Excitation energy transfer and charge separation in photosystem II membranes revisited

We have performed time-resolved fluorescence measurements on photosystem II (PSII) containing membranes (BBY particles) from spinach with open reaction centers. The decay kinetics can be fitted with two main decay components with an average decay time of 150 ps. Comparison with recent kinetic exciton annihilation data on the major light-harvesting complex of PSII (LHCII) suggests that excitation diffusion within the antenna contributes significantly to the overall charge separation time in PSII, which disagrees with previously proposed trap-limited models. To establish to which extent excitation diffusion contributes to the overall charge separation time, we propose a simple coarse-grained method, based on the supramolecular organization of PSII and LHCII in grana membranes, to model the energy migration and charge separation processes in PSII simultaneously in a transparent way. All simulations have in common that the charge separation is fast and nearly irreversible, corresponding to a significant drop in free energy upon primary charge separation, and that in PSII membranes energy migration imposes a larger kinetic barrier for the overall process than primary charge separation.     

Structure-based kinetic modeling of excited-state transfer and trapping in histidine-tagged photosystem II core complexes from synechocystis

Chlorophyll fluorescence decay kinetics in photosynthesis are dependent on processes of excitation energy transfer, charge separation, and electron transfer in photosystem II (PSII). The interpretation of fluorescence decay kinetics and their accurate simulation by an appropriate kinetic model is highly dependent upon assumptions made concerning the homogeneity and activity of PSII preparations. While relatively simple kinetic models assuming sample heterogeneity have been used to model fluorescence decay in oxygen-evolving PSII core complexes, more complex models have been applied to the electron transport impaired but more highly purified D1-D2-cyt b(559) preparations. To gain more insight into the excited-state dynamics of PSII and to characterize the origins of multicomponent fluorescence decay, we modeled the emission kinetics of purified highly active His-tagged PSII core complexes with structure-based kinetic models. The fluorescence decay kinetics of PSII complexes contained a minimum of three exponential decay components at F(0) and four components at F(m). These kinetics were not described well with the single radical pair energy level model, and the introduction of either static disorder or a dynamic relaxation of the radical pair energy level was required to simulate the fluorescence decay adequately. An unreasonably low yield of charge stabilization and wide distribution of energy levels was required for the static disorder model, and we found the assumption of dynamic relaxation of the primary radical pair to be more suitable. Comparison modeling of the fluorescence decay kinetics from PSII core complexes and D1-D2-cyt b(559) reaction centers indicated that the rates of charge separation and relaxation of the radical pair are likely altered in isolated reaction centers.     

Singlet oxygen production in photosystem II and related protection mechanism

High-light illumination of photosynthetic organisms stimulates the production of singlet oxygen by photosystem II (PSII) and causes photo-oxidative stress. In the PSII reaction centre, singlet oxygen is generated by the interaction of molecular oxygen with the excited triplet state of chlorophyll (Chl). The triplet Chl is formed via charge recombination of the light-induced charge pair. Changes in the midpoint potential of the primary electron donor P(680) of the primary acceptor pheophytin or of the quinone acceptor Q(A), modulate the pathway of charge recombination in PSII and influence the yield of singlet oxygen formation. The involvement of singlet oxygen in the process of photoinhibition is discussed. Singlet oxygen is efficiently quenched by beta-carotene, tocopherol or plastoquinone. If not quenched, it can trigger the up-regulation of genes, which are involved in the molecular defence response of photosynthetic organisms against photo-oxidative stress.     

Spectroscopic characterization of triplet forming states in photosystem II.

