Electron spin resonance study of chloroplast photosynthetic activity in the presence of amphiphilic amines

Electron spin resonance spectroscopy (ESR) was used to study the effects of amphiphilic amines of the carbamate, amide, and ester type and amine oxide on the photosynthetic system of spinach chloroplasts. The ESR signal II connected to the photosynthetic center PS II donor side was observed to diminish in the presence of amines, whereas that of PS I remained unchanged. The inhibition of PS II increased with the increasing of amine concentration. In the presence of amines, the light: dark chloroplast ESR signals ratio as well as the intensity of the ESR signal of unbound Mn2+ increased. It is suggested that the amphiphilic amines affect the structure of PS II and the electron transfer to PS I. The effects of the amines tested on the photosynthetic system correlate with their potency to perturb the lipid membrane structure.     

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.     

Determination of the PS I content of PS II core preparations using selective emission: A new emission of PS II at 780 nm

Routinely prepared PS II core samples are often contaminated by a significant (~ 1–5%) fraction of PS I, as well as related proteins. This contamination is of little importance in many experiments, but masks the optical behaviour of the deep red state in PS II, which absorbs in the same spectral range (700–730 nm) as PS I (Hughes et al. 2006). When contamination levels are less than ~ 1%, it becomes difficult to quantify the PS I related components by gel-based, chromatographic, circular dichroism or EPR techniques. We have developed a fluorescence-based technique, taking advantage of the distinctively different low-temperature emission characteristics of PS II and PS I when excited near 700 nm. The approach has the advantage of providing the relative concentration of the two photosystems in a single spectral measurement. A sensitivity limit of 0.01% PS I (or better) can be achieved. The procedure is applied to PS II core preparations from spinach and Thermosynechococcus vulcanus. Measurements made of T. vulcanus PS II preparations prepared by re-dissolving crystallised material indicate a low but measurable PS I related content. The analysis provides strong evidence for a previously unreported fluorescence of PS II cores peaking near 780 nm. The excitation dependence of this emission as well as its appearance in both low PS I cyanobacterial and plant based PS II core preparations suggests its association with the deep red state of PS II.     

Effect of exogenous histidine on restoration of electron transfer on the donor side of Photosystem II depleted of Mn

It is shown that restoration of photoinduced electron flow with added Mn²⁺ (measured by photoreduction of DCPIP and photoinduced change of chlorophyll fluorescence yield) in Mn-depleted Photosystem II (PS II) membrane fragments isolated from spinach chloroplasts, is considerably increased by exogenous histidine (His). The stimulating effect of His is not observed if other electron donors (NH₂OH or diphenylcarbazide) are used instead of Mn²⁺. His added alone does not induce electron transfer in Mn-depleted PS II preparations. Investigation of pH dependence of the stimulating effect of 2 mM His shows that the effect is observed only at pH > 5.0, it gives a 50% activation around pH 6.0 and saturates at pH 7.0–7.5. Nearly 200 μM His is required for a 50 effect at pH 7.0. It is suggested that the added His can be involved in stimulation of electron transfer on the donor side of PS II through direct interaction of Mn²⁺ with deprotonated form(s) of His resulting in formation of Mn–His complexes capable of efficient electron donation to PS II (though it is not excluded that His serves as a base that takes part in proton exchange coupled with redox reactions on the donor side of PS II or as an electron donor to the oxidized Mn).     

EPR characteristics of chloride-depleted photosystem II membranes in the presence of other anions.

Chloride is an essential cofactor for the oxidation of water to oxygen. Anion substitution (Br(-), I(-), NO(2)(-), F(-)) in Cl(-)-depleted PS II membranes brings out significant changes in the EPR signals arising from the S(2) state and from the iron-quinone complex of PS II. On the basis of the changes observed in the S(2) state multiline signal and the Q(A)Fe(3+) EPR signal in Cl(-)-depleted PS II membranes after substituting with various anions, we report a possible binding site of anions such as chloride and bromide at the PS II donor side as well as at the acceptor side.     

