Mode of action of Photosystem II herbicides studied by thermoluminescence

The mode of action of chemically different herbicides (ureas, pyridazinones, phenylcarbamates, triazines, hydroxyquinolines, hydroxybenzonitriles and dinitrophenols) on photosynthetic electron transport was investigated by measurements of oxygen evolution and thermoluminescence. Depending on the particular herbicide used the thermoluminescence band related to Q (the primary acceptor of Photosystem II) appears at +5, 0 or −14°C. It was shown that these three different peak positions can be ascribed to various redox states of Q, the shifts being due to the binding of herbicides to the chloroplast membrane. Both displacement experiments and additive inhibition of herbicide pairs measured by thermoluminescence and oxygen evolution suggested that the sites of action of these herbicides are on the same protein. However, herbicide treatment of trypsinized chloroplasts showed that there were three different binding sites on the same protein, in agreement with the classification of herbicides into three groups based on thermoluminescence measurements. Our results suggest that the primary and secondary acceptors of Photosystem II (Q and B, respectively) are in close proximity and form a common complex with the herbicide-binding protein within the chloroplast membrane.     

Electric-field-induced luminescence emission spectra of Photosystem I and Photosystem II from chloroplasts

The electroluminescence induced by external electric fields in blebs prepared from chloroplasts consists of two kinetically different phases, rapid (R) and slow (S), which were shown to be linked to Photosystem I (PS I) and Photosystem II (PS II) activities, respectively (Symons, M., Korenstein, R. and Malkin, S. (1985) Biochim. Biophys. Acta 806, 305–310). In this report we describe conditions involving heat treatment of broken chloroplasts, which make it possible to observe R phase electroluminescence essentially devoid of any contribution by the S phase. This allowed the precise measurement of the emission spectrum of PS I electroluminescence. The emission spectrum of PS II electroluminescence was obtained using regular broken chloroplasts, which show only S-type emission. The latter emission spectrum is identical to the one obtained for ordinary prompt fluorescence, peaking at 685 nm with a bandwidth of about 25 nm. The PS I emission spectrum is symmetric around 705 nm and is much broader, about 60 nm.     

Charge accumulation and recombination in Photosystem II studied by thermoluminescence. I. Participation of the primary acceptor Q and secondary acceptor B in the generation of thermoluminescence of chloroplasts

In the glow curves of chloroplasts excited by a series of flashes at +1°C the intensity of the main thermoluminescence band appearing at +30°C (B band; B, secondary acceptor of Photosystem II) exhibits a period-4 oscillation with maxima on the 2nd and 6th flashes indicating the participation of the S₃ state of the water-splitting system in the radiative charge recombination reaction. After long-term dark adaptation of chloroplasts (6 h), when the major part of the secondary acceptor pool (B pool) is oxidized, a period-2 contribution with maxima occurring at uneven flash numbers appears in the oscillation pattern. The B band can even be excited at ?160°C as well as by a single flash in which case the water-splitting system undergoes only one transition (S₁ → S₂). The experimental observations and computer simulation of the oscillatory patterns suggest that the B band originates from charge recombination of the S₂B^? and S₃B^? redox states. The half-time of charge recombination responsible for the B band is 48 s. When a major part of the plastoquinone pool is reduced due to prolonged excitation of the chloroplasts by continuous light, a second band (Q band; Q, primary acceptor of Photosystem II) appears in the glow curve at +10°C which overlaps with the B band. In chloroplasts excited by flashes prior to DCMU addition only the Q band can be observed showing maxima in the oscillation pattern at flash numbers 2, 6 and 10. The Q band can also be induced by flashes after DCMU addition which allows only one transition of the water-splitting system (S₁ → S₂). In the presence of DCMU, electrons accumulate on the primary acceptor Q, thus the Q band can be ascribed to the charge recombination of either the S₂Q^? or S₃Q^? states depending on whether the water-splitting system is in the S₂ or the S₃ state. The half-time of the back reaction of Q^? with the donor side of PS II (S₂ or S₃ states) is 3 s. It was also observed that in a sequence of flashes the peak positions of the Q and B bands do not depend on the advancement of the water-splitting system from the S₂ state to the S₃ state. This result implies that the midpoint potential of the water-splitting system remains unmodified during the S₂ → S₃ transition.     

