Photosystem II in a mutant of Chlamydomonas reinhardtii lacking the 23 kDa psbP protein shows increased sensitivity to photoinhibition in the absence of chloride

The psbP gene product, the so called 23 kDa extrinsic protein, is involved in water oxidation carried out by Photosystem II. However, the protein is not absolutely required for water oxidation. Here we have studied Photosystem II mediated electron transfer in a mutant of Chlamydomonas reinhardtii, the FUD 39 mutant, that lacks the psbP protein. When grown in dim light the Photosystem II content in thylakoid membranes of FUD 39 is approximately similar to that in the wild-type. The oxygen evolution is dependent on the presence of chloride as a cofactor, which activates the water oxidation with a dissociation constant of about 4 mM. In the mutant, the oxygen evolution is very sensitive to photoinhibition when assayed at low chloride concentrations while chloride protects against photoinhibition with a dissociation constant of about 5 mM. The photoinhibition is irreversible as oxygen evolution cannot be restored by the addition of chloride to inhibited samples. In addition the inhibition seems to be targeted primarily to the Mn-cluster in Photosystem II as the electron transfer through the remaining part of Photosystem II is photoinhibited with slower kinetics. Thus, this mutant provides an experimental system in which effects of photoinhibition induced by lesions at the donor side of Photosystem II can be studied in vivo.Abbreviations Chl chlorophyll - DCIP 2,6-dichlorophenolindophenol - DPC 2,2-diphenylcarbonic dihydrazide - HEPES 4-(2-hydroxyethyl)-1-piperazinethanesulfonic acid - P₆₈₀ the primary electron donor to PS II - PpBQ phenyl-p-benzoquinone - PS II Photosystem II - Q_A the first quinone acceptor of PS II - Q_B the second quinone acceptor of PS II - SDS sodium dodecyl sulfate - Tris tris(hydroxymethyl)aminomethane - Tyr_D accessory electron donor on the D₂-protein - Tyr_Z tyrosine residue, acting as electron carrier between P₆₈₀ and the water oxidizing system     

Inhibition of Photosystem II in the Green Alga Ankistrodesmus falcatus by Copper

Copper strongly inhibited 2,6-dichloroindophenol (DCIP) photoreduction in the broken cells of the green alga Ankistrodesmus falcatus (C303), and the activity lost could not be restored by adding 1,5-diphenylearbazide (DPC). Inactivation of the DCIP Hill reaction reached 45% after incubation with 10 μM cupric sulfate for 20 min. In the same time, copper (13 μg/mg chlorophyll) was bound to the broken cells. Addition of 10 mM KCl reduced copper binding by about 53%. Fluorescence intensity at room temperature decreased upon addition of cupric sulfate and was partially restored by adding 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), These results suggest that copper inactivates electron transport between the oxidizing side of the reaction center of photosystem II and the electron-donating site of DPC. Further, the effect of light intensity shows that copper mostly affected the reaction rate of the dark step and had less inhibitory effect on the quantum efficiency of the primary reaction of electron transport in photosystem II.     

Site of salicylaldoxime interaction with photosystem II

The reversible inhibition of Photosystem II by salicylaldoxime was studied in spinach D-10 particles by fluorescence, optical absorption, and electron spin resonance spectroscopy. In the presence of 15 mM salicylaldoxime, the initial fluorescence yield was raised to the level of the maximum fluorescence, indicating efficient charge recombination between reduced pheophytin (Ph) and P680⁺. In agreement with the rapid (ns) backreaction expected between Ph^– and P680⁺, the optical absorption transient at 820 mm was not observed. When the particles were washed free of salicylaldoxime, the optical absorption transient resulting from the rereduction of P680⁺ was restored to the µs timescale. These results, along with the previously observed inhibition of electron transport reactions and diminution of the 515-nm absorption change in chloroplasts [Golbeck, J.H. (1980) Arch Biochem Biophys 202, 458–466], are consistent with a site of inhibition between Ph and QA in Photosystem II. ESR Signal IIf and Signal Its were abolished in the presence of 25 mM salicylaldoxime, but both signals could be recovered by washing the D-10 particles free of the inhibitor. The loss of Signal Ilf is most likely a consequence of the inhibition between Ph and Q_A; the rapid charge recombination between Ph^– and P680⁺ would preclude electron transfer from an electron donor on the oxidizing side of Photosystem II. The loss of Signal Its may be due to a change in the environment of the donor complex such that the semiquinone radical giving rise to Signal Its interacts with a nearby reductant.Abbreviations D₁ electron donor to P680⁺ in oxygen-inhibited chloroplasts - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - F₀ prompt chlorophyll a fluorescence yield - F_i initial chlorophyll a fluorescence yield - F_max maximum chlorophyll a fluorescence yield - F_var variable chlorophyll a fluorescence yield - FWHM full width at half maximum - Mes 2-(N-morpholino) ethanesulfonic acid - P680 reaction center chlorophyll a of photosystem II - Ph pheophytin intermediate electron acceptor - Q_A primary quinone electron acceptor - Q_B secondary quinone electron acceptor - Tris tris(hydroxymethyl)aminomethane - Z electron donor to P680⁺     

