Structure and function in the isolated reaction center complex of Photosystem II: energy and charge transfer dynamics and mechanism

The dynamics of energy and charge transfer in the Photosystem II reaction center complex is an area of great interest today. These processes occur on a time scale ranging from femtoseconds to tens of picoseconds or longer. Steady-state and ultrafast spectroscopy techniques have provided a great deal of quantitative and qualitative data that have led to varied interpretations and phenomenological models. More recently, microscopic models that identify specific charge separated states have been introduced, and offer more insight into the charge transfer mechanism. The structure and energetics of PS II reaction centers are reviewed, emphasizing the effects on the dynamics of the initial charge transfer. This revised version was published online in June 2006 with corrections to the Cover Date.     

Determination of the primary charge separation rate in Photosystem II reaction centers at 15 K

We have measured the rate constant for the formation of the oxidized chlorophyll a electron donor (P680⁺) and the reduced electron acceptor pheophytin a ^– (Pheo a ^–) following excitation of isolated Photosystem II reaction centers (PS II RC) at 15 K. This PS II RC complex consists of D₁, D₂, and cytochrome b-559 proteins and was prepared by a procedure which stabilizes the protein complex. Transient absorption difference spectra were measured from 450–840 nm as a function of time with 500fs resolution following 610 nm laser excitation. The formation of P680⁺-Pheo a ^– is indicated by the appearance of a band due to P680⁺ at 820 nm and corresponding absorbance changes at 490, 515 and 546 nm due to the formation of Pheo a ^–. The appearance of the 490 nm and 820 nm bands is monoexponenital with =1.4±0.2 ps. Treatment of the PS II RC with sodium dithionite and methyl viologen followed by exposure to laser excitation results in accumulation of Pheo a ^–. Laser excitation of these prereduced RCs at 15 K results in formation of a transient absorption spectrum assigned to ^1*P680. We observe wavelength-dependent kinetics for the recovery of the transient bleach of the Q_y absorption bands of the pigments in both untreated and pre-reduced PS II RCs at 15K. This result is attributed to an energy transfer process within the PS II RC at low temperature that is not connected with charge separation.Abbreviations PS I Photosystem I - PS II Photosystem II - RC reaction center - P680 primary electron donor in Photosystem II - Chl a chlorophyll a - Pheo a pheophytin a     

Excitation trapping and charge separation in Photosystem II in the presence of an electrical field

To investigate the effects of a membrane potential on excitation trapping and charge separation in Photosystem II we have studied the chlorophyll fluorescence yield in osmotically swollen chloroplasts subjected to electrical field pulses. Significant effects were observed only in those membrane regions where a large membrane potential opposing the photochemical charge separation was built up. When the fluorescence yield was low, close to F₀, a much higher yield, up to F_max, was observed during the presence of the membrane potential. This is explained by an inhibition by the electrical field of electron transfer to the quinone acceptor Q, resulting in a decreased trapping of excitations. A field pulse applied when the fluorescence yield was high, Q and the donor side being in the reduced state, had the opposite effect: the fluorescence was quenched nearly to F₀. This field-induced fluorescence quenching is ascribed to reversed electron transfer from Q^? to the intermediate acceptor, pheophytin. Its field strength dependence suggests that the midpoint potential difference between pheophytin and Q is at most about 300 mV. Even then it must be assumed that electron transfer between pheophytin and Q spans 90% of the potential difference across the membrane.     

The isolated Photosystem II reaction center: first attempts to directly measure the kinetics of primary charge separation

Direct measurements of the intrinsic rate of primary charge separation in the isolated Photosystem II (PS II) reaction center complex had to await the availability of suitable, stabilized reaction center materials as well as sophisticated femtosecond transient absorption spectroscopic techniques. The events that led to the first direct measurements of the primary charge separation act in PS II and discussions of the results thereafter are chronicled in this brief historical review. This revised version was published online in August 2006 with corrections to the Cover Date.     

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.     

Temperature dependence of the primary electron transfer reaction in pigment-modified bacterial reaction centers

The initial electron transfer steps in pigment modified reaction centers, where bacteriopheophytin is replaced by plant pheophytin (R26.Phe-a RCs) have been investigated over a wide temperature range by femtosecond time-resolved spectroscopy. The experimental data obtained in the maximum of the bacteriochlorophyll anion band at 1020 nm show the existence of a high and long-lived population of the primary acceptor P⁺B_A ^– even at 10 K. The data suggest a stepwise electron transfer mechanism with B_A as primary acceptor also in the low temperature domain. A detailed data analysis suggests that the pigment modification leads to a situation with almost isoenergetic primary and secondary acceptor levels, approximately 450 cm^–1 below P^*. A Gaussian distribution (with = 400 cm ^–1) of the G values has to be assumed to account for the strong dispersive character of the kinetics in this sample. Based on these assumptions, a model is presented that reproduces the observed kinetics, heterogeneity and temperature dependence.     

