Photosystem I charge separation in the absence of centers A and B. II. ESR spectral characterization of center ‘X’ and correlation with optical signal ‘A2’

The Photosystem I electron acceptor complex was characterized by optical flash photolysis and electron spin resonance (ESR) spectroscopy after treatment of a subchloroplast particle with lithium dodecyl sulfate (LDS). The following properties were observed after 60 s of incubation with 1% LDS followed by rapid freezing. (i) ESR centers A and B were not observed during or after illumination of the sample at 19 K, although the P-700⁺ radical at g = 2.0026 showed a large, reversible light-minus-dark difference signal. (ii) Center ‘X’, characterized by g factors of 2.08, 1.88 and 1.78, exhibited reversible photoreduction at 8 K in the absence of reduced centers A and B. (iii) The backreaction kinetics at 8 K between P-700, observed at g = 2.0026, and center X, observed at g = 1.78, was 0.30 s. (iv) The amplitudes of the reversible g = 2.0026 radical observed at 19 K and the 1.2 ms optical 698 nm transient observed at 298 K were diminished to the same extent when treated with 1% LDS at room temperature for periods of 1 and 45 min. We interpret the strict correlation between the properties and lifetimes of the optical P-700⁺ A⁻₂ reaction pair and the ESR P-700⁺ center X⁻ reaction pair to indicate that signal A₂ and center X represent the same iron-sulfur center in Photosystem I.     

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.     

Flash photolysis electron spin resonance studies of the electron acceptor species at low temperatures in Photosystem I of spinach subchloroplast particles

The light-induced electron spin resonance signals of Photosystem I spinach subchloroplast particles have been studied at approximately 6 °K. Using the technique of flash photolysis-electron spin resonance with actinic illumination at 647 nm, a kinetic analysis of the previously observed bound ferredoxin ESR signals was carried out. Signal I (P700⁺) exhibits a partial light-reversible behavior at 6 °K so it was expected that if the bound ferredoxin is the primary acceptor of Photosystem I, it should also exhibit a partial reversible behavior. However, none of the bound ferredoxin ESR signals showed any such light reversible behavior. A search to wider fields revealed two components which did exhibit the expected kinetic behavior. These components are very broad (about 80 G) and are centered at g = 1.75 and g = 2.07. These two components exhibit the expected characteristics of the primary electron acceptor. A model is presented to account for the reversible and irreversible photochemical changes in Photosystem I. The possible identity of the primary acceptor responsible for these two new components, is discussed in terms of the available information. The primary acceptor may be an iron-sulfur protein, but not of the type characteristic of the bound or water-soluble ferredoxins found so far in chloroplasts.     

Evidence that the intermediate electron acceptor,A2, in Photosystem I is a bound iron-sulfur protein

Absorption changes accompanying the formation of light-induced P-700⁺ were investigated in a highly enriched Photosystem I preparation where an intermediate electron acceptor preceding P-430 could be detected. In an enriched Photosystem I particle, light-induced reversible absorption changes observed at 700 nm in the presence of dithionite resembled those previously seen at 703 nm and 820 nm [9], thus indicating the presence of a backreaction between P-700⁺ and A^?₂. After this same Photosystem I particle was treated to denature the bound iron-sulfur centers, the photochemical changes that could be attributed to P-700 A₂ were completely lost. These results provide evidence that the intermediate electron acceptor, A₂, is a bound iron-sulfur protein. Additional studies in the 400–500 nm region with Photosystem I particles prepared by sonication indicate that the spectrum of A₂ is different from that of P-430.     

Orientation of Photosystem-I pigments: low temperature linear dichroism spectroscopy of a highly-enriched P700 particle isolated from spinach

The linear dichroism of Photosystem I particles containing 10 chlorophylls per P700 has been investigated at 10 K. The particles were oriented by uniaxial squeezing of polyacrylamide gels. The oxidation state of P700 was altered either by incubation of the gels with redox mediators or by low temperature illumination. The Q_Y transitions of the primary electron donor P700, of the remaining unoxidized chlorophyll in P700⁺ and of a chlorophyll molecule absorbing at 686 nm, which presumably corresponds to the primary electron acceptor A₀, are all preferentially oriented perpendicular to the gel squeezing direction. The Q_Y transition of the chlorophyll forms absorbing at 670 and 675 nm appear tilted at 40 ± 5° from this orientation axis. This orientation of the various chlorophylls is compared to that previously reported for more native Photosystem I particles.Abbreviations PSI Photosystem I - P700 primary electron donor of PSI - A₀ primary electron acceptor of PSI     

Radical pair interactions in spinach chloroplasts

Time-resolved EPR studies were done on broken spinach chloroplasts under reducing conditions at low temperature (10 K). A dramatic dependence of signal dynamics and lineshape in the g 2.00 region on the reduction state of Photosystem I is demonstrated. Computer simulations of the spin-polarized lineshapes obtained in this work suggest that the primary electron acceptor in Photosystem I, a species known as A₁, could be a chlorophyll a dimer.     