Mode of action of 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Evidence that the inhibitor competes with plastoquinone for binding to a common site on the acceptor side of Photosystem II

The kinetics and concentration dependence of the binding of dichlorophenyldimethylurea (DCMU) to Photosystem II (PS II) were monitored through fluorescence measurements. According to whether the acceptor system is in the ‘odd’ state (QB⁻ ag Q⁻B) or ‘even’ state (QB), very different results are obtained. The binding to centers in the even state is rapid ( at [DCMU] = 10⁻⁵ M and [chlorophyll] = 10 μg/ml), with a pH-independent rate. The concentration curve of the bound inhibitor (at equilibrium) corresponds to an association constant of about 3.3·10⁷ M⁻¹·1. The binding of the inhibitor to centers in the odd state is slow ( at pH 7, same DCMU and chlorophyll concentrations as above), and depends on pH. In the pH range 6–8, the lower the pH, the slower the kinetics. The association constant is also diminished by a factor of approx. 20 (at pH 7) compared to the even state centers. It is shown that these effects are in good agreement with predictions from Velthuys' hypothesis (Velthuys, B.R. (1981) FEBS Lett. 126, 277–281) that the mode of action of DCMU is a competition with plastoquinone for the binding to the secondary acceptor site. A large part of PS II photochemical quenching corresponds to acceptors which seem to possess a secondary acceptor distinct from B. They were called ‘non-B-type acceptors’ (Lavergne, J. (1982) Photobiochem. Photobiophys. 3, 257–285) and may be identified with Joliot's ‘Q₂’ (Joliot P. and Joliot, A. (1977) Biochim. Biophys. Acta 462, 559–574). However, the rate at which the inhibition affects these non-B-type acceptors is similar to the rate of DCMU binding on the B site (i.e., slow in the odd state, fast in the even state).     

Thermoluminescence as a probe of Photosystem II photochemistry. The origin of the flash-induced glow peaks

A single flash given at − 15°C to chloroplasts results in charge separation in Photosystem II to form a stable state which, upon warming, recombines giving rise to luminescence. This recombination occurs at 25°C in untreated chloroplasts but is shifted to 0°C in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea or weak concentrations of a reducing agent. The luminescence at 0°C is attributed to recombination of the S₂Q⁻_A state while that at 25°C is attributed to recombination of S₂Q_AQ⁻_B (and S₃Q_AQ⁻_B upon further flash illumination). The identification of the thermoluminescence at 25°C is based upon the following experimental evidence: (1) illumination of chloroplasts in the presence of methyl viologen with 710 nm light before and after flash illumination has no effect on the extent or temperature of the thermoluminescence. This is taken as evidence that the plastoquinone pool is not involved in the recombination reaction. (2) Calculations of the extent of thermoluminescence expected after a number of flashes, assuming that S₂Q_AQ⁻_B and S₃Q_AQ⁻_B are the thermoluminescent reactants, give a good fit to the experimental results. (3) The effect of continuous illumination at 77 K (i.e., donation from cytochrome b-559 to Q_A and thence to Q_B or Q⁻_B) results in predictable changes in the extent of flash-induced thermoluminescence.     

Light-induced quenching of chlorophyll fluorescence at 77 K in leaves,chloroplasts and Photosystem II particles

The light-induced chlorophyll (Chl) fluorescence decline at 77 K was investigated in segments of leaves, isolated thylakoids or Photosystem (PS) II particles. The intensity of chlorophyll fluorescence declines by about 40% upon 16 min of irradiation with 1000 μmol m⁻² s⁻¹ of white light. The decline follows biphasic kinetics, which can be fitted by two exponentials with amplitudes of approximately 20 and 22% and decay times of 0.42 and 4.6 min, respectively. The decline is stable at 77 K, however, it is reversed by warming of samples up to 270 K. This proves that the decline is caused by quenching of fluorescence and not by pigment photodegradation. The quantum yield for the induction of the fluorescence decline is by four to five orders lower than the quantum yield of Q_A reduction. Fluorescence quenching is only slightly affected by addition of ferricyanide or dithionite which are known to prevent or stimulate the light-induced accumulation of reduced pheophytin (Pheo). The normalised spectrum of the fluorescence quenching has two maxima at 685 and 695 nm for PS II emission and a plateau for PS I emission showing that the major quenching occurs within PS II. ‘Light-minus-dark’ difference absorbance spectra in the blue spectral region show an electrochromic shift for all samples. No absorbance change indicating Chl oxidation or Pheo reduction is observed in the blue (410–600 nm) and near infrared (730–900 nm) spectral regions. Absorbance change in the red spectral region shows a broad-band decrease at approximately 680 nm for thylakoids or two narrow bands at 677 and 670–672 nm for PS II particles, likely resulting also from electrochromism. These absorbance changes follow the slow component of the fluorescence decline. No absorbance changes corresponding to the fast component are found between 410 and 900 nm. This proves that the two components of the fluorescence decline reflect the formation of two different quenchers. The slow component of the light-induced fluorescence decline at 77 K is related to charge accumulation on a non-pigment molecule of the PS II complex. This revised version was published online in June 2006 with corrections to the Cover Date.     

