Antenna function of a chlorophyll ab protein complex of Photosystem I

The functional role of a chlorophyll $\text{a}\text{b}$ complex associated with Photosystem I (PS I) has been studied. The rate constant for P-700 photooxidation, K_P-700, which under light-limiting conditions is directly proportional to the size of the functional light-harvesting antenna, has been measured in two PS I preparations, one of which contains the chlorophyll $\text{a}\text{b}$ complex and the other lacking the complex. K_P-700 for the former preparation is half of that of the preparation which has the chlorophyll $\text{a}\text{b}$ complex present. This difference reflects a decrease in the functional light-harvesting antenna in the PS I complex devoid of the chlorophyll $\text{a}\text{b}$ complex. Experiments involving reconstitution of the chlorophyll $\text{a}\text{b}$ complex with the antenna-depleted PS I preparation indicate a substantial recovery of the K_P-700 rate. These results demonstrate that the chlorophyll $\text{a}\text{b}$ complex functions as a light-harvesting antenna in PS I.     

The dependence of reaction center and antenna triplets on the redox state of Photosystem I

The formation of chlorophyll triplet states during illumination of Photosystem I reaction center samples depends upon the redox state of P-700, X and ferredoxin Centers A and B. When the reaction centers are in the states P-700⁺A₁XFd_BFd^?_A and P-700 A₁XFd^?_BFd^?_A prior to illumination, we observe electron paramagnetic resonance (EPR) spectra from a triplet species which has zero-field splitting parameters (|D| and |E|) larger than those of either the chlorophyll a or chlorophyll b monomer triplet, and a polarization which results from population of the triplet spin sublevels by an intersystem crossing mechanism. We interpret this triplet as arising from photoexcited chlorophyll antenna species associated with reaction centers in the states P-700⁺Fd^?_A and P-700⁺X^?, respectively, which undergo de-excitation via intersystem crossing. When the reaction centers are in the states P-700A₁XFd^?_BFd^?_A and P-700A₁X^?Fd^?_BFd^?_A prior to illumination, we observe a triplet EPR signal with a polarization which results from population of the triplet spin sublevels by radical pair recombination, and which has a |D| value similar to that of chlorophyll a monomer. We interpret this triplet (the radical pair-polarized triplet) as arising from ³P-700 which has been populated by the process $\text{P-700}{}^{\mathrm{+}}\text{A}{}^{\mathrm{?}}{}_1\text{→}{}^3\text{P-700}\text{A}{}_1$. We observe both the radical pair-polarized triplet and the chlorophyll antenna triplet when the reaction centers are in the state P-700 A₁XFd^?_BFd^?_A, presumably because the processes P-700⁺A^?₁X → P-700⁺A₁X^? and $\text{P-700}{}^{\mathrm{+}}\text{A}{}^{\mathrm{?}}{}_1\text{X}\text{→}{}^3\text{P-700}\text{A}{}_1\text{X}$ have similar rate constants when Centers A and B are reduced, i.e., the forward electron transfer time from A^?₁ to X is apparently much slower in the redox state P-700 A₁XFd^?_BFd^?_A than it is in state P-700 A₁XFd_BFd_A. The amplitude of the radical pair-polarized triplet EPR signal does not decrease in the presence of a 13.5-G-wide EPR signal centered at g 2.0 which was recorded in the dark prior to triplet measurements in samples previously frozen under intense illumination. This g 2.0 signal, which has been attributed to phototrapped A^?₁ (Heathcote, P., Timofeev, K.N. and Evans, M.C.W. (1979) FEBS Lett. 101, 105–109), corresponds to as many as 12 spins per P-700 and can be photogenerated during freezing without causing any apparent attenuation of the radical pair-polarized triplet amplitude. We conclude that species other than A^?₁ contribute to the g 2.0 signal.     