Primary photochemistry of Photosystem I in chloroplasts. Dynamics of reversible charge separation in open reaction centers at 25 K

The reduction rate of oxidized reaction center chlorophyll of Photosystem I after laser-flash excitation at 25 K has been determined for D-144 subchloroplast fragments and chloroplasts. A maximum of 40% of Photosystem I reaction centers undergo irreversible charge separation (P-700, Cluster A: P-700⁺, Cluster A^?) at 25 K, a percentage which is independent of laser-flash intensity. The remaining reaction centers in chloroplasts and D-144 fragments undergo reversible charge separation with biphasic recombination. Similar amplitudes and time constants (chloroplasts, 49 μs (61%); D-144 fragments, 90 μs (67%)) were obtained for the fast component, while the slower component differed considerably in time (chloroplasts, 2.9 ms; D-144 fragments, 170 ms). It is known that Fe-S Cluster A is photoreduced in less than 1 ms at 25 K. Data obtained support a model for Photosystem I involving a single intermediate in the decay path between the reduced primary electron acceptor (A^?₁) and P-700⁺ and a second intermediate in the decay path between a reduced secondary electron acceptor and P-700⁺. Dual laser-flash experiments to determine rate constants for these processes are included.     

Multiple pathways of charge recombination revealed by the temperature dependence of electron transfer kinetics in cyanobacterial photosystem I

The kinetics of charge recombination in Photosystem I P₇₀₀-F_A/F_B complexes and P₇₀₀-F_X cores lacking the terminal iron?sulfur clusters were studied over a temperatures range of 310 K to 4.2 K. Analysis of the charge recombination kinetics in this temperature range allowed the assignment of backward electron transfer from the different electron acceptors to P₇₀₀⁺. The kinetic and thermodynamic parameters of these recombination reactions were determined. The kinetics of all electron transfer reactions were activation-less below 170 K, the glass transition temperature of the water-glycerol solution. Above this temperature, recombination from [F_A/F_B]^? in P₇₀₀-F_A/F_B complexes was found to proceed along two pathways with different activation energies (E_a). The charge recombination via A_1A has an E_a of ~290 meV and is dominant at temperatures above ~280 K, whereas the direct recombination from F_X^? has an E_a of 22 meV and is prevalent in the 200 K to 270 K temperature range. Charge recombination from the F_X cluster becomes highly heterogeneous at temperatures below 200 K. The conformational mobility of Photosystem I was studied by molecular dynamics simulations. The F_X cluster was found to ‘swing’ by ~30° along the axis between the two sulfur atoms proximal to F_A/F_B. The partial rotation of F_X is accompanied by significant changes of electric potential within the iron?sulfur cluster, which may induce preferential electron localization at different atoms of the F_X cluster. These effects may account for the partial arrest of forward electron transfer and for the heterogeneity of charge recombination observed at the glass transition temperature.     

Bidirectional electron transfer in photosystem I: Replacement of the symmetry-breaking tryptophan close to the PsaB-bound phylloquinone (A1B) with a glycine residue alters the redox properties of A1B and blocks forward electron transfer at cryogenic temperatures

A conserved tryptophan residue located between the A_1B and F_X redox centres on the PsaB side of the Photosystem I reaction centre has been mutated to a glycine in Chlamydomonas reinhardtii, thereby matching the conserved residue found in the equivalent position on the PsaA side. This mutant (PsaB:W669G) was studied using EPR spectroscopy with a view to understanding the molecular basis of the reported kinetic differences in forward electron transfer from the A_1A and the A_1B phyllo(semi)quinones. The kinetics of A₁⁻ reoxidation due to forward electron transfer or charge recombination were measured by electron spin echo spectroscopy at 265 K and 100 K, respectively. At 265 K, the reoxidation kinetics are considerably lengthened in the mutant in comparison to the wild-type. Under conditions in which F_X is initially oxidised the kinetics of charge recombination at 100 K are found to be biphasic in the mutant while they are substantially monophasic in the wild-type. Pre-reduction of F_X leads to biphasic kinetics in the wild-type, but does not alter the already biphasic kinetic properties of the PsaB:W669G mutant. Reduction of the [4Fe-4S] clusters F_A and F_B by illumination at 15 K is suppressed in the mutant. The results provide further support for the bi-directional model of electron transfer in Photosystem I of C. reinhardtii, and indicate that the replacement of the tryptophan residue with glycine mainly affects the redox properties of the PsaB bound phylloquinone A_1B.     