Evidence from thermoluminescence for bicarbonate action on the recombination reactions involving the secondary quinone electron acceptor of Photosystem II

In this paper, we present the first measurements on thermoluminescence from isolated thylakoids to probe the recombination reactions of S₂ (or possibly S₃) with Q^?_B or Q^?_A, after bicarbonate depletion and its readdition. The effects of bicarbonate depletion on the S₂Q^?_B (or S₃O^?_B) thermoluminescence band was (1) a 6–10°C shift to a higher temperature; (2) a reduction in its intensity upon prolonged depletion; and (3) elimination after the first few flashes of the characteristic period four oscillations in its intensity as a function of the flash number. On the other hand, addition of diuron (3-(3′,4′-dichlorophenyl)-1,1-dimethylurea), which blocks electron flow from Q^?_A to Q_B, produced the same thermoluminescence band, at about + 20°C, assigned to S₂Q^?_A recombination, in both depleted and reconstituted samples. These results suggest (1) the initial effect of bicarbonate depletion is to increase the activation energy for S₂(S₃)Q^?_B recombination; (2) with further depletion, the incidence of this recombination decreases and the cycling of the S₂Q^?_B and S₃Q^?_B recombination is inhibited through effects at the Q_B apoprotein; and (3) the depletion effects are fully reversible. It is suggested that a conformational change of the PS II complex in the region of the Q_B apoprotein is responsible for these effects.     

Charge accumulation and recombination in Photosystem II studied by thermoluminescence. II. Oscillation of the C band induced by flash excitation

The characteristics of the thermoluminescence band appearing at +50°C in the glow curve (C band) was investigated in maize chloroplasts. The C band, which had a half-time of 10 min, could be charged in the presence of DCMU, and its amplitude significantly increased if preilluminated chloroplasts were reexcited after DCMU addition. Inactivation of the water-splitting system by hydroxylamine- or Tris-treatment did not abolish the C band. In chloroplasts subjected to various numbers of flashes before DCMU addition, the amplitude of the C band exhibited oscillation patterns which were markedly dependent upon dark adaptation of chloroplasts. Flash excitation of chloroplasts preilluminated by continuous light for 30 s prior to 5 min dark adaptation resulted in a period-4 oscillation with maxima occurring at flash numbers 0, 4, 8, 12. After a 6-h dark-adaptation of chloroplasts the period-4 oscillation was superimposed with a period-2 oscillation. The oscillatory patterns were simulated by model calculations and the possible origin of the C band is discussed.     

Characteristics of the Photosystem II reaction centre. II. Electron donors

The properties of Photosystem II electron donation were investigated by EPR spectrometry at cryogenic temperatures. Using preparations from mutants which lacked Photosystem I, the main electron donor through the Photosystem II reaction centre to the quinone-iron acceptor was shown to be the component termed Signal II. A radical of 10 G line width observed as an electron donor at cryogenic temperatures under some conditions probably arises through modification of the normal pathway of electron donation. High-potential cytochrome b-559 was not observed on the main pathway of electron donation. Two types of PS II centres with identical EPR components but different electron-transport kinetics were identified, together with anomalies between preparations in the amount of Signal II compared to the quinone-iron acceptor. Results of experiments using cells from mutants of Scenedesmus obliquus confirm the involvement of the Signal II component, manganese and high-potential cytochrome b-559 in the physiological process leading to oxygen evolution.     