A new site of bicarbonate effect in Photosystem II of photosynthesis: Evidence from chlorophyll fluorescence transients in spinach chloroplasts

Recent studies on oxygen evolution of corn chloroplast fragments in flashing light [Stemler, A., Babcock, G.T. and Govindjee (1974) Proc. Natl. Acad. Sci. 71, 4679–4683] have shown that the absence of bicarbonate ions increases the turnover time of the Photosystem II reaction center. The rate limiting steps in Photosystem II turnover can be interpreted in terms of reactions either on the oxidizing (electron donor) or reducing (electron acceptor) side of the reaction center. Experiments are reported here that suggest at least one site of bicarbonate action on the reducing side. In Triswashed spinach chloroplasts (incapable of O₂ evolution), the chlorophyll a fluorescence transient in the presence of various artificial electron donors (hydroquinone, diphenylcarbazide, MnCl₂ and NH₂OH) and in the absence of bicarbonate ions shows a rapid initial rise; the addition of 10 mM NaHCO₃ restores the transient to one characteristic of normal chloroplasts. Furthermore, the transients measured as a function of decreasing bicarbonate concentrations are qualitatively similar to those observed with increasing concentrations of 3-(3, 4-dichlorophenyl)-1, 1-dimethyl urea which imposes a block on the reducing side, rather than to transients observed with increasing concentrations of NH₂OH or prolonged heat treatments, which impose a block on the oxidizing side.     

Inhibition of Photosystem II in Isolated Chloroplasts by Lead

Inhibition of photosynthetic electron transport in isolated chloroplasts by lead salts has been demonstrated. Photosystem I activity, as measured by electron transfer from dichlorophenol indophenol to methylviologen, was not reduced by such treatment. However, photosystem II was inhibited by lead salts when electron flow was measured from water to methylviologen and Hill reaction or by chlorophyll fluorescence. Fluorescence induction curves indicated the primary site of inhibition was on the oxidizing side of photosystem II. That this site was between the primary electron donor of photosystem II and the site of water oxidation could be demonstrated by hydroxylamine restoration of normal fluorescence following lead inhibition.     

Interaction of Zn2+ with the donor side of Photosystem II

The inhibitory effect of Zn²⁺ on photosynthetic electron transport was investigated in native and CaCl₂-treated (depleted in extrinsic polypeptides) Photosystem II (PS II) submembrane preparations. Inhibition of 2,6-dichlorophenolindophenol photoreduction by Zn²⁺ was much stronger in protein-depleted preparations in comparison to the native form. It was found that Ca²⁺ significantly reduced the inhibition in the native PS II preparations, as did Mn²⁺ in a combination with H₂O₂ in the protein-depleted counterparts. No other tested monovalent or divalent cations could replace Ca²⁺ or Mn²⁺ in the respective experiments. Diphenylcarbazide could partially relieve (40–45%) the inhibition in both types of preparations. The above indicates the presence of an active Zn²⁺ inhibitory site on the donor side of PS II. However, neither Ca²⁺ nor Mn²⁺ could completely prevent inhibition by high concentrations of Zn²⁺ (>1 mM). We propose that elevated levels of Zn²⁺ strongly perturb the conformation of the PS II core complex and might also affect the acceptor side of the photosystem.Abbreviations PMSF phenylmethanesulfonyl fluoride - MES 2-(N-morpholino)ethane sulphonic acid - Chl chlorophyll - PS II Photosystem II - DCIP 2,6-dichlorophenolindophenol - DPC sym-diphenylcabazide - DCBQ 2,5-dichlorobenzoquinone     