Photosystem I charge separation in the absence of center A and B. III. Biochemical characterization of a reaction center particle containing P-700 and FX

The Photosystem I reaction center is a membrane-bound, multiprotein complex containing a primary electron donor (P-700), a primary electron acceptor (A₀), an intermediate electron acceptor (A₁) and three membrane-bound iron-sulfur centers (F_X, F_B, and F_A). We reported in part I of this series (Golbeck, J.H. and Cornelius, J.M. (1986) Biochim. Biophys. Acta 849, 16–24) that in the presence of 1% lithium dodecyl sulfate (LDS), the reaction center becomes dissociated, resulting in charge separation and recombination between P-700 and F_X without the need for prereduction of F_A and F_B. In this paper, we report (i) the LDS-induced onset of the 1.2-ms ‘fast’ phase of the P-700 absorption transient is time-dependent, attaining a maximum 3:1 ratio of ‘fast’ to ‘slow’ kinetic phases; (ii) the ‘fast’ kinetic phase, corresponding to the P-700⁺ F_X⁻ backreaction, is stabilized indefinitely by dilution of the LDS-treated particle followed by ultrafiltration over a YM-100 membrane; (iii) without stabilization, the P-700⁺ F_X⁻ reaction deteriorates, leading to the rise of the long-lived P-700 triplet formed from the P-700⁺A_O⁻ backreaction; (iv) the ‘slow’ kinetic phase correlates with the redox and ESR properties of F_A and/or F_B, which indicates that in a minority of particles the terminal iron-sulfur protein remains attached to the reaction center core; (v) the ultrafiltered reaction center is severely deficient in all of the low molecular-weight polypeptides, particularly the 19-kDa, 18-kDa and 12-kDa polypeptides relative to the 64-kDa polypeptide(s); (vi) the stabilized particle contains 5.8 mol labile sulfide per mol photoactive P-700, reflecting largely the iron-sulfur content of F_x, but also residual F_A and F_B, on the reaction center; and (vii) the apoproteins of F_A and F_B are physically removed from the reaction center particle as indicated by the presence of protein-bound zero-valence sulfur in the YM-100 filtrate. These results are interpreted in terms of a model for Photosystem I in which F_A and F_B are located on a low-molecular-weight polypeptide and F_X is depicted as a [2Fe-2S] cluster shared between the two high-molecular-weight polypeptides Photosystem I-A1 and Photosystem I-A2.     

Effect of temperature on the photoreduction of centres A and B in Photosystem I,and the kinetics of recombination

When spinach Photosystem I particles, frozen in the dark with ascorbate, are illuminated at low temperatures, one electron is transferred from P-700 to either iron-sulphur centre A or B. It was found that the proportion of centre A or B reduced depended on the temperature of illumination. At 25 K, reduction of centre A, as detected by ESR spectroscopy, was strongly preferred. At higher temperatures, at about 150K, there was an increased proportion of reduced centre B. Reduction of B was more strongly preferred in particles frozen in 50% glycerol. The kinetics of dark reoxidation of A^? and B^? at various temperatures were followed by observing the radical signal of P-700⁺, and also by periodically cooling to 25 K to measure the ESR spectra of the iron-sulphur centres. The recombination of A^? and P-700⁺ occurred at lower temperatures than that at of B^?; at 150–200 K, centre B was the more stable electron trap. Dark reoxidation of both centres was more rapid in samples that were illuminated at 25 K than in samples illuminated at 150–215 K. In no case was net electron transfer between centres A and B observed. Differences in g values of the ESR spectra in particles illuminated at 25 and 200 K indicate that the iron-sulphur centres are in altered conformational states. It is concluded firstly that, in the frozen state, the rates of dark electron transfer decrease in the sequence A^? → P-700⁺ > B^? → P-700⁺ > B^? → A; secondly, that when centres A or B are photoreduced, a temperature-dependent conformational change takes place which slows down the rate of recombination with P-700⁺.     