Excitation energy transfer in Photosystem I from oxygenic organisms

This Review discusses energy transfer pathways in Photosystem I (PS I) from oxygenic organisms. In the trimeric PS I core from cyanobacteria, the efficiency of solar energy conversion is largely determined by ultrafast excitation transfer processes in the core chlorophyll a (Chl a) antenna network and efficient photochemical trapping in the reaction center (RC). The role of clusters of Chl a in energy equilibration and photochemical trapping in the PS I core is discussed. Dimers of the longest-wavelength absorbing (red) pigments with strongest excitonic interactions localize the excitation in the PS I core antenna. Those dimers that are located closer to the RC participate in a fast energy equilibration with coupled pigments of the RC. This suggests that the function of the red pigments is to concentrate the excitation near the RC. In the PS I holocomplex from algae and higher plants, in addition to the red pigments of the core antenna, spectrally distinct red pigments are bound to the peripheral Chl a/b-binding light-harvesting antenna (LHC I), specifically to the Lhca4 subunit of the LHC I-730 complex. Intramonomeric energy equilibration between pools of Chl b and Chl a in Lhca1 and Lhca4 monomers of the LHC I-730 heterodimer are as fast as the energy equilibration processes within the PS I core. In contrast to the structural stability of the PS I core, the flexible subunit structure of the LHC I would probably determine the observed slow excitation energy equilibration processes in the range of tens of picoseconds. The red pigments in the LHC I are suggested to function largely as photoprotective excitation sinks in the peripheral antenna of PS I. This revised version was published online in August 2006 with corrections to the Cover Date.     

UV optical difference spectrum associated with the reduction of electron acceptor A1 in photosystem I of higher plants

Photosystem I particles containing I P700 per 32 chlorophyll molecules were illuminated at cryogenic temperatures in the presence of sodium dithionite. Under conditions which specifically led to reduction of acceptor a₁ (as shown by its characteristic EPR spectrum) optical absorbance changes were detected between 240 and 325 nm. The appearance of these changes correlated closely with the increase in amplitude of the ai EPR signal. The possibility that a quinone-like species is associated with, or directly involved in intermediary PS I electron flow is discussed.     

On the mutual orientation of pigments in Photosystem I particles from green plants

The mutual orientation of pigments in Photosystem I reaction centers from spinach is evaluated by polarized photochemistry. The photoinduced linear dichroism of the absorption changes of chlorophyll a₁ at 701 nm is studied as function of the excitation wavelength. The Photosystem I reaction center particles contain about 100 and if depleted about 40 chlorophylls, respectively. To prevent their rapid Brownian rotation they were immobilized on DEAE-Sephadex.The excitation spectrum of the linear dichroism reveals a high degree of order between the long axis of β-carotene and the Q_y transition moments of those chlorophyll a molecules absorbing at the red end of the spectrum. The latter are the most endangered ones for destructive oxidation via their triplet state. Hence, the location of β-carotene in close proximity to and in parallel with these chlorophylls seems to be most favourable for the protective role of β-carotene within the antennae system I. It is observed that the dichroic ratio of the absorption changes of chlorophyll a₁ does not exceed a figure of $\text{4}\text{3}$, which characterizes a circularly degenerate system, even at far red excitation (724 nm). This will hit selectively those few chlorophyll a molecules with their peak absorption at about 700 nm (including the photooxidizable dimer). We conclude, if the dimer is the only species peaking at 700 nm then the two chlorophyll a within the dimer have their y-axes oriented perpendicular to each other. If there are some antennae in addition to the dimer, the y-axes of all chlorophyll-a peaking at 700 nm form a star which accounts for the circular degeneracy of absorption.     

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.     