EPR and optical changes of the photosystem II reaction center produced by low temperature illumination

Electron paramagnetic resonance (EPR) and absorption spectroscopy have been used to study the low temperature photochemical behavior of the Photosystem II D-1/D-2/ cytochrome b559 reaction center complex. The reaction center displays large triplet state EPR signals which are attenuated after actinic illumination at low temperatures in the presence of sodium dithionite. Concomitant with the triplet attenuation is the buildup of a structured radical signal with an effective g value of 2.0046 and a peak-to-peak width of 11.9 G. The structure in the signal is suggestive of it being comprised in part of the anion radical of pheophytin a. This assignment is corroborated by low temperature optical absorbance measurements carried out after actinic illumination at the low temperatures which show absorption bleachings at 681 nm, 544 nm and 422 nm and an absorbance buildup at 446 nm indicating the formation of reduced pheophytin.Abbreviations EPR electron paramagnetic resonance     

Pheophytin-mediated energy storage of photosystem II particles detected by photoacoustic spectroscopy

The photoacoustic (PA) characteristics (energy storage and heat dissipation) of photosystem II (PSII) core-enriched particles from barley were studied (i) in conditions where there was electron flow, i.e., in the presence of a combination of the electron acceptor K₃ Fe (CN)₆, referred to as FeCN, and the electron donor diphenylcarbazide (DPC), and (ii) in conditions where electron flow was suppressed, i.e., in the absence of FeCN and DPC. The experimental data show that a decrease of heat dissipation with a minimum at 540 nm can be interpreted as energy storage resulting from the presence of pheophytin (Pheo) in the PSII particles. On account of the capability of the PA method to measure the energy absorbed by the chromophores which is converted to heat, it is suggested that the PA detection of Pheo present in the PSII complex will permit to clarify the function of processes involving non-radiative relaxation of excited states in P680-Pheo-Q_A interactions.Abbreviations -Car -Carotene - Chl Chlorophyll - DPC Diphenylcarbazide - EPR Electron Paramagnetic Resonance - FeCN potassium ferricyanide - HEPES N-2-hydroxyethylenepiperazine-N-2-ethanesulfonate - P680 reaction center of PSII - PA Photoacoustic - Pheo pheophytin - PSI photosystem I - PSII photosystem II - Q_A primary electron acceptor of PSII     

Structural differences in the inner part of Photosystem II between higher plants and cyanobacteria

A detailed comparison of key components in the Photosystem II complexes of higher plants and cyanobacteria was carried out. While the two complexes are overall very similar, significant differences exist in the relative orientation of individual components relative to one another. We compared a three-dimensional map of the inner part of plant PS II at 8 Å resolution, and a 5.5 Å projection map of the same complex determined by electron crystallography, to the recent 3.5–3.8 Å X-ray structures of cyanobacterial complexes. The largest differences were found in the rotational alignment of the cyt b_^559 subcomplex, and of the CP47 core antenna with respect to the D1/D2 reaction centre. Within the D1/D2 proteins, there are clear differences between plants and cyanobacteria at the stromal ends of membrane-spanning helices, even though these proteins are highly homologous. Notwithstanding these differences in the protein scaffold, the distances between the critical photosynthetic pigment cofactors seem to be precisely conserved. The different protein arrangements in the two complexes may reflect an adaptation to the two very different antenna systems, membrane-extrinsic phycobilisomes for cyanobacteria, and membrane-embedded chlorophyll a/b proteins in plants.     

Measurement of the extent of electron transfer to the bacteriopheophytin in the M-subunit in reaction centers of Rhodopseudomonas viridis

We have measured the extent of flash-induced electron transfer from the bacteriochlorophyll dimer, P, to the bacteriopheophytin in the M-subunit, H_M, in reaction centers of Rhodopseudomonas viridis. This has been done by measuring the transient states produced by excitation of reaction centers trapped in the PH_L ^–H_M state at 90 K. Under these conditions the normal forward electron transfer to the bacteriopheophytin in the L-subunit, H_L, is blocked and the yield of transient P⁺H_M ^– can be estimated with respect to the lifetime of P^*. Under these conditions flash induced absorbance decreases of the bacteriochlorophyll dimer 990 nm band suggest that a transient P⁺ state is formed with a quantum yield of 0.09±0.06 compared to that formed during normal photochemistry. These transient measurements provide an upper limited on the yield of a transient P⁺ H_M ^– state. An estimate of 0.09 as the yield of the P⁺ H_M ^– state is consistent with all current observations. This estimate and the lifetime of P^* suggest that the electron transfer rate from P^* to H_M, k_M, is about 5 × 10⁹ sec^–1 (_M = 200ps). These measurements suggest that the a branching ratio k_L/k_M is on the order of 200. The large value of the branching ratio is remarkable in view of the structural symmetry of the reaction center. This measurement should be useful for electron transfer calculations based upon the reaction center structure.     