Transmembrane distribution of phospholipids and their involvement in electron transport,as revealed by phospholipase A2 treatment of spinach thylakoids

Thylakoid membranes were treated with either pancreatic or snake venom phospholipase A₂, and the residual phospholipid content of these membranes was determined and compared to the rates of Photosystem II and/or Photosystem I electron transports. The hydrolysis curves of both phosphatidylglycerol and phosphatidylcholine displayed a first, rapid phase which was almost temperature-insensitive, followed by a second, slower phase which depended strongly on the temperature. When pancreatic phospholipase A₂ had access either to the outer face or to both faces of the thylakoid membrane, either only part of or all the phospholipids, respectively, could be hydrolysed. These results were interpreted as indicating an asymmetric distribution of phospholipids across the thylakoid membrane, phosphatidylglycerol and phosphatidylcholine being preferentially located in the outer and the inner layer, respectively. When acting on uncoupled thylakoid membranes, phospholipase A₂ exerted an inhibitory effect on Photosystem II activity and a stimulatory effect on Photosystem I activity. The involvement of phosphatidylcholine and of phosphatidylglycerol in electron transport activities of Photosystem II and of Photosystem I are discussed with special reference to the role of the external and internal pools of these phospholipids.     

Flash photolysis electron spin resonance studies of the electron acceptor species at low temperatures in photosystem I of spinach subchloroplast particles.

The light-induced electron spin resonance signals of Photosystem I spinach subchloroplast particles have been studied at approximately 6 degrees K. Using the technique of flash photolysis-electron spin resonance with actinic illumination at 647 nm, a kinetic analysis of the previously observed bound ferredoxin ESR signals was carried out. Signal I (P700+) exhibits a partial light-reversible behavior at 6 degrees K so it was expected that if the bound ferredoxin is the primary acceptor of Photosystem I, it should also exhibit a partial reversible behavior. However, none of the bound ferredoxin ESR signals showed any such light reversible behavior. A search to wider fields revealed two components which did exhibit the expected kinetic behavior. These components are very broad (about 80 G) and are centered at g equals to 1.75 and g equals to 2.07. These two components exhibit the expected characteristics of the primary electron acceptor. A model is presented to account for the reversible and irreversible photochemical changes in Photosystem I. The possible identity of the primary acceptor responsible for these two new components, is discussed in terms of the available information. The primary acceptor may be an iron-sulfur protein, but not of the type characteristic of the bound or water-soluble ferredoxins found so far in chloroplasts.     

Extraction of electron acceptor A1 from pea photosystem I

Photosystem I particles were washed twice in either 0, 50 or 100% water-saturated diethyl ether. It was found that the characteristic electron spin resonance signal associated with electron acceptor A₁ was progressively lost with increasing percentage saturation of the ether. Light-induced electron flow to the terminal iron-sulphur acceptors was inhibited although these acceptors were still present and could be chemically reduced. The kinetics of optical measurements of P700⁺ re-reduction at 820 nm following ether washing were consistent with removal of electron acceptor A₁.     

A study of chemically induced dynamic electron polarization (CIDEP) in Photosystem I of whole algal cells at ambient temperatures

Time-resolved electron paramagnetic resonance (EPR) studies were carried out at room temperature and at 273 K on whole-cell samples of the photosynthetic algae: Anacystis nidulans and Scenedesmus obliquus, the latter being 97% deuterated from the growing medium. These photosynthetic organisms show greatly enhanced EPR signals which result from the generation of nonequilibrium spin populations, a phenomenon known as chemically induced dynamic electron polarization (CIDEP). We report magnetic-field profiles of the early transient signals of Photosystem I which are very similar to those observed at low temperatures. The results suggest that one or more early reduced electron acceptors in Photosystem I are being observed at ambient physiological temperatures.     

An FTIR study on the structure of the oxygen-evolving Mn-cluster of Photosystem II in different spin forms of the S2 state

The S₂ state of the oxygen-evolving Mn-cluster of Photosystem II (PS II) is known to have different forms that exhibit the g =2 multiline and g = 4.1 EPR signals. These two spin forms are interconvertible at > 200 K and the relative amplitudes of the two signals are dependent on the species of cryoprotectant and alcohol contained in the medium. Also, it was recently found that the mutiline form can be converted to the g = 4.1 form by absorption of near-infrared light by the Mn-cluster itself at around 150 K [Boussac et al. (1996) Biochemistry 35: 6984–6989]. We have used light-induced Fourier transform infrared (FTIR) difference spectroscopy to study the structural difference in these two S₂ forms. FTIR difference spectra for S₂/S₁ as well as for S₂Q_A ^-/S₁Q_A measured at cryogenic temperatures using PS II membranes in the presence of various cryoprotectants, and monohydric alcohols did not show any specific differences except for intensities of amide I bands, which were larger when ethylene glycol or glycerol was present in addition to sucrose. This result was interpreted due to more flexible movement of the protein backbones upon S₂ formation with a higher cryoprotectant content. Light-induced difference spectra measured at 150 K using either blue light without near-infrared light or red plus near-infrared light also did not show any detectable difference. In addition, a different spectrum upon near-infrared illumination at 150 K of the PS II sample in which the S₂ state had been photogenerated at 200 K exhibited no meaningful signals. These results indicate that the two S₂ forms that give rise to the multiline and g = 4.1 signals have only minor differences, if any, in the structures of amino-acid ligands and polypeptide backbones. This conclusion suggests that conversion between the two spin states is caused by a spin-state transition in the Mn(III) ion rather than valence swapping within the Mn-cluster that would considerably affect the vibrations of ligands.This revised version was published online in October 2005 with corrections to the Cover Date.     