Probing the lowest energy chlorophyll a states of Photosystem II via selective spectroscopy: new insights on P680

We present the wavelength dependence of homogeneous holewidths of persistent spectral holes burnt in O₂-evolving Photosystem II core complexes isolated from spinach, in the temperature range 2.5–8 K. The data supports the assignment that those chlorophylls which undergo persistent spectral hole-burning are specific CP43 and CP47-trap states that transfer their excitation energy to the reaction center. The lifetime-limited holewidths show that when PS II is in the S₁(Q_A⁻) (closed) state, the CP43/CP47-trap states have excited-state lifetimes in the range from 70 to 270 ps. These lifetimes correspond to excitation transfer rates to the reaction center, and are far slower than required for models in which the PS II reaction center (P680) acts as a ‘shallow-trap’ for excitations. For wavelengths at which both traps absorb, the hole shape is clearly a composite of two Lorentzians, corresponding to hole-burning in both states simultaneously. The temperature dependence of the homogeneous holewidth does not follow the usual T^1.3 dependence found in many chlorophyll–protein systems. Our data indicates T ² temperature dependence, typically found in crystalline systems where the chromophore is coupled to localized phonon modes.     

Influence of temperature on Photosystem II electron transfer reactions

The influence of temperature on the rate of reduction of P-680⁺, the primary donor of Photosystem II, has been studied in the range 5–294 K, in chloroplasts and subchloroplasts particles. P-680 was oxidized by a short laser flash. Its oxidation state was followed by the absorption level at 820 nm, and its reduction attributed to two mechanisms: electron donation from electron donor D₁ and electron return from the primary plastoquinone (back-reaction).Between 294 and approx. 200 K, the rate of the back-reaction, on a logarithmic scale, is a linear function of the reciprocal of the absolute temperature, corresponding to an activation energy between 3.3 and 3.7 kcal · mol^?1, in all of the materials examined (chloroplasts treated at low pH or with Tris; particles prepared with digitonin). Between approx. 200 K and 5 K the rate of the back-reaction is temperature independent, with $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 1.6}\text{ms}$. In untreated chloroplasts we measured a $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of 1.7 ms for the back-reaction at 77 and 5 K.The rate of electron donation from the donor D₁ has been measured in darkadapted Tris-treated chloroplasts, in the range 294–260 K. This rate is strongly affected by temperature. An activation energy of 11 kcal · mol^?1 was determined for this reaction.In subchloroplast particles prepared with Triton X-100 the signals due to P-680 were contaminated by absorption changes due to the triplet state of chlorophyll a. This triplet state has been examined with pure chlorophyll a in Triton X-100. An Arrhenius plot of its rate of decay shows a temperature-dependent region (292–220 K) with an activation energy of 9 kcal · mol^?1, and a temperature-independent region (below 200 K) with $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 1.1}\text{ms}$.     

Evidence for slow turnover in a fraction of Photosystem II complexes in thylakoid membranes

We used two different techniques to measure the recovery time of Photosystem II following the transfer of a single electron from P-680 to Q_A in thylakoid membranes isolated from spinach. Electron transfer in Photosystem II reaction centers was probed first by spectroscopic measurements of the electrochromic shift at 518 nm due to charge separation within the reaction centers. Using two short actinic flashes separated by a variable time interval we determined the time required after the first flash for the electrochromic shift at 518 nm to recover to the full extent on the second flash. In the second technique the redox state of Q_A at variable times after a saturating flash was monitored by measurement of the fluorescence induction in the absence of an inhibitor and in the presence of ferricyanide. The objective was to determine the time required after the actinic flash for the fluorescence induction to recover to the value observed after a 60 s dark period. Measurements were done under conditions in which (1) the electron donor for Photosystem II was water and the acceptor was the endogenous plastoquinone pool, and (2) Q₄₀₀, the Fe²⁺ near Q_A, remained reduced and therefore was not a participant in the flash-induced electron-transfer reactions. The electrochromic shift at 518 nm and the fluorescence induction revealed a prominent biphasic recovery time for Photosystem II reaction centers. The majority of the Photosystem II reaction centers recovered in less than 50 ms. However, approx. one-third of the Photosystem II reaction centers required a half-time of 2–3 s to recover. Our interpretation of these data is that Photosystem II reaction centers consist of at least two distinct populations. One population, typically 68% of the total amount of Photosystem II as determined by the electrochromic shift, has a steady-state turnover rate for the electron-transfer reaction from water to the plastoquinone pool of approx. 250 e⁻ / s, sufficiently rapid to account for measured rates of steady-state electron transport. The other population, typically 32%, has a turnover rate of approx. 0.2 e⁻ / s. Since this turnover rate is over 1000-times slower than normally active Photosystem II complexes, we conclude that the slowly turning over Photosystem II complexes are inconsequential in contributing to energy transduction. The slowly turning over Photosystem II complexes are able to transfer an electron from P-680 to Q_A rapidly, but the reoxidation of Q⁻_A is slow (t1/2 = 2 s). The fluorescence induction measurements lead us to conclude that there is significant overlap between the slowly turning over fraction of Photosystem II complexes and PS II_β reaction centers. One corollary of this conclusion is that electron transfer from P-680 to Q_A in PS II_β reaction centers results in charge separation across the membrane and gives rise to an electrochromic shift.     