Fluorescence and electron paramagnetic resonance (EPR) measurements have been applied to characterize chlorophyll triplet formation in the reaction center of photosystem II (PSII). A highly triplet forming state was generated in PSII membranes by chemical double reduction of the primary electron acceptor QA. In triplet forming PSII centers, the steady-state yield of chlorophyll fluorescence decreased to about 70% of the maximal fluorescence yield observed in closed PSII centers in which QA is singly reduced. The results are well interpreted in the framework of a model where the charge state of QA electrostatically controls the yield of primary charge separation [Schatz, G. H., Brock, H., & Holzwarth, A. R. (1988) Biophys. J. 54, 397-405]. Thus, high triplet yield and decreased, although still quite high, fluorescence indicate a charge-neutralized state of PSII in which QA is singly or doubly reduced and protonated or absent. The EPR signal of the triplet primary chlorophyll donor, 3P680, is suppressed by illumination at 77 K concomitant with the formation of a cationic radical (g = 2.0025-2.0027, and 0.92 mT wide) that is stable in the dark. This is attributed to the oxidation of an accessory chlorophyll (Chl) in the vicinity of P680. Electrostatic repulsion between Chl+ and P680+ is likely to prevent primary charge separation, and in turn triplet formation, providing a further example of electrostatic control of primary charge separation. The triplet P680 EPR signal is also suppressed in the presence of oxygen. This effect, which is almost completely reversible by removing the oxygen, is attributed to the interaction of triplet P680 with triplet O2.     

Magnetic field protects plants against high light by slowing down production of singlet oxygen

Recombination of the primary radical pair of photosystem II (PSII) of photosynthesis may produce the triplet state of the primary donor of PSII. Triplet formation is potentially harmful because chlorophyll triplets can react with molecular oxygen to produce the reactive singlet oxygen (1O?). The yield of 1O? is expected to be directly proportional to the triplet yield and the triplet yield of charge recombination can be lowered with a magnetic field of 100-300 mT. In this study, we illuminated intact pumpkin leaves with strong light in the presence and absence of a magnetic field and found that the magnetic field protects against photoinhibition of PSII. The result suggests that radical pair recombination is responsible for significant part of 1O? production in the chloroplast. The magnetic field effect vanished if leaves were illuminated in the presence of lincomycin, an inhibitor of chloroplast protein synthesis, or if isolated thylakoid membranes were exposed to light. These data, in turn, indicate that 1O? produced by the recombination of the primary charge pair is not directly involved in photoinactivation of PSII but instead damages PSII by inhibiting the repair of photoinhibited PSII. We also found that an Arabidopsis thaliana mutant lacking α-tocopherol, a scavenger of 1O?, is more sensitive to photoinhibition than the wild-type in the absence but not in the presence of lincomycin, confirming that the target of 1O? is the repair mechanism.     

Deregulation of electron flow within photosystem II in the absence of the PsbJ protein

The photosystem II (PSII) complex of photosynthetic oxygen evolving membranes comprises a number of small proteins whose functions remain unknown. Here we report that the low molecular weight protein encoded by the psbJ gene is an intrinsic component of the PSII complex. Fluorescence kinetics, oxygen flash yield, and thermoluminescence measurements indicate that inactivation of the psbJ gene in Synechocystis 6803 cells and tobacco chloroplasts lowers PSII-mediated oxygen evolution activity and increases the lifetime of the reduced primary acceptor Q(A)(-) (more than a 100-fold in the tobacco DeltapsbJ mutant). The decay of the oxidized S(2,3) states of the oxygen-evolving complex is considerably accelerated, and the oscillations of the Q(B)(-)/S(2,3) recombination with the number of exciting flashes are damped. Thus, PSII can be assembled in the absence of PsbJ. However, the forward electron flow from Q(A)(-) to plastoquinone and back electron flow to the oxidized Mn cluster of the donor side are deregulated in the absence of PsbJ, thereby affecting the efficiency of PSII electron flow following the charge separation process.     