Deciphering the 820 nm signal: redox state of donor side and quantum yield of Photosystem I in leaves

By recording leaf transmittance at 820 nm and quantifying the photon flux density of far red light (FRL) absorbed by long-wavelength chlorophylls of Photosystem I (PS I), the oxidation kinetics of electron carriers on the PS I donor side was mathematically analyzed in sunflower (Helianthus annuus L.), tobacco (Nicotiana tabacum L.) and birch (Betula pendula Roth.) leaves. PS I donor side carriers were first oxidized under FRL, electrons were then allowed to accumulate on the PS I donor side during dark intervals of increasing length. After each dark interval the electrons were removed (titrated) by FRL. The kinetics of the 820 nm signal during the oxidation of the PS I donor side was modeled assuming redox equilibrium among the PS I donor pigment (P700), plastocyanin (PC), and cytochrome f plus Rieske FeS (Cyt f + FeS) pools, considering that the 820 nm signal originates from P700⁺ and PC⁺. The analysis yielded the pool sizes of P700, PC and (Cyt f + FeS) and associated redox equilibrium constants. PS I density varied between 0.6 and 1.4 μmol m⁻². PS II density (measured as O₂ evolution from a saturating single-turnover flash) ranged from 0.64 to 2.14 μmol m⁻². The average electron storage capacity was 1.96 (range 1.25 to 2.4) and 1.16 (range 0.6 to 1.7) for PC and (Cyt f + FeS), respectively, per P700. The best-fit electrochemical midpoint potential differences were 80 mV for the P700/PC and 25 mV for the PC/Cyt f equilibria at 22 °C. An algorithm relating the measured 820 nm signal to the redox states of individual PS I donor side electron carriers in leaves is presented. Applying this algorithm to the analysis of steady-state light response curves of net CO₂ fixation rate and 820 nm signal shows that the quantum yield of PS I decreases by about half due to acceptor side reduction at limiting light intensities before the donor side becomes oxidized at saturating intensities. Footnote: This revised version was published online in August 2006 with corrections to the Cover Date.     

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.     

Influence of protein phosphorylation on the electron-transport properties of Photosystem II

Many of the core proteins in Photosystem II (PS II) undergo reversible phosphorylation. It is known that protein phosphorylation controls the repair cycle of Photosystem II. However, it is not known how protein phosphorylation affects the partial electron transport reactions in PS II. Here we have applied variable fluorescence measurements and EPR spectroscopy to probe the status of the quinone acceptors, the Mn cluster and other electron transfer components in PS II with controlled levels of protein phosphorylation. Protein phosphorylation was induced in vivo by varying illumination regimes. The phosphorylation level of the D1 protein varied from 10 to 58% in PS II membranes isolated from pre-illuminated spinach leaves. The oxygen evolution and Q_A ⁻ to Q_B(Q_B ⁻) electron transfer measured by flash-induced fluorescence decay remained similar in all samples studied. Similar measurements in the presence of DCMU, which reports on the status of the donor side in PS II, also indicated that the integrity of the oxygen-evolving complex was preserved in PS II with different levels of D1 protein phosphorylation. With EPR spectroscopy we examined individual redox cofactors in PS II. Both the maximal amplitude of the charge separation reaction (measured as photo-accumulated pheophytin⁻) and the EPR signal from the Q_A ⁻ Fe²⁺ complex were unaffected by the phosphorylation of the D1 protein, indicating that the acceptor side of PS II was not modified. Also the shape of the S₂ state multiline signal was similar, suggesting that the structure of the Mn-cluster in Photosystem II did not change. However, the amplitude of the S₂ multiline signal was reduced by 35% in PS II, where 58% of the D1 protein was phosphorylated, as compared to the S₂ multiline in PS II, where only 10% of the D1 protein was phosphorylated. In addition, the fraction of low potential Cyt b ₅₅₉ was twice as high in phosphorylated PS II. Implications from these findings, were precise quantification of D1 protein phosphorylation is, for the first time, combined with high-resolution biophysical measurements, are discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Carotenoid distribution and deepoxidation in thylakoid pigment-protein complexes from cotton leaves and bundle-sheath cells of maize

