 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (DCMU) to CO₂-deprived cells raises the fluorescence yield approximately 4-fold, that is to the same high level as cells supplied with CO₂ and DCMU.

6. 6. The effects of CO₂ provide a new example of a marked parallelism between photosynthetic electron transport and fluorescence. To explain such parallelism, it seems necessary to postulate large changes in the de-excitation processes within Photosystem II units or in the distribution of excitation between Photosystems I and II.

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.     

Light-induced slow changes in chlorophyll a fluorescence in isolated chloroplasts: Effects of magnesium and phenazine methosulfate

We have investigated the possible relationships between the cation-induced and phenazine methosulfate (PMS)-induced fluorescence changes and their relation to light induced conformational changes of the thylakoid membrane.1. In isolated chloroplasts, PMS markedly lowers the quantum yield of chlorophyll a fluorescence (φ_f) when added either in the presence or the absence of dichloro-phenyldimethylurea (DCMU). In contrast, Mg²⁺ causes an increase in φ_f. However, these effects are absent in isolated chloroplasts fixed with glutaraldehyde that retain (to a large extent) the ability to pump protons, suggesting that structural alteration of the membrane—not the pH changes—is required for the observed changes in φ_f. The PMS triggered decrease in φ_f is not accompanied by any changes in the emission (spectral) characteristics of the two pigment systems, whereas room temperature emission spectra with Mg²⁺ and Ca²⁺ show that there is a relative increase of System II to System I fluorescence.2. Washing isolated chloroplasts with 0.75 mM EDTA eliminates (to a large extent) the PMS-induced quenching and Mg²⁺-induced increase of φ_f, and these effects are not recovered by the further addition of dicyclohexyl carbodiimide. It is known that washing with EDTA removes the coupling factor, and thus, it seems that the coupling factor is (indirectly) involved in conformational change of thylakoid membranes leading to fluorescence yield changes.3. In purified pigment System II particles, neither PMS nor Mg²⁺ causes any change in φ_f. Our data, taken together with those of the others, suggest that a structural modification of the thylakoid membranes (not macroscopic volume changes of the chloroplasts) containing both Photosystems I and II is necessary for the PMS-induced quenching and Mg²⁺-induced increase of φ_f. These two effects can be explained with the assumption that the PMS effect is due to an increase in the rate of internal conversion (k_h), whereas the Mg²⁺ effect is due to a decrease in the rate of energy transfer (k_t), between the two photosystems.4. From the relative ratio of φ_f with DCMU and DCMU plus Mg²⁺, we have calculated k_t (the rate constant of energy transfer between Photosystems II and I to be 4.2·10⁸ s^?1, and φ_t (quantum yield of this transfer) to be 0.12.     

Biogenesis of chloroplast membranes X. Changes in the photosynthetic specific activity and the relationship between the light harvesting system and photosynthetic electron transfer chain during greening of Chlamydomonas reinhardi y-1 cells

Specific activities of photophosphorylation and light-dependent pH rise at different stages of the greening process, have been measured as a function of the illumination intensity.     

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.     

The site of KCN inhibition in the photosynthetic electron transport pathway

Treatment of chloroplasts with high concentrations of KCN inhibits reactions which involve Photosystem I (e.g. electron transport from water or diaminodurene to methylviologen), but not those assumed to by-pass Photosystem I (e.g. electron transport from water to quinonediimides). The spectrophotometric experiments described in this paper showed that KCN inhibits the oxidation of cytochrome f by far-red light without blocking its reduction by red light. Both optical and EPR experiments indicated that KCN does not inhibit the photooxidation of P700 but markedly slows down the subsequent dark decay (reduction). Reduction of P700 by Photosystem II is prevented by KCN. It is concluded that KCN blocks electron transfer between cytochrome f and P700, i.e. the reaction step which is believed to be mediated by plastocyanin. In KCN-poisoned chloroplasts the slow dark reduction of P700 following photooxidation is greatly accelerated by reduced 2,6-dichlorophenolindophenol or by reduced N-methylphenazonium methosulfate (PMS), but not by diaminodurene. It appears that the reduced indophenol dye and reduced PMS are capable of donating electrons directly to P700, at least partially by-passing the KCN block.     

Fluorescence yield kinetics in the microsecond-range in Chlorella pyrenoidosa and spinach chloroplasts in the presence of hydroxylamine

The kinetics of fluorescence yield inChlorella pyrenoidosa and spinach chloroplasts were studied in the time range of 0.5 μs to several hundreds of microseconds in the presence of hydroxylamine. Fluorescence was excited with a just-saturating xenon flash with a halfwidth of 13 μs (λ = 420 nm). The fast rise of the fluorescence yield which was limited by the rate of light influx, was, in the presence of 10⁻³–10⁻² M hydroxylamine, replaced by a slow component which had a half risetime of 25 μs in essence independent of light intensity. This slow fluorescence yield increase reflects a dark reaction on the watersplitting side of Photosystem II. Simultaneous oxygen evolution measurements suggested that a fast fluorescence component is only present in organisms with intact O₂-evolving system, whereas a slow rise predominantly occurs in organisms with the watersplitting system irreversibly inhibited by hydroxylamine.

The results can be explained by the following hypotheses: (a) The primary donor of Photosystem II in its oxidized state, P⁺, is a fluorescence quencher. (b) Hydroxylamine prevents the secondary electron donor Z from reducing the oxidized reaction center pigment P⁺ rapidly. This inhibition is dependent on hydroxylamine concentration and is complete at a concentration of 10⁻² M. (c) A second donor (not transporting electrons from water) transfers electrons to P⁺ with a half time of roughly 25 μs.     

Photosynthetic electron transport,ATP synthesis and nitrogenase activity in isolated heterocysts of Anabaena cylindrica

Isolated heterocysts of the N₂-fixing blue-green alga Anabaena cylindrica contain the Photosystem I components P-700, bound and soluble ferredoxins and ferredoxin-NADP reductase. They also show Photosystem I activity being able to photoreduce both methylviologen and NADP when ascorbate+dichlorophenol-indophenol acts as reductant. They photophosphorylate (64 μmol ATP produced/mg chlorophyll $\text{a}\text{h}$) and carry out oxidative phosphorylation (8.7 μmol ATP produced/mg chlorophyll $\text{a}\text{h}$). Ninety per cent of the total cell-free extract nitrogenase activity is located in the heterocyst fraction of aerobic cultures.     

The existence of a high photochemical turnover rate at the reaction centers of System II in Tris-washed chloroplasts

In Tris-washed chloroplasts the kinetics of the primary electron acceptor X 320 of reaction center II has been investigated by fast repetitive flash spectroscopy with a time resolution of $\text{≈ 1 μs}$. It has been found that X 320 is reduced by a flash in $\text{? 1 μs}$. The subsequent reoxidation in the dark occurs mainly by a reaction with a 100–200 μs kinetics. The light-induced difference spectrum confirms X 320 to be the reactive species. From these results it is concluded that in Tris-washed chloroplasts the reaction centers of System II are characterized by a high photochemical turnover rate mediated either via rapid direct charge recombination or via fast cyclic electron flow.