Desiccation tolerant lichens facilitate in vivo H/D isotope effect measurements in oxygenic photosynthesis

We have used the desiccation-tolerant lichen Flavoparmelia caperata, containing the green algal photobiont Trebouxia gelatinosa, to examine H/D isotope effects in Photosystem II in vivo. Artifact-free H/D isotope effects on both PSII primary charge separation and water oxidation yields were determined as a function of flash rate from chlorophyll-a variable fluorescence yields. Intact lichens could be reversibly dehydrated/re-hydrated with H₂O/D₂O repeatedly without loss of O₂ evolution, unlike all isolated PSII preparations. Above a threshold flash rate, PSII charge separation decreases sharply in both D₂O and H₂O, reflecting loss of excitation migration and capture by PSII. Changes in H/D coordinates further slow charge separation in D₂O (?23% at 120?Hz), attributed to reoxidation of the primary acceptor Q_A^?. At intermediate flash rates (5–50?Hz) D₂O decreases water oxidation efficiency (O₂ evolution) by ?2–5%. No significant isotopic difference is observed at slow flash rates (<5?Hz) where charge recombination dominates. Slower D₂O diffusion, changes in hydrogen bonding networks, and shifts in the pK_a's of ionizable residues may all contribute to these systematic variations of H/D isotope effects. Lichens' reversible desiccation tolerance allows highly reproducible H/D exchange kinetics in PSII reactions to be studied in vivo for the first time.     

CHROMATIC REGULATION OF QUANTUM YIELDS FOR PHOTOSYSTEM II CHARGE SEPARATION,OXYGEN EVOLUTION,AND CARBON FIXATION IN HETEROCAPSA PYGMAEA (PYRROPHYTA)1

Operational and maximum quantum yields for system (PSII) charge separation, oxygen evolution, and carbon fixation were quantified and compared for Heterocapsa pygmaea Loeblich, Schmidt et Sherley populations chromatically adapted to white, green, blue, and red light. Significant variability in quantum yields was induced by chromatic adaptation alone or when chromatically adapted cells were suddenly exposed to biased light fields (i.e. white light). Results indicated a close coupling between the variability in quantum yields for PSII charge separation and oxygen evolution, but not between quantum yields for oxygen evolution and carbon fixation. But not between quantum yields for oxygen evolution and carbon fixation. The ability to regrlate and optimize light energy distribution between PSII and photosystem I (PSI) appears to be the mechanism underlying chromatic adaptation for PSII charge separation and oxygen evolution. Conceptually, the resulting impacts on PSI cyclic electron transport rates could account for observed variability in quantum yields for oxygen evolution and some variability in quantum yields for carbon fixation. Similarly, enzymatic processes associated with organic carbon synthesis appeared to be variably dependent on spectral growth irradiances and contributing to the observed variability in quantum yields for carbon fixation. The relevance of these findings to the in situ primary production is discussed.     

Primary light-energy conversion in tetrameric chlorophyll structure of photosystem II and bacterial reaction centers: II. Femto- and picosecond charge separation in PSII D1/D2/Cyt b559 complex

In Part I of the article, a review of recent data on electron-transfer reactions in photosystem II (PSII) and bacterial reaction center (RC) has been presented. In Part II, transient absorption difference spectroscopy with 20-fs resolution was applied to study the primary charge separation in PSII RC (DI/DII/Cyt b 559 complex) excited at 700 nm at 278 K. It was shown that the initial electron-transfer reaction occurs within 0.9 ps with the formation of the charge-separated state P680(+)Chl(D1)(-), which relaxed within 14 ps as indicated by reversible bleaching of 670-nm band that was tentatively assigned to the Chl(D1) absorption. The subsequent electron transfer from Chl(D1)(-) within 14 ps was accompanied by a development of the radical anion band of Pheo(D1) at 445 nm, attributable to the formation of the secondary radical pair P680(+)Pheo(D1)(-). The key point of this model is that the most blue Q(y) transition of Chl(D1) in RC is allowing an effective stabilization of separated charges. Although an alternative mechanism of charge separation with Chl(D1)* as a primary electron donor and Pheo(D1) as a primary acceptor can not be ruled out, it is less consistent with the kinetics and spectra of absorbance changes induced in the PSII RC preparation by femtosecond excitation at 700 nm.     

