Charge recombination in photosystem I at low temperatures kinetics of electron tunneling

Absorption changes accompanying light-induced P-700 oxidation and the decay of P-700+ in the dark were measured in the temperature range 294-5 K over a broad time scale (three to four orders of magnitude). Two qualitatively different types of kinetics for the dark decay of P-700⁺ were observed. In the 294-240K region, a usual exponential kinetics is observed with the rate constant κ = 1 · 10¹⁰ · exp(-16 000/RT) s^?1, with R in cal/mol per degree. Below 220 K, a rather unusual logarithmic or near-logarithmic kinetics are observed. These kinetics can be explained quantitatively if one assumes for the various (P-700+ ··· X^-) pairs a broad rectangular or near-rectangular distribution over the values of the rate constant. The following kinetic equation corresponding to this model was obtained: n_t/n_o = [In(κ_max/κ_min)]-1 - [In(1/κ_min)? In t] where no and nt are respectively the initial concentration of P-700⁺ and its concentration at time t, and kmax and kmin the maximum and minimum values of the rate constant, respectively. The decay processes observed can be ascribed to electron tunneling. Distribution over the values of k can be accounted for by different environments or different mutual orientations of P-700⁺ and X^?, or by different distances between them in the various reacting pairs.The corresponding distribution function was reconstructed from the experimentally measured P-700+-decay curves. The rate of tunneling was found to be temperature dependent. In the 160-80-K region, the temperature dependence corresponds to an activation energy of 2.9 kcal/mol. Below 80 K, new modes of P-700+ decay with lower activation energy become operative. The tunneling distance for the majority of the (P-700+ ··· X^?) pairs was estimated from the EPR linewidth of P-700+ to exceed 13.2 A.     

The rate of charge recombination in Photosystem II

Loss by recombination of the charge separated state P₆₈₀⁺Q_A⁻ limits the performance of Photosystem II (PS II) as a photochemical energy converter. Time constants reported in literature for this process are mostly either near 0.17 ms or near 1.4 ms. The shorter time is found in plant PS II when reduction of P₆₈₀⁺ by the secondary electron donor Tyrosine Z cannot occur because Y_Z is already oxidized. The 1.4 ms recombination is seen in Y_Z-less mutants of the cyanobacterium Synechocystis. However, the rate of P₆₈₀⁺Q_A⁻ recombination that actually competes with the stabilization of the charge separation has not been previously reported. We have measured the kinetics of the flash-induced fluorescence yield changes in the microsecond time domain in Tris-washed spinach chloroplasts. In this way the kinetics and yield of P₆₈₀⁺ reduction by Y_Z were obtained, and the rate of the competing P₆₈₀⁺Q_A⁻ recombination could be evaluated. The recombination time was less than 0.5 ms; the best-fitting time constant was 0.1 ms. The presence of Y_Z^ox slightly decreased the efficiency of excitation trapping but did not seem to accelerate P₆₈₀⁺Q_A⁻ recombination. The two P₆₈₀⁺Q_A⁻ lifetimes in the literature probably reflect a significant difference between plant and cyanobacterial PS II.     

Effect of the redox state of QB on electric field-induced charge recombination in Photosystem II

Electric field-induced charge recombination in Photosystem II (PS II) was studied in osmotically swollen spinach chloroplasts (blebs) by measurement of the concomitant chlorophyll luminescence emission (electroluminescence). A pronounced dependence on the redox state of the two-electron gate Q_B was observed and the earlier failure to detect it is explained. The influence of the Q_B/Q_B ^– oscillation on electroluminescence was dependent on the redox state of the oxygen evolving complex; at times around one millisecond after flash illumination a large effect was observed in the states S₂ and S₃, but not in the state S₄ (actually Z⁺S₃). The presence of the oxidized secondary electron donor, tyrosine Z⁺, appeared to prevent expression of the Q_B/Q_B ^– effect on electroluminescence, possibly because this effect is primarily due to a shift of the redox equilibrium between Z/Z⁺ and the oxygen evolving complex.Abbreviations BSA bovine serum albumin - EDTA ethylene-diaminetetraacetic acid - EL electroluminescence - FCCP carbonylcyanide p-trifluoromethyloxyphenyl-hydrazone - HEPESI 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - I primary electron acceptor - MOPS 3-(N-morpholino) propane sulfonic acid - P680 primary electron donor of Photosystem II - P700 primary electron donor of Photosystem I - Q_A and Q_B secondary and tertiary electron acceptors of Photosystem II - Z secondary electron donor (D1 Tyr 161)     