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

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

Revisiting the Supramolecular Organization of Photosystem II in Chlamydomonas reinhardtii

Photosystem II (PSII) is a multiprotein complex that splits water and initiates electron transfer in photosynthesis. The central part of PSII, the PSII core, is surrounded by light-harvesting complex II proteins (LHCIIs). In higher plants, two or three LHCII trimers are seen on each side of the PSII core whereas only one is seen in the corresponding positions in Chlamydomonas reinhardtii, probably due to the absence of CP24, a minor monomeric LHCII. Here, we re-examined the supramolecular organization of the C. reinhardtii PSII-LHCII supercomplex by determining the effect of different solubilizing detergents. When we solubilized the thylakoid membranes with n-dodecyl-β-d-maltoside (β-DM) or n-dodecyl-α-d-maltoside (α-DM) and subjected them to gel filtration, we observed a clear difference in molecular mass. The α-DM-solubilized PSII-LHCII supercomplex bound twice more LHCII than the β-DM-solubilized supercomplex and retained higher oxygen-evolving activity. Single-particle image analysis from electron micrographs of the α-DM-solubilized and negatively stained supercomplex revealed that the PSII-LHCII supercomplex had a novel supramolecular organization, with three LHCII trimers attached to each side of the core.     

Refined purification and further characterization of oxygen-evolving and Tris-treated Photosystem II particles from the thermophilic Cyanobacterium synechococcus sp.

Highly active, monomeric and dimeric Photosystem II complexes were purified from the thermophilic cyanobacterium Synechococcus sp. by two sucrose density gradients, and the size, shape and mass of these complexes have been estimated (Rögner, M., Dekker, J.P., Boekema, E.J. and Witt, H.T. (1987) FEBS Lett. 219, 207–311). (1) Further purification could be obtained by ion-exchange chromatography, by which the 300 kDa monomer could be separated into a highly active, O₂-evolving fraction, and a fraction without O₂-evolving capacity, which has lost its extrinsic 34 kDa protein. Both showed very high reaction center activities as measured by the photoreduction of the primary quinone acceptor, Q_A, at 320 nm, being up to one reaction center per 31 Chl a molecules. (2) Tris-treatment yielded homogeneous 300 kDa particles which had lost their extrinsic 34 kDa polypeptide. Electron microscopy of this complex revealed very similar dimensions compared to the oxygen-evolving 300 kDa particle, except that the smallest dimension was decreased from about 6.5 nm to about 5.8 nm. This difference is attributed to the missing extrinsic 33 kDa protein, and the smallest dimension is attributed to the distance across the membrane. (3) Experiments are presented, allowing an estimation for the contribution of detergent to the other dimensions being about 2 × 1.5 nm for dodecyl β- -maltoside. This leads to dimensions, corrected for detergent size, of 12.3 × 7.5 nm for the monomeric form of PS II and 12 × 15.5 nm for the dimeric form. (4) From some extracts a 35 kDa, chlorophyll-binding complex could be isolated which lacks the characteristic absorbance changes of Q_A and of Chl a_II (P-680) and is therefore supposed to be a light-harvesting complex of cyanobacteria. (5) A model for the in vivo organization of PS II in cyanobacteria is discussed.     