Modulating the redox potential of the stable electron acceptor, Q(B), in mutagenized photosystem II reaction centers

One of the unique features of electron transfer processes in photosystem II (PSII) reaction centers (RC) is the exclusive transfer of electrons down only one of the two parallel cofactor branches. In contrast to the RC core polypeptides (psaA and psaB) of photosystem I (PSI), where electron transfer occurs down both parallel redox-active cofactor branches, there is greater protein-cofactor asymmetry between the PSII RC core polypeptides (D1 and D2). We have focused on the identification of protein-cofactor relationships that determine the branch along which primary charge separation occurs (P(680)(+)/pheophytin(-)(Pheo)). We have previously shown that mutagenesis of the strong hydrogen-bonding residue, D1-E130, to less polar residues (D1-E130Q,H,L) shifted the midpoint potential of the Pheo(D1)/Pheo(D1)(-) couple to more negative values, reducing the quantum yield of primary charge separation. We did not observe, however, electron transfer down the inactive branch in D1-E130 mutants. The protein residue corresponding to D1-E130 on the inactive branch is D2-Q129 which presumably has a reduced hydrogen-bonding interaction with Pheo(D2) relative to the D1-E130 residue with Pheo(D1). Analysis of the recent 2.9 ? cyanobacterial PSII crystal structure indicated, however, that the D2-Q129 residue was too distant from the Pheo(D2) headgroup to serve as a possible hydrogen bond donor and directly impact its midpoint potential as well as potentially determine the directionality of electron transfer. Our objective was to characterize the function of this highly conserved inactive branch residue by replacing it with a nonconservative leucine or a conservative histidine residue. Measurements of Chl fluorescence decay kinetics and thermoluminescence studies indicate that the mutagenesis of D2-Q129 decreases the redox gap between Q(A) and Q(B) due to a lowering of the redox potential of Q(B). The resulting increased yield of S(2)Q(B)(-) charge recombination in the D2-Q129 mutants leads to an increased susceptibility to photoinhibitory light presumably due to (3)P(680)-mediated oxidative damage. The results indicate that the D2-Q129 residue plays a critical role in stabilizing the charge-separated state in PSII and further documents the structural and functional asymmetry between the two cofactor branches in PSII.     

Deletion mutagenesis in Synechocystis sp. PCC6803 indicates that the Mn-stabilizing protein of photosystem II is not essential for O2 evolution

The photosystem II (PSII) reaction center complex coordinates a cluster of Mn atoms that are involved in the accumulation of oxidizing equivalents generated by light-induced charge separations within the intrinsic portion of the PSII complex. A 33-kDa extrinsic protein, termed the Mn-stabilizing protein (MSP), has been implicated in the stabilization of two of the four Mn atoms of the cluster, yet the precise role of this protein in O2 evolution remains to be elucidated. Here we describe the construction of a mutant of the cyanobacterium Synechocystis sp. PCC6803 in which the entire gene encoding MSP has been deleted. Northern and immunoblot analyses indicate that other PSII proteins are expressed and accumulated, despite the absence of MSP. Fluorescence emission spectra at 77 K indicate PSII assembles in the mutant, but that the binding of MSP is required for the normal fluorescence characteristics of the PSII complex, and suggest a specific interaction between MSP and CP47. Fluorescence induction measurements indicate a reduced rate of forward electron transport to the primary electron donor, P680, in the mutant. It is concluded that in contrast to previous reports, MSP is not required for the assembly of active PSII complexes nor is it essential for H2O-splitting activity in vivo.     

Spectroscopic studies of photosystem II in chlorophyll d-containing Acaryochloris marina

Photosystem II (PSII) electron transfer (ET) in the chlorophyll d-containing cyanobacterium Acaryochloris marina (A. marina) was studied by time-resolved electron paramagnetic resonance (EPR) spectroscopy at room temperature, chlorophyll fluorescence, and low-temperature optical spectroscopy. To maximize the ability to measure PSII ET in the intact cells of this organism, growth conditions were optimized to provide the highest specific O(2) activity and the instrumental parameters for the EPR measurements of tyrosine Z (Y(Z)) reduction were adjusted to give the best signal-to-noise over spectral resolution. Analysis of the Y(Z)(*) reduction kinetics revealed that ET to the oxygen-evolving complex on the donor side of PSII in A. marina is indistinguishable from that in higher plants and other cyanobacteria. Likewise, the charge recombination kinetics between the first plastoquinone acceptor Q(A) and the donor side of PSII monitored by the chlorophyll fluorescence decay on the seconds time scale are not significantly different between A. marina and non-chlorophyll d organisms, while low-temperature optical absorption spectroscopy identified the primary electron acceptor in A. marina as pheophytin a. The results indicate that, if the PSII primary electron donor in A. marina is made up of chlorophyll d instead of chlorophyll a, then there must be very different interactions with the protein environment to account for the ET properties, which are similar to higher plants and other cyanobacteria. Nevertheless, the water oxidation mechanism in A. marina is kinetically unaltered.     