The detection,isolation and characterization of a light-harvesting complex which is specifically associated with Photosystem I

10% of the chlorophyll associated with a ‘native’ Photosystem (PS) I complex (110 chlorophylls/P-700) is chlorophyll (Chl) b. The Chl b is associated with a specific PS I antenna complex which we designate as LHC-I (i.e., a light-harvesting complex serving PS I). When the native PS I complex is degraded to the core complex by LHC-I extraction, there is a parallel loss of Chl b, fluorescence at 735 nm, together with 647 and 686 nm circular dichroism spectral properties, as well as a group of polypeptides of 24-19 kDa. In this paper we present a method by which the LHC-I complex can be dissociated from the native PS I. The isolated LHC-I contains significant amounts of Chl b (Chl $\text{a}\text{b ? 3.7}$). The long-wavelength fluorescence at 730 nm and circular dichroism signal at 686 nm observed in native PS I are maintained in this isolated complex. This isolated fraction also contains the low molecular weight polypeptides lost in the preparation of PS I core complex. We conclude that we have isolated the PS I antenna in an intact state and discuss its in vivo function.     

Evidence for a new early acceptor in Photosystem I of plants. An ESR investigation of reaction center triplet yield and of the reduced intermediary acceptors

The yield of the triplet state of the primary electron donor of Photosystem I of photosynthesis (P^T-700) and the characteristic parameters (g value, line shape, saturation behavior) of the ESR signal of the photoaccumulated intermediary acceptor A have been measured for two types of Photosystem I subchloroplast particles: Triton particles (TSF 1, about 100 chlorophyll molecules per P-700) that contain the iron-sulfur acceptors F_X, F_B and F_A, and lithium dodecyl sulfate (LDS) particles (about 40 chlorophyll molecules per P-700) that lack these iron-sulfur acceptors. The results are: (i) In Triton particles the yield of P^T-700 upon illumination is independent of the redox state of A and of F_X,B,A and is maximally about 5% of the active reaction centers at 5 K. The molecular sublevel decay rates are k_x = 1100 s^?1 ± 10%, k_y = 1300 s^?1 ± 10% and k_z = 83 s^?1 ± 20%. In LDS particles the triplet yield decreases linearly with concentration of reduced intermediary acceptors, the maximal yield being about 4% at 5 K assuming full P-700 activity. (ii) In Triton particles the acceptor complex A consists of two acceptors A₀ and A₁, with A₀ preceding A₁. In LDS particles at temperatures below ?30°C only A₀ is photoactive. (iii) The spin-polarized ESR signal found in the time-resolved ESR experiments with Triton particles is attributed to a polarized P-700-A^?₁ spectrum. The decay kinetics are complex and are influenced by transient nutation effects, even at low microwave power. It is concluded that the lifetime at 5 K of P-700A₀A^?₁ must exceed 5 ms. We conclude that P^T-700 originates from charge recombination of P-700A^?₀, and that in Triton particles A₀ and A₁ are both photoaccumulated upon cooling at low redox potential in the light. Since the state P-700AF^?_X does not give rise to triplet formation the 5% triplet yield in Triton particles is probably due to centers with damaged electron transport.     

Delayed fluorescence observed in the nanosecond time region at 77 K originates directly from the photosystem II reaction center

The excited-state dynamics of delayed fluorescence in photosystem (PS) II at 77 K were studied by time-resolved fluorescence spectroscopy and decay analysis on three samples with different antenna sizes: PS II particles and the PS II reaction center from spinach, and the PS II core complexes from Synechocystis sp. PCC 6803. Delayed fluorescence in the nanosecond time region originated from the 683-nm component in all three samples, even though a slight variation in lifetimes was detected from 15 to 25 ns. The relative amplitude of the delayed fluorescence was higher when the antenna size was smaller. Energy transfer from the 683-nm pigment responsible for delayed fluorescence to antenna pigment(s) at a lower energy level was not observed in any of the samples examined. This indicated that the excited state generated by charge recombination was not shared with antenna pigments under the low-temperature condition, and that delayed fluorescence originates directly from the PS II reaction center, either from chlorophyll a_D1 or P680. Supplemental data on delayed fluorescence from spinach PS I complexes are included.     