Simultaneous detection of spin-coupled and decoupled QA– EPR-signals in Photosystem II complexes isolated with isoelectric focusing

The Photosystem II multisubunit protein complex can be extracted from thylakoid membranes with non-ionic detergents and subjected to various spectroscopical and biochemical investigations. This paper shows that after extraction with dodecyl--D-maltoside, several Photosystem II complexes could be resolved by isoelectric focusing. Structurally, the various Photosystem II complexes differed from each other in polypeptide composition, especially with regard to the chlorophyll a/b-binding proteins, which gave rise to differing isoelectric points. Functionally, the various Photosystem II complexes differed from each other on the acceptor side, as judged by acceptor side-dependent electron transfer and electron paramagnetic resonance (EPR). The Q_A ^- Fe2+-signal (g = 1.84), arising from Q_A ^- spin-coupled to the acceptor-side iron, and a radical signal arising from decoupled Q_A ^- (g = 2.0045) could be detected simultaneously in some of the Photosystem II complexes, and the amount of each of the two signals were inversely related. The results are discussed in relation to previously known heterogeneities in Photosystem II.     

Light-induced oxidation-reduction reactions in a cell-free preparation from the blue-green alga Nostoc muscorum: The role of cytochrome f, cytochrome b558, C550, and P700 in noncyclic electron transport

1. Cytochrome f (λ_max = 554 nm, E_m = +0.35 V) and cytochrome b₅₅₈ (λ_max = 558 nm, E_m = +0.35 V) were photooxidized by Photosystem I and photoreduced by Photosystem II in a cell-free preparation from the blue-green alga Nostoc muscorum. The steady-state oxidation levels of both cytochromes were affected by noncyclic electron acceptors and by inhibitors of noncyclic electron transport. These results are consistent with the hypothesis that the mechanism of NADP reduction by water involves a Photosystem II and a Photosystem I light reaction operating in series and linked by a chain of electron carriers that includes cytochrome f and cytochrome b₅₅₈.2. Phosphorylation cofactors shifted the steady-state of cytochrome f to a more reduced level under conditions of noncyclic electron transport but had no effect on cytochrome b₅₅₈. These observations suggest that the noncyclic phosphorylation site lies before cytochrome f (on the Photosystem II side) and that cytochrome f is closer to this site than is cytochrome b₅₅₈.3. A Photosystem II photoreduction of C550 at 77 °K was observed, suggesting that in blue-green algae, as in other plants, C550 is closely associated with the primary electron acceptor for Photosystem II. A Photosystem I photooxidation of P700 at 77 °K was observed, consistent with P700 serving as the primary electron donor of Photosystem I.     

Chlorophyll composition of Photosystems IIα, IIβ and I in tobacco chloroplasts

The antenna composition of the Photosystems II_α, II_β and I was studied in tobacco chloroplasts. Absorbance spectra, recorded at 4 K, were analyzed for the wild type and the mutants Su/su and Su/su var. Aurea, containing higher concentrations of the photosystems. With chloroplasts of Su/su we measured the action spectra of the three photosystems from 625 to 690 nm. Above 675 nm absorption by Photosystem I dominated. This sytem had a maximum at 678 nm and a shoulder at 660 nm. Of the long-wavelength chlorophyll a forms, absorbing at 690, 697 and 705 nm at 4 K, which are generally assigned to Photosystem I, the 697 nm form occurred in an amount of four molecules per reaction center of Photosystem I in each type of chloroplast. The Photosystem II_α spectrum was characterized by maxima at 650 and 672 nm, showing clearly the participation of the chlorophyll a and b containing light-harvesting complex. In the mutants the light-harvesting complex has a chlorophyll a to chlorophyll b ratio of more than 1; the amount of the 672 nm chlorophyll a was normal, whereas the amount of chlorophyll b was markedly decreased in the mutants relative to the wild type. The Photosystem II_β spectrum mainly consisted of a band at 683 nm.