Fluorescence Induction Kinetics in Membrane Preparations of Photosystem II with Heterogeneous Metal Clusters (Mn/Fe) in the Oxygen-Evolving Complex

Transport of electrons in spinach photosystem II (PSII) whose oxygen-evolving complex (OEC) contains heterogeneous metal clusters 2Mn2Fe and 3Mn1Fe was studied by measuring the fluorescence induction kinetics (FIK). PSII(2Mn,2Fe) and PSII(3Mn,1Fe) preparations were produced using Cadepleted PSII membranes (PSII(–Ca)). It was found that FIK in PSII(2Mn,2Fe) membranes is similar in form to FIK in PSII(–Ca) samples, but the fluorescence yield is lower in PSII(2Mn,2Fe). The results demonstrate that, just as in PSII(–Ca) preparations, there is electron transfer from the metal cluster in the OEC to the primary plastoquinone electron acceptor Q_A. They also show that partial substitution of Mn cations with Fe has no effect on the electron transport on the acceptor side of PSII. Thus, these data demonstrate the possibility of water oxidation either by the heterogeneous metal cluster or just by the manganese dimer. We established that FIK in PSII(3Mn,1Fe) preparations are similar in form to FIK in PSII(2Mn,2Fe) membranes but PSII(3Mn,1Fe) is characterized by a slightly higher maximal fluorescence yield, F_max. The electron transfer rate in PSII(3Mn,1Fe) preparations significantly (by a factor of two) increases in the presence of Ca²⁺, whereas Ca²⁺ has hardly any effect on the electron transport in PSII(2Mn,2Fe) membranes. In Mndepleted PSII membranes, FIK reaches its maximum (the so-called peak K), after which the fluorescence yield starts to decrease as the result of two factors: the oxidation of reduced primary plastoquinone Q _A ^? and the absence of electron influx from the donor side of PSII. The replacement of Mn cations by Fe in PSII(?Mn) preparations leads to fluorescence saturation and disappearance of the K peak. This is possibly due to the deceleration of the charge recombination process that takes place between reduced primary electron acceptor Q _A ^? and oxidized tyrosine Y _Z ^+. which is an electron carrier between the OEC and the primary electron donor P680.     

Heterogeneity of photosystem II reaction centers as influenced by heat treatment of barley leaves

Three functionally distinct populations of PSII reaction centers differing in the ability to keep the primary acceptors in a reduced state and to transfer electrons to PSI were estimated using chlorophyll fluorescence measurements in primary barley leaves exposed to elevated temperatures in the range of 37–51°C. The capacity of the PSII reaction centers to perform at least one light-induced charge separation was not affected by a 5-min heat treatment at temperatures up to 51°C. The first population containing Q_B-non-reducing centers corresponded to 15–20% of the total PSII activity up to 45°C. In a second population, PSII reaction centers maintained Q_A reduction under light in the presence of oxygen, but not in the presence of a strong artificial PSI electron acceptor, methyl viologen. In a third population that gradually increases from zero at 37°C to about 60% at 45°C, the PSII centers were not able to keep Q_A in the reduced state even in the presence of oxygen as the sole electron acceptor. Three electron transport pathways, the pseudocyclic one involving both PSII and PSI, the NAD(P)H-dependent pathway mediated by PSI alone after the loss of activity in some PSII centers, and the PSI-driven ferredoxin-dependent route enhanced by weakly efficient PSII centers that are able to provide only catalytic amounts of electrons, are suggested to create a proton gradient in chloroplasts of heat-stressed leaves thus protecting PSII reaction centers from photodamage.     