Purification of His6-tagged Photosystem I from Chlamydomonas reinhardtii

We have developed a rapid method for isolation of the Photosystem I (PS1) complex from Chlamydomonas reinhardtii using epitope tagging. Six histidine residues were genetically added to the N-terminus of the PsaA core subunit of PS1. The His₆-tagged PS1 could be purified with a yield of 80–90% from detergent-solubilized thylakoid membranes within 3 h in a single step using a Ni-nitrilotriacetic acid (Ni-NTA) column. Immunoblots and low-temperature fluorescence analysis indicated that the His₆-tagged PS1 preparation was highly pure and extremely low in uncoupled pigments. Moreover, the introduced tag appeared to have no adverse effect upon PS1 structure/function, as judged by photochemical assays and EPR spectroscopy of isolated particles, as well as photosynthetic growth tests of the tagged strain. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Photosystem II reaction center with altered pigment-composition: reconstitution of a complex containing five chlorophyll a per two pheophytin a with modified chlorophylls

Pigment-depleted Photosystem II reaction centers (PS II-RCs) from a higher plant (pea) containing five chlorophyll a (Chl) per two pheophytin a (Phe), were treated with Chl and several derivatives under exchange conditions [FEBS Lett. 434 (1998) 88]. The resulting reconstituted complexes were compared to those obtained by pigment exchange of “conventional” PS II-RCs containing six Chl per two Phe. (1) The extraction of one Chl is fully reversible. (2) The site of extraction is the same as the one into which previously extraneous pigments have been exchanged, most likely the peripheral D1-H118. (3) Introducing an efficient quencher (Ni-Chl) into this site results in only 25% reduction of fluorescence, indicating incomplete energy equilibration among the “core” and peripheral chlorophylls.     

Orientation of pigments in the thylakoid membrane and in the isolated chlorophyll-protein complexes of higher plants. II. Linear dichroism spectra of isolated pigment-protein complexes oriented in polyacrylamide gels at 300 and 100 K

The absorption and linear dichroism (LD) spectra (380–780 nm) of isolated light-harvesting complex (LHC), Photosystem I (PS I), Photosystem II (PS II), as well as intact thylakoids have been determined at 300 and 100 K. The samples were oriented in squeezed polyacrylamide gel. The low-temperature spectra of LHC and PS I present LD signals which are characteristic enough to be recognized in the LD spectrum of thylakoids. Tentative assignments of the various features of the LD spectra to the major photosynthetic pigments are discussed. A shoulder in the low-temperature absorption spectra is observed at about 673 nm in all the systems under investigation. The absence of an associated LD signal suggests that this ubiquitous chlorophyll (Chl) a form is non-dichroic. Furthermore, in the three isolated chlorophyll-protein complexes described in this study the sign of the LD signal indicates that both the Q_y transition of the Chl a and the carotenoid molecules are preferentially oriented parallel to the largest dimension(s) of the particles.     

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.     

Photoinhibition of Photosystem I electron transfer activity in isolated Photosystem I preparations with different chlorophyll contents

Photoinhibition of the light-induced Photosystem I (PS I) electron transfer activity from the reduced dichlorophenol indophenol to methyl viologen was studied. PS I preparations with Chl/P700 ratios of about 180 (PS I-180), 100 (PS I-100) and 40 (PS I(HA)-40) were isolated from spinach thylakoid membranes by the treatments with Triton X-100, followed by sucrose density gradient centrifugation and hydroxylapatite column chromatography. White light irradiation (1.1 × 10⁴E m^–2 s^–1) of PS I-180 for 2 hours bleached 50% of the chlorophyll and caused a 58% decrease in the electron transfer activity with virtually no loss of the primary donor, P700. The flash-induced absorbance change showed the decay phase with a half time of about 10 s that was attributed to the P700 triplet, suggesting that the photoinhibitory light treatment caused the destruction of the PS I acceptor(s), F_x and possibly A₁. PS I-100 was similarly photobleached by the irradiation and the electron transfer activity decreased. There was, however, no apparent photoinhibition of the electron transport activity in PS I(HA)-40. Photoinhibition similar to that seen in PS I-180 also occurred in membrane fragments that were isolated without any detergent from a PS II-deficient mutant strain of the cyanobacterium Synechocystis sp. PCC 6803. PS I-180 was not photoinhibited under anaerobic conditions. The production of superoxide and fatty acid hydroperoxide during white light irradiation was significantly greater in PS I-180 than in PS I(HA)-40. The mechanism of photoinhibition in PS I preparations is discussed in relation to the formation of toxic oxygen molecules.Abbreviations A₀,A₁ primary and secondary electron acceptors of PS I - CD circular dichroism - DCPIP 2,6-dichlorophenol indophenol - F_A, F_B, F_X iron-sulfur centers A, B, X - HA hydroxylapatite - LHCI lightharvesting complex of PS I - MDA malondialdehyde - MV methyl viologen - Na-Asc sodium L-ascorbate - P700 primary electron donor of PS I - PFD photon flux density - PS I-A and PS I-B psaA and psaB gene products - TBA thiobarbituric acid     