Far-red photosynthesis: Two charge separation pathways exist in plant Photosystem II reaction center

An alternative charge separation pathway in Photosystem II under the far-red light was proposed by us on the basis of electron transfer properties at 295 K and 5 K. Here we extend these studies to the temperature range of 77–295 K with help of electron paramagnetic resonance spectroscopy. Induction of the S₂ state multiline signal, oxidation of Cytochrome b₅₅₉ and Chlorophyll_Z was studied in Photosystem II membrane preparations from spinach after application of a laser flashes in visible (532 nm) or far-red (730–750 nm) spectral regions. Temperature dependence of the S₂ state signal induction after single flash at 730–750 nm (T_inhibition ~ 240 K) was found to be different than that at 532 nm (T_inhibition ~ 157 K). No contaminant oxidation of the secondary electron donors cytochrome b₅₅₉ or chlorophyll_Z was observed. Photoaccumulation experiments with extensive flashing at 77 K showed similar results, with no or very little induction of the secondary electron donors. Thus, the partition ratio defined as (yield of Y_Z/CaMn₄O₅-cluster oxidation):(yield of Cytb₅₅₉/Chl_Z/Car_D2 oxidation) was found to be 0.4 at under visible light and 1.7 at under far-red light at 77 K. Our data indicate that different products of charge separation after far-red light exists in the wide temperature range which further support the model of the different primary photochemistry in Photosystem II with localization of hole on the Chl_D1 molecule.     

Identity of the Photosystem II reaction center polypeptide

A Photosystem-II (PS-II)-enriched chloroplast submembrane fraction has been subjected to non-denaturing gel-electrophoresis. Two chlorophyll a (Chl a)-binding proteins associated with the core complex were isolated and spectrally characterized. The Chl protein with apparent apoprotein mass of 47 kDa (CP47) displayed a 695 nm fluorescence emission maximum (77 K) and light-induced absorption characteristics indicating the presence of the reaction center Chl, P-680, and its primary electron acceptor, pheophytin. A Chl protein of apparent apoprotein mass of 43 kDa (CP43) displayed a fluorescence emission maximum at 685 nm. We conclude that CP43 serves as an antenna Chl protein and the PS II reaction center is located in CP47.     

Coupling of exciton motion in the core antenna and primary charge separation in the reaction center

The relation between exciton motion in the LH1 antenna and primary charge separation in the reaction center of purple bacteria is briefly reviewed. It is argued that in models based on hopping excitons described strictly by Förster theory, transfer-to-trap-limited kinetics is quite unlikely according to the relation between the exciton trapping kinetics and N, the size of the photosynthetic unit in such models. Because the results of several recent experiments have been interpreted in terms of transfer-to-trap limited kinetics, this presents a conflict between these experimental interpretations and strictly Förster-based theoretical models. Two possible resolutions are proposed. One arises from the random phase-redistribution trapping kinetics of partially coherent excitons, a kinetics uniquely independent of both N and the rate constant for primary charge separation in the reaction center. The other comes from multiple-pathways models of the multipicosecond nonexponentiality of the decay of P^*, the electronically excited primary electron donor in the reaction center. In these models, because it depends only on a certain averaged electron-transfer time constant, the exciton lifetime may be relatively insensivive to variations of individual electrontransfer rate constants-thereby undercutting the argument appearing in recent literature that by default the exciton kinetics must be transfer-to-trap limited.     

Electron donation to Photosystem II in reaction center preparations

The room-temperature EPR characteristics of Photosystem II reaction center preparations from spinach, pokeweed and Chlamydomonas reinhardii have been investigated. In all preparations a light-induced increase in EPR Signal II, which arises from the oxidized form of a donor to P-680⁺, is observed. Spin quantitation, with potassium nitrosodisulfonate as a spin standard, demonstrates that the Signal II species, Z^?, is present in approx. 60% of the reaction centers. In response to a flash, the increase in Signal II spin concentration is complete within the 98 μs response time of our instrument. The decay of Z^? is dependent on the composition of the particle suspension medium and is accelerated by addition of either reducing agents or lipophilic anions in a process which is first order in these reagents. Comparison of these results with optical data reported previously (Diner, B.A. and Bowes, J.M. (1981) in Proceedings of the 5th International Congress on Photosynthesis (Akoyunoglou, G., ed.), Vol. 3, pp. 875–883, Balaban, Philadelphia), supports the identification of Z with the P-680⁺ donor, D₁. From the polypeptide composition of the particles used in this study, we conclude that Z is an integral component of the reaction center and use this conclusion to construct a model for the organization of Photosystem II.     