Functional heterogeneity of photosystem II in domain specific regions of the thylakoid membrane of spinach (Spinacia oleracea L.)

A mild sonication and phase fractionation method has been used to isolate five regions of the thylakoid membrane in order to characterize the functional lateral heterogeneity of photosynthetic reaction centers and light harvesting complexes. Low-temperature fluorescence and absorbance spectra, absorbance cross-section measurements, and picosecond time-resolved fluorescence decay kinetics were used to determine the relative amounts of photosystem II (PSII) and photosystem I (PSI), to determine the relative PSII antenna size, and to characterize the excited-state dynamics of PSI and PSII in each fraction. Marked progressive increases in the proportion of PSI complexes were observed in the following sequence: grana core (BS), whole grana (B3), margins (MA), stroma lamellae (T3), and purified stromal fraction (Y100). PSII antenna size was drastically reduced in the margins of the grana stack and stroma lamellae fractions as compared to the grana. Picosecond time-resolved fluorescence decay kinetics of PSII were characterized by three exponential decay components in the grana fractions, and were found to have only two decay components with slower lifetimes in the stroma. Results are discussed in the framework of existing models of chloroplast thylakoid membrane lateral heterogeneity and the PSII repair cycle. Kinetic modeling of the PSII fluorescence decay kinetics revealed that PSII populations in the stroma and grana margin fractions possess much slower primary charge separation rates and decreased photosynthetic efficiency when compared to PSII populations in the grana stack.     

Toxic Effects of Copper on Photosystem II of Spinach Chloroplasts

The room temperature fluorescence induction of chloroplasts was utilized as a probe to locate the site of inhibition on PSII by copper. It was found that, while the initial fluorescence yield was hardly affected, the variable fluorescence yield was lowered without significant change in its kinetics. Addition of DCMU, or abolishing oxygen evolution capability by Tris treatment, did not alter this basic inhibition pattern. Copper was also found to lower the fluorescence yield of chloroplasts treated with linolenic acid which inhibited the secondary electron transport on both oxidizing and reducing sides of PSII. The data indicate that copper adversely affects the primary charge separation at the PSII reaction center. We suggest that the inhibition is due to creation of a lesion close to the reaction center, leading to increased dissipation of incoming excitation energy to heat.     

Probing the events of photoinhibition by altering electron-transport activity and light-harvesting capacity in chloroplast thylakoids

Abstract. The effect of photoinhibition on the activity of photosystem II (PSII) in spinach chloroplasts was investigated. Direct light-induced absorbance change measurements at 320 nm (Δ A ₃₂₀) provided a measure of the PSII charge separation reaction and revealed that photoinhibition prevented the stable photoreduction of the primary quinone acceptor Q_A. Sensitivity to photoinhibition was substantially enhanced by treatment of thylakoids with NH₂OH which extracts manganese from the H₂O-splitting enzyme and prevents electron donation to the reaction centre. Incubation with 3-(3,4,-dichlorophenyl)-1,1-dimethylurea (DCMU) during light exposure did not affect the extent of photoinhibitory damage. The chlorophyll (Chl) b -less chlorina (2 mutant of barley displayed a significantly smaller light-harvesting antenna size of PSII (about 20% of that in wild type chloroplasts) and, simultaneously, a lower sensitivity to photoinhibition. These observations suggest that photoinhibition depends on the amount of light absorbed by PSII and that the process of photoinhibition is accelerated when electron donation to the reaction centre is prevented. It is postulated that the probability of photoinhibition is greater when excitation energy is trapped by P680⁺, the oxidized form of the PSII reaction centre. The results are discussed in terms of the D1/D2 heterodimer which contains the functional PSII components P680, pheophytin, Q_A and Q_B.     