In response to excess light, the xanthophyll violaxanthin (V) is deepoxidized to zeaxanthin (Z) via antheraxanthin (A) and the degree of this deepoxidation is strongly correlated with dissipation of excess energy and photoprotection in PS II. However, little is known about the site of V deepoxidation and the localization of Z within the thylakoid membranes. To gain insight into this problem, thylakoids were isolated from cotton leaves and bundle-sheath strands of maize, the pigment protein-complexes separated on Deriphat gels, electroeluted, and the pigments analyzed by HPLC. In cotton thylakoids, 30% of the xanthophyll cycle pigments were associated with the PS I holocomplex, including the PS I light-harvesting complexes and PS I core complex proteins (CC I), and about 50% with the PS II light-harvesting complexes (LHC II). The Chl was evenly distributed between PS I and PS II. Less than 2% of the neoxanthin, about 18% of the lutein, and as much as 76% of the -carotene of the thylakoids were associated with PS I. Exposure of pre-darkened cotton leaves to a high photon flux density for 20 min prior to thylakoid isolation caused about one-half of the V to be converted to Z. The distribution of Z among the pigment-protein complexes was found to be similar to that of V. The distribution of the other carotenoids was unaffected by the light treatment. Similarly, in field-grown maize leaves and in the bundle-sheath strands isolated from them, about 40% of the V present at dawn had been converted to Z at solar noon. Light treatment of isolated bundle-sheath strands which initially contained little Z caused a similar degree of conversion of V to Z. As in cotton thylakoids, about 30% the V+A+Z pool in bundle-sheath thylakoids from maize was associated with the PS I holocomplex and the CC I bands and 46% with the LHC II bands, regardless of the extent of deepoxidation. These results demonstrate that Z is present in PS I as well as in PS II and that deepoxidation evidently takes place within the pigment-protein complexes of both photosystems.Abbreviations A antheraxanthin - CC I, CC II Core or reaction center complex of PS I, PS II - CP Chl protein - EPS epoxidation state - F_m Chl fluorescence at closed PS II reaction centers - IEF isoelectric focussing gels - LHC I, LHC II light-harvesting complex of PS I, PS II - OE oxygen evolving polypeptide - PFD photon flux density - PS I^* PS I holocomplex - V violaxanthin - Z zeaxanthin - antibody against C.I.W.-D.P.B. Publication No. 1127.     

Functional characterization of monomeric photosystem II core preparations from Thermosynechococcus elongatus with or without the Psb27 protein

Two monomeric fractions of photosystem II (PS II) core pacticles from the thermophilic cyanobacterium Thermosynechococcus elongatus have been investigated using flash-induced variable fluorescence kinetics and EPR spectroscopy. One fraction was highly active in oxygen evolution and contained the extrinsic protein subunits PsbO, PsbU, and PsbV. The other monomeric fraction lacked oxygen evolving activity as well as the three extrinsic subunits, but the luminally located, extrinsic Psb27 lipoprotein was present. In the monomeric fraction with bound Psb27, flash-induced variable fluorescence showed an absence of oxidizable Mn on the donor side of PS II and impaired forward electron transfer from the primary quinone acceptor, QA. These results were confirmed with EPR spectroscopy by the absence of the "split S1" interaction signal from YZ* and the CaMn4 cluster and by the absence of the S2-state multiline signal. A different protein composition on the donor side of PS II monomers with Psb27 was also supported by the lack of an EPR signal from cytochrome c550 (in the PsbV subunit). In addition, we did not observe any oxidation of cytochrome b559 at low temperature in this fraction. The presence of Psb27 and the absence of the CaMn4 cluster did not affect the protein matrix around YD or the acceptor side quinones as can be judged from the appearance of the corresponding EPR signals. The diminished electron transport capabilities on both the donor and the acceptor side of PS II when Psb27 is present give further indications that this PS II complex is involved in the earlier steps of the PS II repair cycle.     

Time sequence of the damage to the acceptor and donor sides of photosystem II by UV-B radiation as evaluated by chlorophyll a fluorescence

The effects of ultraviolet-B (UV-B) radiation on photosystem II (PS II) were studied in leaves of Chenopodium album. After the treatment with UV-B the damage was estimated using chlorophyll a fluorescence techniques. Measurements of modulated fluorescence using a pulse amplitude modulated fluorometer revealed that the efficiency of photosystem II decreased both with increasing time of UV-B radiation and with increasing intensity of the UV-B. Fluorescence induction rise curves were analyzed using a mechanistic model of energy trapping. It appears that the damage by UV-B radiation occurs first at the acceptor side of photosystem II, and only later at the donor side.     

Interactions of Chloride and Formate at the Donor and the Acceptor Side of Photosystem II

Chloride is required for the maximum activity of the oxygen evolving complex (OEC) while formate inhibits the function of OEC. On the basis of the measurements of oxygen evolution rates and the S₂ state multiline EPR signal, an interaction between the action of chloride and formate at the donor side of PS II has been suggested. Moreover, the Fe₂+Q–A EPR signals were measured to investigate a common binding site of both these anions at the PS II acceptor side. Other monovalent anions like bromide, nitrate etc. could influence the effects of formate to a small extent at the donor side of PS II, but not significantly at the acceptor side of PS II. The results presented in this paper clearly suggest a competitive binding of formate and chloride at the PS II acceptor side.     