Effects of selective destruction of iron-sulfur center B on electron transfer and charge recombination in Photosystem I

Incubation of spinach thylakoids with HgCl₂ selectively destroys Fe–S center B (F_B). The function of electron acceptors in F_B-less PS I particles was studied by following the decay kinetics of P700⁺ at room temperature after multiple flash excitation in the absence of a terminal electron acceptor. In untreated particles, the decay kinetics of the signal after the first and the second flashes were very similar (t _1/22.5 ms), and were principally determined by the concentration of the artificial electron donor added. The decay after the third flash was fast (t _1/20.25 ms). In F_B-less particles, although the decay after the first flash was slow, fast decay was observed already after the second flash. We conclude that in F_B-less particles, electron transfer can proceed normally at room temperature from F_X to F_A and that the charge recombination between P700⁺ and F_X ^-/A₁ ^- predominated after the second excitation. The rate of this recombination process is not significantly affected by the destruction of F_B. Even in the presence of 60% glycerol, F_B-less particles can transfer electrons to F_A at room temperature as efficiently as untreated particles.Abbreviations DCIP 2, 6-dichlorophenol indophenol - F_A, F_B, F_X iron-sulfur center A, B and X, respectively - PMS phenazine methosulfate     

Femtosecond nuclear oscillations under charge separation in reaction centers of photosynthesis

Results are presented of a study of primary processes of formation of the charge separated states P⁺B_A ^- and P⁺H_A ^- (where P is the primary electron donor, B_A and H_A the primary and secondary electron acceptors) in native and pheophytin-modified reaction centers (RCs) of Rhodobacter sphaeroides R-26 by methods of femtosecond spectroscopy of absorption changes at low temperature. Coherent oscillations were studied in the kinetics at 935 nm (P* stimulated emission band), at 1020 nm (B_A ^- absorption band), and at 760 nm (H_A absorption band). It was found that when the wavepacket created under femtosecond light excitation approaches the intersection between P* and P⁺B_A ^- potential surfaces at 120- and 380-fsec delays, the formation of two electron states emitting light at 935 nm (P*) and absorbing light at 1020 nm (P⁺B_A ^-) takes place. At the later time the wavepacket motion has a frequency of 32 cm^-1 and is accompanied by electron transfer from P* to B_A in pheophytin-modified and native RCs and further to H_A in native RCs. It was shown that electron transfer processes monitored by the 1020-nm absorption band development as well as by bleaching of 760-nm absorption band have the enhanced 32 cm^-1 mode in the Fourier transform spectra.     

Unifying model for the photoinactivation of Photosystem II in vivo under steady-state photosynthesis

We present a unifying mechanism for photoinhibition based on current obsevations from in vivo studies rather than from in vitro studies with isolated thylakoids or PS II membranes. In vitro studies have limited relevance for in vivo photoinhibition because very high light is used with photon exposures rarely encountered in nature, and most of the multiple, interacting, protective strategies of PS II regulation in living cells are not functional. It is now established that the photoinactivation of Photosystem II in vivo is a probability and light-dosage event which depends on the photons absorbed and not the irradiance per se. As the reciprocity law is obeyed and target theory analysis strongly suggests that only one photon is required, we propose that a single dominant molecular mechanism occurs in vivo with one photon inactivating PS II under limiting, saturating or sustained high light. Two mechanisms have been proposed for photoinhibition under high light, acceptor-side and donor-side photoinhibition [see Aro et al. (1994) Biochim Biophys Acta 1143: 113–134], and another mechanism for very low light, the low-light syndrome [Keren et al. (1995) J Biol Chem 270: 806–814]. Based on the exciton-radical pair equilibrium model of exciton dynamics, we propose a unifying mechanism for the photoinactivation of PS II in vivo under steady-state photosynthesis that depends on the generation and maintenance of increased concentrations of the primary radical pair, P680⁺Pheo_–, and the different ways charge recombination is regulated under varying environmental conditions [Anderson et al. (1997) Physiol Plant 100: 214–223]. We suggest that the primary cause of damage to D1 protein is P680⁺, rather than singlet O₂ formed from triplet P680, or other reactive oxygen species.     