Lactoperoxidase-catalyzed iodination of chloroplast membranes. I. Analysis of surface-localized proteins

Lactoperoxidase-catalyzed iodination of chloroplast membranes has been employed to characterize the vectorial distribution of lamellar proteins. The enzymatic reaction is highly specific for only the outermost membrane components (Phillips, D. R. and Morrison, M. (1971) Biochemistry 10, 1766–1771); we have determined the distribution of ¹²⁵I label and changes in photochemical activities after iodination in an effort to identify these components. Three major conclusions are evident:

1. 1. The coupling factor for photophosphorylation is highly exposed and is selectively and rapidly inhibited by the iodination reaction.

2. 2. A loss of Photosystem I activity (NADP reduction) resulted from iodination. Partial reactions indicated the effect was on electron-transport components on the reducing side of Photosystem I. There was also a limited inhibition of methyl viologen reduction.

3. 3. Iodination of intact membranes caused a reduction in rates of Photosystem II-dependent Hill reaction activity. This inhibition could not be explained solely on the basis of iodination effects on electron-transport components involved in the oxidation of water. The implications of these data with respect to previous chloroplast-membrane models are discussed.

Photosystem I-dependent cyclic electron transport is important in controlling Photosystem II activity in leaves under conditions of water stress

Leaves of the C₃ plant Brassica oleracea were illuminated with red and/or far-red light of different photon flux densities, with or without additional short pulses of high intensity red light, in air or in an atmosphere containing reduced levels of CO₂ and/or oxygen. In the absence of CO₂, far-red light increased light scattering, an indicator of the transthylakoid proton gradient, more than red light, although the red and far-red beams were balanced so as to excite Photosystem II to a comparable extent. On red background light, far-red supported a transthylakoid electrical field as indicated by the electrochromic P₅₁₅ signal. Reducing the oxygen content of the gas phase increased far-red induced light scattering and caused a secondary decrease in the small light scattering signal induced by red light. CO₂ inhibited the light-induced scattering responses irrespective of the mode of excitation. Short pulses of high intensity red light given to a background to red and/or far-red light induced appreciable additional light scattering after the flashes only, when CO₂ levels were decreased to or below the CO₂ compensation point, and when far-red background light was present. While pulse-induced light scattering increased, non-photochemical fluorescence quenching increased and F₀ fluorescence decreased indicating increased radiationless dissipation of excitation energy even when the quinone acceptor Q_A in the reaction center of Photosystem II was largely oxidized. The observations indicate that in the presence of proper redox poising of the chloroplast electron transport chain cyclic electron transport supports a transthylakoid proton gradient which is capable of controlling Photosystem II activity. The data are discussed in relation to protection of the photosynthetic apparatus against photoinactivation.Abbreviations F, F_M, F'_M, F"_M, F₀, F'₀ chlorophyll fluorescence levels - _exc quantum efficiency of excitation energy capture by open Photosystem II - _{PS II} quantum efficiency of electron flow through Photosystem II - P₅₁₅ field indicating rapid absorbance change peaking at 522 nm - P₇₀₀ primary donor of Photosystem I - Q_A primary quinone acceptor in Photosystem II - Q_N non-photochemical fluorescence quenching - Q_q photochemical quenching of chlorophyll fluorescence     