Specific 125I labeling of D1 (herbicide-binding protein): An indication that D1 functions on both the donor and acceptor sides of photosystem II

When ¹²⁵I⁻ was given as an artificial electron donor to non-O₂-evolving thylakoids of spinach, a 29 kDa polypeptide was specifically tagged by ¹²⁵I due to its photooxidation by PS II [(1985) Plant Cell Physiol. 26, 1093–1100]. We examined precisely the ¹²⁵I-labeling pattern in comparison with azido[¹⁴C]atrazine photoaffinity labeling of D1 and immunoblotting with anti-D1 and anti-D2, and found that D1 (herbicidebinding protein) of PS II reaction center complex is specifically tagged by ¹²⁵I in three different species of higher plants (spinach, pea and wheat) and a thermophilic cyanobacterium (Synechococcus vulcanus). It was suggested that D1 bears the photooxidation site or has a domain very close to the photooxidation site on the donor side of PS II, in addition to the well established binding site for Q_b and herbicides on the acceptor side of PS II.     

Formate-induced inhibition of the water-oxidizing complex of photosystem II studied by EPR

The effects of various formate concentrations on both the donor and the acceptor sides in oxygen-evolving PS II membranes (BBY particles) were examined. EPR, oxygen evolution and variable chlorophyll fluorescence have been observed. It was found that formate inhibits the formation of the S(2) state multiline signal concomitant with stimulation of the Q(A)(-)Fe(2+) signal at g = 1.82. The decrease and the increase in intensities of the multiline and Q(A)(-)Fe(2+) signals, respectively, had a linear relation for formate concentrations between 5 and 500 mM. The g = 4.1 signal formation measured in the absence of methanol was not inhibited by formate up to 250 mM in the buffer. In the presence of 3% methanol the g = 4.1 signal evolved as formate concentration increased. The evolved signal could be ascribed to the inhibited centers. Oxygen evolution measured in the presence of an electron acceptor, phenyl-p-benzoquinone, was also inhibited by formate proportionally to the decrease in the multiline signal intensity. The inhibition seemed to be due to a retarded electron transfer from the water-oxidizing complex to Y(Z)(+), which was observed in the decay kinetics of the Y(Z)(+) signal induced by illumination above 250 K. These results show that formate induces inhibition of water oxidation reactions as well as electron transfer on the PS II acceptor side. The inhibition effects of formate in PS II were found to be reversible, indicating no destructive effect on the reaction center induced by formate.     

Measurement of the relative electron-transport capacity of Photosystem I and Photosystem II in spinach chloroplasts

The ratio of Photosystem (PS) II to PS I electron-transport capacity in spinach chloroplasts was compared from reaction-center and steady-state rate measurements. The reaction-center electron-transport capacity was based upon both the relative concentrations of the PS II_α, PS II_β and PS I centers, and the number of chlorophyll molecules associated with each type of center. The reaction-center ratio of total PS II to PS I electron-transport capacity was about 1.8:1. Steady-state electron-transport capacity data were obtained from the rate of light-induced absorbance-change measurements in the presence of ferredoxin-NADP⁺, potassium ferricyanide and 2,5-dimethylbenzoquinone (DMQ). A new method was developed for determining the partition of reduced DMQ between the thylakoid membrane and the surrounding aqueous phase. The ratio of membrane-bound to aqueous DMQH₂ was experimentally determined to be 1.3:1. When used at low concentrations (200 μM), potassium ferricyanide is shown to be strictly a PS I electron acceptor. At concentrations higher than 200 μM, ferricyanide intercepted electrons from the reducing side of PS II as well. The experimental rates of electron flow through PS II and PS I defined a PS II/PS I electron-transport capacity ratio of 1.6:1.     