In vivo bicarbonate requirement for water oxidation by Photosystem II in the hypercarbonate-requiring cyanobacterium Arthrospira maxima

While the presence of inorganic carbon in the form of (bi)carbonate has been known to be important for activity of Photosystem II (PSII), the vast majority of studies on this "bicarbonate effect" have been limited to in vitro studies of isolated thylakoid membranes and PSII complexes. Here we report an in vivo requirement for bicarbonate that is both reversible and selective for this anion for efficient water oxidation activity in the hypercarbonate-requiring cyanobacterium Arthrospira (Spirulina) maxima, originally isolated from highly alkaline soda lakes. Using a non-invasive internal probe of PSII charge separation (variable fluorescence), primary electron acceptor (Q(A)(-)/Q(A)) reoxidation rate, and flash-induced oxygen yield, we report the largest reversible bicarbonate effect on PSII activity ever observed, which is due to the requirement for bicarbonate at the water-oxidizing complex. Temporal separation of this donor side bicarbonate requirement from a smaller effect of bicarbonate on the Q(A)(-) reoxidation rate was observed. We expect the atypical way in which Arthrospira manages intracellular pH, sodium, and inorganic carbon concentrations relative to other cyanobacteria is responsible for this strong in vivo bicarbonate requirement.     

Light harvesting in photosystem II

Water oxidation in photosynthesis takes place in photosystem II (PSII). This photosystem is built around a reaction center (RC) where sunlight-induced charge separation occurs. This RC consists of various polypeptides that bind only a few chromophores or pigments, next to several other cofactors. It can handle far more photons than the ones absorbed by its own pigments and therefore, additional excitations are provided by the surrounding light-harvesting complexes or antennae. The RC is located in the PSII core that also contains the inner light-harvesting complexes CP43 and CP47, harboring 13 and 16 chlorophyll pigments, respectively. The core is surrounded by outer light-harvesting complexes (Lhcs), together forming the so-called supercomplexes, at least in plants. These PSII supercomplexes are complemented by some “extra” Lhcs, but their exact location in the thylakoid membrane is unknown. The whole system consists of many subunits and appears to be modular, i.e., both its composition and organization depend on environmental conditions, especially on the quality and intensity of the light. In this review, we will provide a short overview of the relation between the structure and organization of pigment-protein complexes in PSII, ranging from individual complexes to entire membranes and experimental and theoretical results on excitation energy transfer and charge separation. It will become clear that time-resolved fluorescence data can provide invaluable information about the organization and functioning of thylakoid membranes. At the end, an overview will be given of unanswered questions that should be addressed in the near future.     

Photosystem II peripheral accessory chlorophyll mutants in Chlamydomonas reinhardtii. Biochemical characterization and sensitivity to photo-inhibition

In addition to the four chlorophylls (Chls) involved in primary charge separation, the photosystem II (PSII) reaction center polypeptides, D1 and D2, coordinate a pair of symmetry-related, peripheral accessory Chls. These Chls are axially coordinated by the D1-H118 and D2-H117 residues and are in close association with the proximal Chl antennae proteins, CP43 and CP47. To gain insight into the function(s) of each of the peripheral Chls, we generated site-specific mutations of the amino acid residues that coordinate these Chls and characterized their energy and electron transfer properties. Our results demonstrate that D1-H118 and D2-H117 mutants differ with respect to: (a) their relative numbers of functional PSII complexes, (b) their relative ability to stabilize charge-separated states, (c) light-harvesting efficiency, and (d) their sensitivity to photo-inhibition. The D2-H117N and D2-H117Q mutants had reduced levels of functional PSII complexes and oxygen evolution capacity as well as reduced light-harvesting efficiencies relative to wild-type cells. In contrast, the D1-H118Q mutant was capable of near wild-type rates of oxygen evolution at saturating light intensities. The D1-H118Q mutant also was substantially more resistant to photo-inhibition than wild type. This reduced sensitivity to photo-inhibition is presumably associated with a reduced light-harvesting efficiency in this mutant. Finally, it is noted that the PSII peripheral accessory Chls have similarities to a to a pair of Chls also present in the PSI reaction center complex.