Energy transfers from Photosystem II to Photosystem I in Cryptomonas rufescens (Cryptophyceae)

In Cryptomonas rufescens (Cryptophyceae), phycoerythrin located in the thylakoid lumen is the major accessory pigment. Oxygen action spectra prove phycoerythrin to be efficient in trapping light energy.The fluorescence excitation spectra at ?196°C obtained by the method of Butler and Kitajima (Butler, W.L. and Kitajima, M. (1975) Biochim. Biophys. Acta 396, 72–85) indicate that like in Rhodophycease, chlorophyll a is the exclusive light-harvesting pigment for Photosystem I.For Photosystem II we can observe two types of antennae: (1) a light-harvesting chlorophyll complex connected to Photosystem II reaction centers, which transfers excitation energy to Photosystem I reaction centers when all the Photosystem II traps are closed. (2) A light-harvesting phycoerythrin complex, which transfers excitation energy exclusively to the Photosystem II reaction complexes responsible for fluorescence at 690 nm.We conclude that in Cryptophyceae, phycoerythrin is an efficient light-harvesting pigment, organized as an antenna connected to Photosystem II centers, antenna situated in the lumen of the thylakoid. However, we cannot afford to exclude that a few parts of phycobilin pigments could be connected to inactive chlorophylls fluorescing at 690 nm.     

Potentiometric titration of Photosystem II fluorescence decay kinetics in spinach chloroplasts

The fluorescence yield of chloroplasts reflects the redox state of the electron acceptor of the Photosystem II reaction center, with increasing yield as the acceptor is reduced. Chemical reductive titrations of fluorescence yield in chloroplasts at room temperature indicate two distinct midpoint potentials, suggesting the possibility of Photosystem II electron acceptor heterogeneity. We have carried out a potentiometric titration of the fluorescence decay kinetics in spinach chloroplasts using a continuous mode-locked dye laser with low-intensity excitation pulses and a picosecond-resolution single-photon timing system. At all potentials the fluorescence decay is best described by three exponential components. As the potential is lowered, the slow phase changes 30-fold in yield with two distinct midpoint potentials, accompanied by a modest (3-fold) increase in the lifetime. The titration curve for the slow component of the fluorescence decay of spinach chloroplasts is best characterized by two single-electron redox reactions with midpoint potentials at pH 8.0 of +119 and ?350 mV, with corresponding relative contributions to the fluorescence yield of 49 and 51%, respectively. There is little change in the fast and middle components of the fluorescence decay. We found that the oxidized form of the redox mediator 2-hydroxy-1,4-naphthoquinone preferentially quenches the fluorescence, causing an anomalous decrease in the apparent midpoint of the high-potential transition. This effect accounts for a significant difference between the midpoint potentials that we observe and some of those previously reported. The selective effect of reduction potentials on particular fluorescence decay components provides useful information about the organization and distribution of the Photosystem II electron acceptor.     

Kinetics of EPR signal IIvf in chloroplast Photosystem II

The risetime of EPR signal II_vf (S II_vf) has been measured in oxygen-evolving Photosystem II particles from spinach chloroplasts at pH 6.0. The EPR signal shows an instrument-limited rise upon induction ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{? 3 μ}\text{s}$). These data are consistent with a model where the species Z responsible for S II_vf is the immediate electron donor to P-680⁺ in spinach chloroplasts. A new, faster decay component of S II_vf has also been detected in these experiments.     

Two possible 3-(3,4-dichlorophenyl)-1,1-dimethylurea-insensitive sites in Photosystem II of spinach chloroplasts

Two possible 3-(3,4-dichlorophenyl)-1,1-dimethylurea-insensitive sites were found in PS II of spinach chloroplasts, depending on the pH of the assay medium used. The low site (pH 6) can be inhibited by certain quinolines, such as 8-hydroxyquinoline at concentrations less than 50 μM. The high pH site (pH 8) can be inhibited by disodium cyanamide, folic acid, or 5,6-benzoquinoline at concentrations from 50 μM to 5 mM. With the exception of orthophenanthroline, which stimulates the high pH site but does not show much inhibition at low pH, all other inhibitors gave opposite effects at the pH values used, i.e., they stimulated at low pH or inhibited at high pH, or vice versa. Several mechanisms for the observed effects are discussed.