Computer simulation study of pH-dependent regulation of electron transport in chloroplasts

A mathematical model is presented that describes the key steps of photosynthetic electron transport and transmembrane proton transfer in chloroplasts. Numerical modeling has been performed with due regard for regulatory processes at the donor and acceptor parts of photosystem (PS) I. The influence of pH-dependent activation of the Calvin cycle enzymes and energy dissipation in PS II (nonphotochemical quenching of chlorophyll fluorescence) on the light-induced redox transients of P₇₀₀, plastoquinone, and NADP as well as on the changes in intrathylakoid pH and ATP level is examined. It is demonstrated that pH-dependent regulatory processes alter the distribution of electron fluxes on the acceptor side of PS I and the total rate of electron flow between PS II and PS I. The light-induced activation of the Calvin cycle leads to significant enhancement of the electron flow from PS I to NADP⁺ and attenuation of the electron flow to molecular oxygen.     

Inhibition of photosystem II of spinach by the respiration inhibitors piericidin A and thenoyltrifluoroacetone

The effects of several respiration inhibitors on photosystem II (PS II) were investigated. Among the agents tested, piericidin A and thenoyltrifluoroacetone (TTFA) inhibited the photosynthetic electron transport of spinach as measured from chlorophyll (Chl) fluorescence parameters (Fm'-F)/Fm' and Fv/Fm. Using specific donors and acceptors of electrons, we identified the sites of inhibition in and around the PS II complex; the site of inhibition by TTFA was between QA, primary quinone acceptor in PS II, and QB, secondary quinone acceptor, in the acceptor side of P680, the reaction center Chl of PS II, while inhibition by piericidin A of the acceptor side was downstream of Q(B), out of the PS II complex. Both agents also inhibited the donor side of P680, probably between tyrosine-161 of the reaction center protein of PS II and P680.     

The effects of ultraviolet irradiation on P680+ reduction in PS II core complexes measured for individual S-states and during repetitive cycling of the oxygen-evolving complex

Flash-induced absorbance measurements at 830 nm on both nanosecond and microsecond timescales have been used to characterise the effect of ultraviolet light on Photosystem II core particles. A combination of UV-A and UV-B, closely simulating the spectrum of sunlight below 350 nm, was found to have a primary effect on the donor side of P680. Repetitive measurements indicated reductions in the nanosecond components of the absorbance decay with a concomitant appearance and increase in the amplitude of a component with a 10 s time constant attributed to slow reduction of P680⁺ by Tyr_z when the function of the oxygen evolving complex is inhibited. Single-flash measurements show that the nanosecond components have amplitudes which vary with S-state. Increasing UV irradiation inhibited the amplitude of these components without changing their S-state dependence. In addition, UV irradiation resulted in a reduction in the total amplitude, with no change in the proportion of the 10 s contribution.Abbreviations BBY- PS II membrane fragments - P680- primary electron donor of PS II - PS II- Photosystem II - Q_A and Q_B– primary and secondary quinone electron acceptors of PS II - S-state- redox state of the oxygenevolving complex - Tyr_z– tyrosine residue in PS II - UV-A- ultraviolet radiation 320–400 nm - UV-B- ultraviolet radiation 280–320 nm     

Defining the Far-red Limit of Photosystem I: THE PRIMARY CHARGE SEPARATION IS FUNCTIONAL TO 840 nm*

The far-red limit of photosystem I (PS I) photochemistry was studied by EPR spectroscopy using laser flashes between 730 and 850 nm. In manganese-depleted spinach thylakoid membranes, the primary donor in PS I, P₇₀₀, was oxidized simultaneously with tyrosine Z, the secondary donor in PS II. It was found that at 295 K PS I photochemistry, observed as P₇₀₀⁺ formation, was functional up to 840 nm. This is 30 nm further to the red region than was reported for PS II photochemistry (Thapper, A., Mamedov, F., Mokvist, F., Hammarström, L., and Styring, S. (2009) Plant Cell 21, 2391–2401). The same far-red limit for the P₇₀₀⁺ formation was observed in a PS I reaction center core preparation from Nostoc punctiforme. The reduction of the acceptor side of PS I, observed as reduction of the iron-sulfur centers F_A and F_B by low temperature EPR measurements, was also functional at 15 K with light up to >830 nm. Taken together, these results, obtained from both plants and cyanobacteria, most likely rule out involvement of the red-absorbing antenna chlorophylls in this reaction. Instead we propose the existence of weak charge transfer bands absorbing in the far-red region in the ensemble of excitonically coupled chlorophyll a molecules around P₇₀₀ similar to what has been found in the reaction center of PS II. These charge transfer bands could be responsible for the far-red light absorption leading to PS I photochemistry at wavelengths up to 840 nm.