Photosystem I reaction center from Mastigocladus laminosus. Correlation between reduction state of the iron-sulfur centers and the triplet formation mechanisms

EPR study of reduced ground and photoexcited triplet state of Photosystem I reaction center in the thermophylic cyanobacterium Mastigocladus laminosus at 8 K is reported. In the reduced ground state preparation, the iron-sulfur EPR spectra are found to be similar to that of Photosystem I reaction center of higher plants. Two types of transient photoexcited triplets are observed and are correlated to the reduction state of the iron-sulfur centers. When electrons can be transferred freely through the acceptors chain, a polarized triplet spectrum is observed, typical of spin-orbit intersystem crossing mechanism with lifetime of approx. 2 ms and is attributed to chlorophyll a, either at the antenna or at A₁ in the electron-transport chain. When the iron-sulfur centers are reduced the triplet spectrum is typical of a radical-pair intersystem crossing mechanism with triplet lifetime shorter than 1 ms, and is attributed to P-700. Both species have similar spectroscopic zero field splitting parameters identifying both as chlorophyll a.     

Function and organization of Photosystem I polypeptides

Photosystem I functions as a plastocyanin:ferredoxin oxidoreductase in the thylakoid membranes of chloroplasts and cyanobacteria. The PS I complex contains the photosynthetic pigments, the reaction center P700, and five electron transfer centers (A₀, A₁, F_X, F_A, and F_B) that are bound to the PsaA, PsaB, and PsaC proteins. In addition, PS I complex contains at least eight other polypeptides that are accessory in their functions. Recent use of cyanobacterial molecular genetics has revealed functions of the accessory subunits of PS I. Site-directed mutagenesis is now being used to explore structure-function relations in PS I. The overall architecture of PSI complex has been revealed by X-ray crystallography, electron microscopy, and biochemical methods. The information obtained by different techniques can be used to propose a model for the organization of PS I. Spectroscopic and molecular genetic techniques have deciphered interaction of PS I proteins with the soluble electron transfer partners. This review focuses on the recent structural, biochemical and molecular genetic studies that decipher topology and functions of PS I proteins, and their interactions with soluble electron carriers.Abbreviation NHS N-hydroxysuccinamide This review is dedicated to Prof. J. Philip Thornber, in whose laboratory PRC was introduced to the green world of chlorophyllproteins.     

Cobalt ions inhibit electron-transport activity of Photosystem II without affecting Photosystem I

Investigations on photosynthesis have greatly benefited by the use of specific inhibitors that affect a specific site of inhibition on the electron-transport chain. We show here for the first time that cobalt (Co²⁺) ions can be used specifically to inactivate electron donation to the reaction centre of Photosystem (PS) II without affecting PS I reactions. This conclusion is based on the following observations: (1) addition of exogenous electron donors such as NH₂OH does not relieve Co²⁺-induced inactivation of photoelectron transport or the lowering of steady-state chlorophyll a fluorescence yield; this suggests that the inhibition is beyond the NH₂OH donation site and before the fluorescence quencher Q, i.e., on the reaction centre complex itself. (2) Washing of Co²⁺-pretreated chloroplasts with isolation buffer to remove Co²⁺ does not relieve Co²⁺-induced inhibition of Hill activity, suggesting that the Co²⁺ effect is irreversible. (3) Co²⁺ did not alter the PS I reactions. Thus, Co²⁺-treated chloroplasts can be used to study PS I functions free from PS II reactions in isolated chloroplasts.     