Chlorophyll a fluorescence as a monitor of nanosecond reduction of the photooxidized primary donor P-680 of Photosystem II

1. Changes in the fluorescence yield of aerobic Chlorella vulgaris have been measured in laser flashes of 15 ns, 30 ns and 350 ns half time. The kinetics after the first flash given after a 3 min dark period could be simulated on a computer using the hypothesis that the oxidized acceptor Q and primary donor P⁺ are fluorescence quenchers, and Q⁻ is a weak quencher, and that the reduction time for P⁺ is 20–35 ns.

2. The P⁺ reduction time for at least an appreciable part of the reaction centers was found to be longer after the second and subsequent flashes. In the first 5 flashes an oscillation was observed. Under steady state conditions, with a pulse separation of 3 s, a reduction time for P⁺ of about 400 ns for all reaction centers gave the best correspondence between computed and experimental fluorescence kinetics.     

Photoreduction of cytochrome b559 in a Photosystem-II subchloroplast particle

A Photosystem-II reaction-center particle derived from spinach chloroplasts by Triton treatment contains only one kind of cytochrome, namely, cytochrome b₅₅₉, in the amount of slightly more than 2 per 100 total chlorophyll molecules. Cytochrome b₅₅₉ is present in the oxidized form, has a standard redox potential of 58 mV, and undergoes photoreduction.     

Different temperature dependencies of the charge recombination reaction in photoreaction centers isolated from different bacterial species

We compared the temperature dependency of the rate of the charge recombination reaction in photoreaction centers isolated from Ectothiorhodospira sp. and from Rhodospirillum rubrum G9. We also examined the temperature dependency of the bandwidth and peak wavelength of their far-red absorption band. In both preparations, the peak wavelength and the bandwidth vary monotonically with temperature between 80 and 300 K. However, the rate of the charge recombination reaction has a quite different temperature dependency. In the preparation from R. rubrum, the reaction is accelerated 5-fold in a typical sigmoidal fashion as the temperature is lowered from 300 to 80 K. In the preparation from Ectothiorhodospira sp., the reaction is accelerated monotonically only about 1.5-fold in the same temperature range. At temperatures below 100 K, the rates are similar in the two preparations. We interpret the temperature dependency of the charge recombination reaction in terms of an activationless electron-transfer model formulated by Jortner (Jortner, J. (1980) Biochim. Biophys. Acta 394, 193–230). The minimal model provides a good fit for the temperature dependency of charge recombination in the preparation from Ectothiorhodospira sp. However, to fit the temperature dependency of the R. rubrum preparation with the same model, we must further postulate that the electronic coupling factor varies with temperature in this preparation. We find that, in both preparations, the temperature dependency of the far-red absorption bandwidth is consistent with the assumption that similar vibrational modes are involved in electron transfer and in electronic excitation.     

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.     

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.     

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.     

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.     

A demonstration of the physiological role of membrane phosphorylation in chloroplasts, using the bipartite and tripartite models of photosynthesis