Regulation of electron transport in Photosystem-II fragments by magnesium ions

In Photosystem-II reaction-center particles (TSF-IIa) fractionated from spinach chloroplasts by Triton X-100 treatment, divalent cations appear to regulate electron-transport reactions. Oxidation of cytochrome b-559 after illumination of the particles was accelerated by the presence of Mg²⁺, whereas photoreduction of 2,6-dichlorophenolindophenol (DCIP) by diphenyl carbazide was inhibited, both at a half-effective concentration of Mg²⁺ of approx. 0.1 mM.The site of regulation was shown to be on the oxidizing side of Photosystem II, near P-680, based on the effects of actinic-light intensity and nature of the electron donors on DCIP photoreduction. Mg²⁺ was effective in quenching chlorophyll fluorescence in TSF-IIa particles, but the quenching was sensitive to the presence of 3(3,4-dichloropheny)-1,1-dimethylurea. In the reactioncenter (core) complex of Photosystem II, where the light-harvesting chlorophyll-protein complex is absent, there seems to be no regulation by Mg²⁺ on excitation-energy distribution.     

Genetic variation in soybean photosynthetic electron transport capacity is related to plastocyanin concentration in the chloroplast

Fifteen ancestral genotypes of United States soybean cultivars were screened for differences in photosynthetic electron transport capacity using isolated thylakoid membranes. Plants were grown in controlled environment chambers under high or low irradiance conditions. Thylakoid membranes were isolated from mature leaves. Photosynthetic electron transport was assayed as uncoupled Hill activity using 2,6-dichlorophenolindophenol (DCIP). Soybean electron transport activity was dependent on genotype and growth irradiance and ranged from 6 to 91 mmol DCIP reduced [mol chlorophyll]^–1 s^–1. Soybean plastocyanin pool size ranged from 0.1 to 1.3 mol plastocyanin [mol Photosystem I]^–1. In contrast, barley and spinach electron transport activities were 140 and 170 mmol DCIP reduced [mol chlorophyll]^–1 s^–1, respectively, with plastocyanin pool sizes of 3 to 4 mol plastocyanin [mol Photosystem I]^–1. No significant differences in the concentrations of Photosystem II, plastoquinone, cytochrome b₆f complexes, or Photosystem I were observed. Thus, genetic differences in electron transport activity were correlated with plastocyanin pool size. The results suggested that plastocyanin pool size can vary significantly and may limit photosynthetic electron transport capacity in certain species such as soybean. Soybean plastocyanin consisted of two isoforms with apparent molecular masses of 14 and 11 kDa, whereas barley and spinach plastocyanins each consisted of single polypeptides of 8 and 12 kDa, respectively.Abbreviations DAP days after planting - DCIP 2,6-dichlorophenolindophenol - LiDS lithium dodecyl sulfate - PPFD photosynthetic photon flux density (mol photons m^–2 s^–1) - PS I Photosystem I - PS II Photosystem II - P700 reaction center of Photosystem I The US Government right to retain a non-exclusive, royalty free licence in and to any copyright is acknowledged.     

The Effect of Cadmium on Photosystem II in Spinach Chloroplasts

Cadmium ions, as an environmental pollution factor, significantly inhibited the photosynthesis especially, photosystem Ⅱ activity in isolated spinach chloroplasts. The presence of 5 mmol/l Cd2+ inhibited the O2-evolution to 53%. Cd2+ reduced the activity of photoreduction of DCIP and the variable fluorescence of chloroplasts and PSⅡ preparation. The inhibited DCIP photoreduction activity could only be restored slightly by the addition of an artificial electron donor of PSII, DPC, and the inhibited variable fluorescence could not be obviously recovered by the addition of NH2OH, another artificial electron donor of PSⅡ. It is considered that, besides the oxidizing side of PSI1, Cd2+ could also inhibit directly the PSⅡ reaction center. The inhibitory effect of Cd2+ on the whole chain electron transport (H2O→MV) was more serious than on O2-evolution (H2O→DCMU). It is suggested that the oxidizing side of PSⅡ is not the only site for Cd2+ action. There may be another site inhibited by Cd2+ in the electron transport chain between PSⅠ and PSⅡ.     