The origin of split EPR signals in the Ca2+-depleted Photosystem II

A light-driven reaction model for the Ca²⁺-depleted Photosystem (PS) II is proposed to explain the split signal observed in electron paramagnetic resonance (EPR) spectra based on a comparison of EPR assignments with recent x-ray structural data. The split signal has a splitting linewidth of 160 G at around g = 2 and is seen upon illumination of the Ca²⁺-depleted PS II in the S₂ state associated with complete or partial disappearance of the S₂ state multiline signal. Another g=2 broad ESR signal with a 110 G linewidth was produced by 245 K illumination for a short period in the Ca²⁺-depleted PS II in S₁ state. At the same time a normal Y_Z^· radical signal was also efficiently trapped. The g=2 broad signal is attributed to an intermediate S₁X^· state in equilibrium with the trapped Y_Z^· radical. Comparison with x-ray structural data suggests that one of the split signals (doublet signal) is attributable to interaction between His 190 and the Y_Z^· radical, and other signals is attributable to interaction between His 337 and the manganese cluster, providing further clues as to the mechanism of water oxidation in photosynthetic oxygen evolution.     

EPR spectra of Photosystem I and other iron protein components in intact cells of cyanobacteria

Electron paramagnetic resonance (EPR) spectra were recorded of whole filaments of the cyanobacteria Nostoc muscorum and Anabaena cylindrica. Signals due to manganese were removed by freezing and thawing the cells in EDTA. EPR spectra were assigned on the basis of their g values, linewidths, temperature dependence and response to dithionite and light treatments. The principal components identified were: (i) rhombic Fe³⁺ (signal at g = 4.3), probably a soluble storage form of iron; (ii) iron-sulfur centers A and B of Photosystem I; (iii) the photochemical electron acceptor ‘X’ of Photosystem I; this component was also observed for the first time in isolated heterocysts; (iv) soluble ferredoxin which was present at a concentration of 1 molecule per 140 ± 20 chlorophyll molecules; (v) a membrane-bound iron-sulfur protein (g = 1.92). A signal g = 6 in the oxidized state was probably due to an unidentified heme compound. During deprivation of iron the rhombic Fe³⁺, centers A, B and X of Photosystem I, and soluble ferredoxin were all observed to decrease.     

EPR evidence for a modified S-state transition in chloride-depleted Photosystem II

The role of chloride on the S-state transition in spinach Photosystem II (PS II) particles was investigated by EPR spectroscopy at low temperature and the following results were obtained. (1) After excitation by continuous light at 200 K, chloride-depleted particles did not show the EPR multiline signal associated with the S₂ state, but only showed the broad signal at g = 4.1. The S₂ multiline signal was completely restored upon chloride repletion. (2) In the absence of chloride the S₂ multiline signal was not induced by a single flash excitation at 0°C. However, upon addition of chloride after the flash the signal was developed in darkness. (3) The amplitude of the multiline S₂ signal thus developed upon chloride addition after flash illumination did not show oscillations dependent upon flash number. These results indicate that the O₂-evolving complex in chloride-depleted PS II membranes is able to store at least one oxidizing equivalent, a modified S₂ state, which does not give rise to the multiline signal. Addition of chloride converts this oxidizing equivalent to the normal S₂ state which gives rise to the multiline signal. The modified S₂ state is more stable than the normal S₂ state, showing decay kinetics about 20-times slower than those of the normal S₂ state, and the formation of higher S states is blocked.