Using the equations derived from the bipartite and tripartite models for photosynthetic organization in green plants, we have been able to characterize the effect of membrane phosphorylation on energy transduction. Phosphorylation reversibly increases (the proportion of absorbed quanta going directly to Photosystem (PS) I). This increase in we believe to be due to a decrease in the coupling between the PS II core and its associated light-harvesting complex [ΨT(3,2)·ΨT(2,3)]. Phosphorylation also reversibly increases the transfer of energy from PS II to PS I [ψT_(II→I)]. We propose that membrane phosphorylation provides the in vivo control of , ΨT(3,2)·ΨT(2,3) and ψT_(II→I). From the data we present it is clear that the changes caused in energy distribution as a result of phosphorylation are large enough to induce real changes in electron-transfer reactions. The effects of phosphorylation on these parameters are distinct from those of Mg²⁺ depletion. We have discussed changes in ΨT(3,2)·ΨT(2,3) (the coupling term) with respect to the ‘connected package’ model of photosynthetic units (Butler, W.L. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 4697–4701) and the proposed - and β-centers of PS II (Melis, A. and Homann, P.H. (1976) Photochem. Photobiol. 23, 345–350). The demonstration of changes in reversible coupling [ΨT(3,2)·ΨT(2,3)] strongly supports a connected package model in which the degree of ‘connectivity’ is under physiological control.     

Membrane-surface electric properties of Triton-fractionated spinach subchloroplast fragments

Surface charge density of subchloroplast fragments fractionated from spinach by Triton X-100 treatment was estimated from cation-induced quenching of chlorophyll fluorescence, with the premise that the fluorescence yield is dependent on the surface electric potential of the preparations. Application of the Gouy-Chapman theory of diffuse double layer to the subchloroplast preparations, or treating the surface of the preparations under electric charge regulation conditions yielded a result suggesting the Photosystem II reaction-center preparation (TSF-IIa) to be more negatively charged than the Photosystem I reaction-center preparation (TSF-I). Isoelectric points of the subchloroplast fragments were determined by measuring 90° light scattering and more directly by gel isoelectric focusing. Isoelectric points of TSF-I and -IIa were estimated to be 4.8 and 4.0 from light-scattering experiments, and 4.5 and 4.1 from gel electrophoresis, respectively. The TSF-II preparation that contains both a light-harvesting complex and the reaction-center (core) complex showed a small cation-induced quenching of chlorophyll fluorescence. This fluorescence quenching may be ascribed mostly to the regulation of energy transfer in the preparation (Yamamoto, Y. and Ke, B. (1980) Biochim. Biophys. Acta 592, 296–302). Furthermore, the TSF-II preparation showed a broad and indefinite peak in light scattering in the pH range 3–8, suggesting that the complex probably carries a small amount of charge in this pH range. The physiological role of the membrane surface charge of the subchloroplast preparations in membrane structure and cation regulated processes in chloroplast is discussed.     

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.     

The effect of pH on the reduction kinetics of P-680 in tris-treated chloroplasts

The primary donor of Photosystem II (PS II), P-680, was photo-oxidized by a short flash and its rate of reduction was measured at different pH values by following the recovery of the absorption change at 820 nm in chloroplasts pretreated with a high concentration of Tris. The re-reduction is biphasic with a fast phase (dominant after the first flash) attributed to the donation by a donor, D₁, and a slow phase (usually dominant after the second flash) attributed to a back-reaction with the primary acceptor.

It is found that pH has a strong influence on the donation from D₁ (τ = 2 μs at pH 9, 44 μs at pH 4), but no influence on the back reaction (τ ≈ 200 μs). pH also influences the stability of the charge separation since the contribution of donation from D₁ at the second flash increases at lower pH, getting close to 100% at pH 4.     

A photoactive Photosystem-II reaction-center complex lacking a chlorophyll-binding 40 kilodalton subunit from the thermophilic cyanobacterium Synechococcus sp.

The Photosystem-II reaction-center complex of the thermophilic cyanobacterium Synechococcus sp. was resolved into two complemental chlorophyll-protein complexes, CP2b which contained a chlorophyll-binding 47 kDa polypeptide, two polypeptides in the 28–31 kDa region and a 9 kDa polypeptide, and CP2c which had only a chlorophyll-binding 40 kDa polypeptide. CP2b was found to be highly active in photoreduction of 2,6-dichlorophenolindophenol with diphenylcarbazide as an electron donor. The activity was insensitive to 3-(3,4-dichlorophenyl)-1,1-dimethylurea and ioxynil but was half inactivated by the treatment of the complex at 50°C for 5 min, or on addition of 0.001% sodium dodecyl sulfate, indicating its dependence on the protein conformation. CP2c also showed a low activity of the dye photoreduction, which was insensitive to heat and enhanced at high concentrations of sodium dodecyl sulfate. The quantum yield of the photoreduction was estimated to be 0.12 for CP2b and 0.002 for CP2c. It is concluded that the 47 kDa polypeptide is the site of the Photosystem-II reaction center and the 40 kDa polypeptide is not required for the Photosystem-II-driven electron transport.     