Light-induced oxidation-reduction reactions in a cell-free preparation from the blue-green alga Nostoc muscorum: The role of cytochrome f, cytochrome b558, C550, and P700 in noncyclic electron transport

1. Cytochrome f (λ_max = 554 nm, E_m = +0.35 V) and cytochrome b₅₅₈ (λ_max = 558 nm, E_m = +0.35 V) were photooxidized by Photosystem I and photoreduced by Photosystem II in a cell-free preparation from the blue-green alga Nostoc muscorum. The steady-state oxidation levels of both cytochromes were affected by noncyclic electron acceptors and by inhibitors of noncyclic electron transport. These results are consistent with the hypothesis that the mechanism of NADP reduction by water involves a Photosystem II and a Photosystem I light reaction operating in series and linked by a chain of electron carriers that includes cytochrome f and cytochrome b₅₅₈.2. Phosphorylation cofactors shifted the steady-state of cytochrome f to a more reduced level under conditions of noncyclic electron transport but had no effect on cytochrome b₅₅₈. These observations suggest that the noncyclic phosphorylation site lies before cytochrome f (on the Photosystem II side) and that cytochrome f is closer to this site than is cytochrome b₅₅₈.3. A Photosystem II photoreduction of C550 at 77 °K was observed, suggesting that in blue-green algae, as in other plants, C550 is closely associated with the primary electron acceptor for Photosystem II. A Photosystem I photooxidation of P700 at 77 °K was observed, consistent with P700 serving as the primary electron donor of Photosystem I.     

Alteration in the acceptor side of photosystem II of chloroplast by high light

The effect of high light on the acceptor side of photosystem II of chloroplasts and core particles of spinach was studied. BothV _max and apparentK _m for DCIP were altered in photoinhibited photosystem II core particles. The double reciprocal plot analysis as a function of actinic light showed increased slope in chloroplasts photoinhibited in the presence of DCMU. Exposure of chloroplasts to high light in the presence of DCMU did not protect the chloroplast against high light induced decrease in F_m, level. Further the high light stress induced decrease inF _m level was not restored by the addition of DCMU. These results suggest that the high light stress induced damage to chloroplast involves alteration in the binding site forQ _B on the DI protein on the acceptor side of photosystem II     

Hydroxylamine,hydrazine and methylamine donate electrons to the photooxidizing side of Photosystem II in leaves inhibited in oxygen evolution due to water stress

When leaf discs are water stressed, they lose the capacity for photosynthetic oxygen evolution and variable (chlorophyll a) fluorescence. Such a loss of variable fluorescence was previously reported by Govindjee et al. (Plant Sci. Lett. 20 (1981) 191–194). The later activity is not lost if prior to the water-stress treatment the leaf is incubated with typical water analogs known to act as electron donors to Photosystem II, such as hydroxylamine and hydrazine. Methylamine also acts in the same fashion. These results indicate that one of the sites of drought damage is the oxidizing side of Photosystem II, and that electron donors can restore electron transport, at least to the plastoquinone pool, similar to their effect in Tris treatment of isolated chloroplasts.     

Cooperativity between Photosystem II centers at the level of primary electron transfer

1. Spinach chloroplasts, but not whole Chlorella cells, show an acceleration of the Photosystem II turnover time when excited by non-saturating flashes (exciting 25 % of centers) or when excited by saturating flashes for 85–95 % inhibition by 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Following dark adaptation, the turnover is accelerated after a non-saturating flash, preceded by none or several saturating flashes, and primarily after a first saturating flash for 3-(3,4-dichlorophenyl)-1,1-dimethylurea inhibition. A rapid phase ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ approx. 0.75 s) is observed for the deactivation of State S₂ in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea.2. These accelerated relaxations suggest that centers of Photosystem II are interconnected at the level of the primary electron transfer and compete for primary oxidizing equivalents in a saturating flash. The model in best agreement with the experimental data consists of a paired interconnection of centers.3. Under the conditions mentioned above, an accelerated turnover may be observed following a flash for centers in S₀, S₁ or S₂ prior to the flash. This acceleration is interpreted in terms of a shift of the rate-limiting steps of Photosystem II turnover from the acceptor to the donor side.     

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.