Energy transfer to low energy chlorophyll species prior to trapping by P700 and subsequent electron transfer

It is found that the two singlet state lifetimes observed in medium sized isolated Photosystem One reaction centres belong to two distinct sets of particles. The nanosecond lifetime is due to PS1 particles in which P700 does not trap excitation energy, and the excitation energy is homogeneously distributed within the antennae of these particles. The spectral features of the picosecond component show that excitation energy in the antenna has become largely concentrated in one or more low energy (red) chlorophyll species within 3.5 ps. Antennae which have become decoupled from P700 also appear to be decoupled from these red ancillary chlorophylls, and this suggests that some substructure or level of organisation links them to P700.The rate of quenching of antenna singlet states appears to be independent of the redox state of P700 under the conditions used here, and oxidising P700 does not prevent excitation energy from reaching the red chlorophyll species in the antenna.We find no evidence in the data presented here of a chlorophyll molecule acting as a metastable primary acceptor (A₀). The lower limit for the detection of such a species in these data is 20% of the optical density of the transient P700 bleach.Abbreviations Chl chlorophyll - PS1 Photosystem One - P700 primary electron donor - A₀ primary electron acceptor     

The involvement of lipids in light-induced charge separation in the reaction center of photosystem II

Extraction of PS II particles with 50 mM cholate and 1 M NaCl releases several proteins (33-, 23-, 17- and 13 kDa) and lipids from the thylakoid membrane which are essential for O₂ evolution, dichlorophenolindophenol (DCIP) reduction and for stable charge separation between P680⁺ and Q_A ^-. This work correlates the results on the loss of steady-state rates for O₂ evolution and PS II mediated DCIP photo-reduction with flash absorption changes directly monitoring the reaction center charge separation at 830 nm due to P680⁺, the chlorophyll a donor. Reconstitution of the extracted lipids to the depleted membrane restores the ability to photo-oxidize P680 reversibly and to reduce DCIP, while stimulating O₂ evolution minimally. Addition of the extracted proteins of masses 33-, 23- and 17- kDa produces no further stimulation of DCIP reduction in the presence of an exogenous donor like DPC, but does enhance this rate in the absence of exogenous donors while also stimulating O₂ evolution. The proteins alone in the absence of lipids have little influence on charge separation in the reaction center. Thus lipids are essential for stable charge separation within the reaction center, involving formation of P680⁺ and Q_A ^-.Abbreviations A₈₃₀ Absorption change at 830 nm - Chl Chlorophyll - D₁ primary electron donor to P680 - DCIP 2,6-dichlorophenolindophenol - DPC 1,5-diphenylcarbazide - MOPS 3-(N-morpholino)propanesulfonic acid - P680 reaction center chlorophyll a molecule of photosystem II - PPBQ Phenyl-p-benzoquinone - PS II Photosystem II - Q_A, Q_B first and second quinone acceptors in PS II - V-DCIP rate of DCIP reduction - V-O₂ rate of oxygen evolution - Y water-oxidizing enzyme system - CHAPS 3-Cyclohexylamino-propanesulfonic acid     

Primary charge separation in Photosystem II

In this Minireview, we discuss a number of issues on the primary photosynthetic reactions of the green plant Photosystem II. We discuss the origin of the 683 and 679 nm absorption bands of the PS II RC complex and suggest that these forms may reflect the single-site spectrum with dominant contributions from the zero-phonon line and a pronounced ∼80 cm⁻¹ phonon side band, respectively. The couplings between the six central RC chlorins are probably very similar and, therefore, a `multimer' model arises in which there is no `special pair' and in which for each realization of the disorder the excitation may be dynamically localized on basically any combination of neighbouring chlorins. The key features of our model for the primary reactions in PS II include ultrafast (<500 fs) energy transfer processes within the multimer, `slow' (∼20 ps) energy transfer processes from peripheral RC chlorophylls to the RC multimer, ultrafast charge separation (<500 fs) with a low yield starting from the singlet-excited `accessory' chlorophyll of the active branch, cation transfer from this `accessory' chlorophyll to a `special pair' chlorophyll and/or charge separation starting from this `special pair' chlorophyll (∼8 ps), and slow relaxation (∼50 ps) of the radical pair by conformational changes of the protein. The charge separation in the PS II RC can probably not be described as a simple trap-limited or diffusion-limited process, while for the PS II core and larger complexes the transfer of the excitation energy to the PS II RC may be rate limiting. This revised version was published online in June 2006 with corrections to the Cover Date.