The deactivation of Photosystem II in Chlorella as a second-order process

The kinetics of deactivation of the S₃ state in Chlorella have been observed under a variety of conditions. The S₃ state appears to decline in a dark period coming after a sequence of 30 saturating flashes in a second-order reaction, the rate constant of which is 0.132/[S*₃] s⁻¹ and which involves an electron donor, D₁, of concentration 1.25[S*₃] where [S*₃] is the concentration of the S₃ state when the oxygen yield of the light flashes is constant. If a 1 min period of 650 nm illumination is employed after the sequence of flashes, the subsequent S₃ state deactivation kinetics are more complex. There is an initial phase of S₃ state deactivation, accounting for about 35% of the original S₃ state, which is complete in less than 100 ms. The remaining 65% of the S₃ state appears to deactivate in a second-order reaction, the rate constant of which is 1.36/[S*₃] s⁻¹ and which involves an electron donor of initial concentration 0.58[S*₃]. If a 1 min period of 710 nm illumination comes after the 30 flashes, at least 98% of the S₃ state deactivates according to first-order kinetics. It is shown that this can be explained using a second-order model if there is an electron donor present of which the concentration is large compared with [S*₃]. However, S₃ state deactivation observed after 5 min of dark and two saturating flashes can be described neither by a first-order model nor a second-order model. Deactivation of the S₂ state after a 5 min dark period and one saturating flash follows second-order kinetics with a rate constant of 0.2/[S*₃] s⁻¹ and appears to involve an electron donor of initial concentration 1.3[S*₃]. Arguments are presented which tend to rule out the primary electron acceptor to Photosystem II as being any of the electron donors but it appears quite possible that the large plastoquinone pool is involved.     

Singlet oxygen production in herbicide-treated photosystem II

Photo-generated reactive oxygen species in herbicide-treated photosystem II were investigated by spin-trapping. While the production of .OH and O2-* was herbicide-independent, 1O2 with a phenolic was twice that with a urea herbicide. This correlates with the reported influence of these herbicides on the redox properties of the semiquinone QA-* and fits with the hypothesis that 1O2 is produced by charge recombination reactions that are stimulated by herbicide binding and modulated by the nature of the herbicide. When phenolic herbicides are bound, charge recombination at the level of P+*Pheo-* is thermodynamically favoured forming a chlorophyll triplet and hence 1O2. With urea herbicides this pathway is less favourable.     

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.     

Regulation of electron transport in Photosystem-II fragments by magnesium ions

In Photosystem-II reaction-center particles (TSF-IIa) fractionated from spinach chloroplasts by Triton X-100 treatment, divalent cations appear to regulate electron-transport reactions. Oxidation of cytochrome b-559 after illumination of the particles was accelerated by the presence of Mg²⁺, whereas photoreduction of 2,6-dichlorophenolindophenol (DCIP) by diphenyl carbazide was inhibited, both at a half-effective concentration of Mg²⁺ of approx. 0.1 mM.The site of regulation was shown to be on the oxidizing side of Photosystem II, near P-680, based on the effects of actinic-light intensity and nature of the electron donors on DCIP photoreduction. Mg²⁺ was effective in quenching chlorophyll fluorescence in TSF-IIa particles, but the quenching was sensitive to the presence of 3(3,4-dichloropheny)-1,1-dimethylurea. In the reactioncenter (core) complex of Photosystem II, where the light-harvesting chlorophyll-protein complex is absent, there seems to be no regulation by Mg²⁺ on excitation-energy distribution.