Recognition of interaction between the donor electron-transfer chains of Photosystem II under conditions of partial inhibition of oxygen evolution

The electron-transfer pathway on the donor side of Photosystem (PS) II has been examined using unfractionated and inside-out thylakoid membrane vesicles. A number of treatments are identified which result in the inhibition of light-dependent oxygen evolution. The differential capacities of the exogenous donors diphenylcarbazide and NH₂OH to restore the PS II-mediated reduction of 2,6-dichlorophenolindophenol (DCIP) in the inhibited membranes is discussed in terms of multiple donor sites for the electron-transfer pathway on the oxidising side of PS II. We also present data which indicate that the donor chains are not isolated from each other but that an individual PS II reaction centre may be able to interact with several oxygen-evolving complexes. The implication of such an interaction to the mechanism of oxygen evolution is discussed.     

Isolation and characterization of Photosystem I and II membrane particles from the blue-green alga, Synechococcus cedrorum

Fractions enriched in either Photosystem I or Photosystem II activity have been isolated from the blue-green alga, Synechococcus cedrorum after digitonin treatment. Sedimentation of this homogenate on a 10–30% sucrose gradient yielded three green bands: the upper band was enriched in Photosystem II, the lowest band was enriched in Photosystem I, while the middle band contained both activities. Large quantities of both particles were isolated by zonal centrifugation, and the material was then further purified by chromatography on DEAE-cellulose.The resulting Photosystem II particles carried out light-induced electron transport from semicarbizide to ferricyanide of over 2000 μmol/mg Chlorophyll per h (which was sensitive to 3-(3,4-dichlorophenyl)-1,1-dimethylurea), and was nearly devoid of Photosystem I activity. This particle contains β-carotene, very little phycocyanin, has a chlorophyll absorption maximum at 675 nm, and a liquid N₂ fluorescence maximum at 685 nm. The purest Photosystem II particles have a chlorophyll to cytochrome $\text{b-559}$ ratio of 50 : 1. The Photosystem I particle is highly enriched in $\text{P-700}$, with a chlorophyll to $\text{P-700}$ ratio of 40 : 1. The physical structure of the two Photosystem particles has also been studied by gel electrophoresis and electron microscopy. These results indicate that the size and protein composition of the two particles are distinctly different.     

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).     

Heat sensitivity of chloroplasts and leaves: Leakage of protons from thylakoids and reversible activation of cyclic electron transport

In illuminated intact spinach chloroplasts, warming to and beyond 40 °C increased the proton permeability of thylakoids before linear electron transport through Photosystem II was inhibited. Simultaneously, antimycin A-sensitive cyclic electron transport around Photosystem II was activated with oxygen or CO2, but not with nitrite as electron acceptors. Between 40 to 42 °C, activation of cyclic electron transport balanced the loss of protons so that a sizeable transthylakoid proton gradient was maintained. When the temperature of darkened spinach leaves was slowly increased to 40°C, reduction of the quinone acceptor of Photosystem II, Q_A, increased particularly when respiratory CO2 production and autoxidation of plastoquinones was inhibited by decreasing the oxygen content of the atmosphere from 21 to 1%. Simultaneously, Photosystem II activity was partially lost. The enhanced dark Q_A reduction disappeared after the leaf temperature was decreased to 20 °C. No membrane energization was detected by light-scattering measurements during heating the leaf in the dark. In illuminated spinach leaves, light scattering and nonphotochemical quenching of chlorophyll fluorescence increased during warming to about 40 °C while Photosystem II activity was lost, suggesting extra energization of thylakoid membranes that is unrelated to Photosystem II functioning. After P700 was oxidized by far-red light, its reduction in the dark was biphasic. It was accelerated by factors of up to 10 (fast component) or even 25 (slow component) after short heat exposure of the leaves. Similar acceleration was observed at 20 °C when anaerobiosis or KCN were used to inhibit respiratory oxidation of reductants. Methyl viologen, which accepts electrons from reducing side of Photosystem II, completely abolished heat-induced acceleration of P700+ reduction after far-red light. The data show that increasing the temperature of isolated chloroplasts or intact spinach leaves to about 40 °C not only inhibits linear electron flow through Photosystem II but also activates Photosystem I-driven cyclic electron transport pathways capable of contributing to the transthylakoid proton gradient. Heterogeneity of the kinetics of P700+ reduction after far-red oxidation is discussed in terms of Photosystem I-dependent cyclic electron transport in stroma lamellae and grana margins.