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.     

The effect of temperature on the formation and decay of the multiline EPR signal species associated with photosynthetic oxygen evolution

We have investigated the effects of temperature on the formation and decay of the light-induced multiline EPR signal species associated with photosynthetic oxygen evolution (Dismukes, G.C. and Siderer, Y. (1980) FEBS Lett. 121, 78–80). (1) The decay rate following illumination is temperature dependent: at 295 K the half-time of decay is about 40 s, at 253 K the half-time is approx. 40 min. (2) A single intense flash of light becomes progressively less effective in generating the multiline signal below about 240 K. (3) Continuous illumination is capable of generating the signal down to almost 160 K. (4) Continuous illumination after a preilluminating flash generates less signal above 200 K than at lower temperatures. Our results support the conclusion of Dismukes and Siderer that the S₂ state gives rise to this multiline signal; we find that the S₁ state can be fully advanced to the S₂ state at temperatures as low as 160 K. The S₂ state is capable of further advancement at temperatures above about 210 K, but not below that temperature.     

Partial disintegration and reconstitution of the photosynthetic oxygen evolution system. Binding of 24 kilodalton and 18 kilodalton polypeptides

Treatment with 1 M NaCl almost totally removed two polypeptides of 24 and 18 kDa from the Photosystem II particles of spinach chloroplasts and reduced the oxygen-evolution activity by about half. Both polypeptides were able to rebind to the NaCl-treated particles in a low-salt medium. The rebinding of the 24 kDa polypeptide showed a saturation curve whose maximum level was close to that naturally occurring in the untreated particles. In parallel with the amount of rebound 24 kDa polypeptide, the oxygen-evolution activity was recovered. The 18 kDa polypeptide bound to the NaCl-treated particles without saturation. When the 18 kDa polypeptide was added to the particles previously treated with NaCl and then supplemented with a saturating amount of 24 kDa polypeptide, there appeared, in addition to the binding without saturation, another binding of the 18 kDa polypeptide with saturation to a maximum level close to that naturally occurring in the untreated particles. The 18 kDa polypeptide did not restore the oxygen-evolution activity. These findings suggest that there are specific binding sites; one for the 24 kDa polypeptide located on the Photosystem II particles, and the other for the 18 kDa polypeptide on the 24 kDa polypeptide.     

An improved purification method and a further characterization of the 33-kilodalton protein of spinach chloroplasts

The 33-kDa protein was purified in a high yield from thylakoid membranes of spinach chloroplasts. The extinction coefficient and A^1%_1cm value at 276 nm of the protein were 22000 M^?1·cm^?1 and 6.8, respectively. The 33-kDa protein and a polypeptide appearing at 32 kDa in the SDS-polyacrylamide gel electrophoresis of thylakoid membranes were compared by peptide mapping after limited proteolysis. This indicates that the 32-kDa band is entirely due to the 33-kDa protein. The molar ratio of chlorophyll to the 33-kDa protein in the chloroplasts was estimated to be 300. This suggests that one photosynthetic unit possesses one or two molecules of the 33-kDa protein.     

Kinetics of EPR signal IIvf in chloroplast Photosystem II

The risetime of EPR signal II_vf (S II_vf) has been measured in oxygen-evolving Photosystem II particles from spinach chloroplasts at pH 6.0. The EPR signal shows an instrument-limited rise upon induction ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{? 3 μ}\text{s}$). These data are consistent with a model where the species Z responsible for S II_vf is the immediate electron donor to P-680⁺ in spinach chloroplasts. A new, faster decay component of S II_vf has also been detected in these experiments.     

CIDEP observations in photosystem I of green plant and algal photosynthesis.

Flash photolysis experiments with electron paramagnetic resonance detection were carried out between 10 K and 300 K on samples of green plant and algal species. Chemically induced dynamic electron polarization was evident for the signals observed in the g = 2.0 region for 100 KHz modulated detection and also for a system with no magnetic field modulation. The light reversible signals decaying in about 1 ms at low temperatures are interpreted as arising from photosystem I of the green plant and algal samples. Evidence is presented which indicates that the origin of the electron spin polarization is the well established radical-pair mechanism.     

The effects of mono- and divalent salts on the O2-evolution activity and low temperature multiline EPR spectrum of Photosystem II preparations from spinach

The ability of salts to inhibit the O₂-evolution activity of PS II preparations is shown to parallel closely the Hofmeister series, suggesting that inhibition is related to the solubility of the 16, 24 and 33 kDa proteins in these salt solutions. An examination of the effect of salt inactivation on the low temperature multiline EPR signal indicates that the release of either the 16 and 24 kDa proteins, or additionally the 33 kDa protein blocks or greatly reduces the efficiency of the advancement of the water-splitting complex to the S₂-state; under some conditions, this inhibition is reversible.     

Influence of temperature on Photosystem II electron transfer reactions

The influence of temperature on the rate of reduction of P-680⁺, the primary donor of Photosystem II, has been studied in the range 5–294 K, in chloroplasts and subchloroplasts particles. P-680 was oxidized by a short laser flash. Its oxidation state was followed by the absorption level at 820 nm, and its reduction attributed to two mechanisms: electron donation from electron donor D₁ and electron return from the primary plastoquinone (back-reaction).Between 294 and approx. 200 K, the rate of the back-reaction, on a logarithmic scale, is a linear function of the reciprocal of the absolute temperature, corresponding to an activation energy between 3.3 and 3.7 kcal · mol^?1, in all of the materials examined (chloroplasts treated at low pH or with Tris; particles prepared with digitonin). Between approx. 200 K and 5 K the rate of the back-reaction is temperature independent, with $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 1.6}\text{ms}$. In untreated chloroplasts we measured a $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of 1.7 ms for the back-reaction at 77 and 5 K.The rate of electron donation from the donor D₁ has been measured in darkadapted Tris-treated chloroplasts, in the range 294–260 K. This rate is strongly affected by temperature. An activation energy of 11 kcal · mol^?1 was determined for this reaction.In subchloroplast particles prepared with Triton X-100 the signals due to P-680 were contaminated by absorption changes due to the triplet state of chlorophyll a. This triplet state has been examined with pure chlorophyll a in Triton X-100. An Arrhenius plot of its rate of decay shows a temperature-dependent region (292–220 K) with an activation energy of 9 kcal · mol^?1, and a temperature-independent region (below 200 K) with $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 1.1}\text{ms}$.     

Calcium ions can be substituted for the 24-kDa polypeptide in photosynthetic oxygen evolution

Photosystem II particles were prepared from spinach chloroplasts with Triton X-100, and treated with 1.0 M NaCl to remove polypeptides of 24 kDa and 18 kDa and to reduce the photosynthetic oxygen-evolution activity by about half. Oxygen-evolution activity was restored almost to the original level with 10 mM Ca²⁺, in a similar manner to the rebinding of 24-kDa polypeptide. Other cations such as magnesium, sodium and manganese ions could not restore any oxygen-evolution activity. These observations, together with a kinetic analysis, suggest that Ca²⁺ can be substituted for the 24-kDa polypeptide in photosynthetic oxygen evolution in Photosystem II particles.     

Evidence that the low-potential (−700 mV) electron acceptor (X) in Photosystem I has two iron-sulphur centres

The Photosystem I reaction centre contains two groups of iron-sulphur centres: Fe-S_A and Fe-S_B with redox potentials between ?510 and ?590 mV, and Fe-S_X with redox potential about ?700 mV. Spin quantitation (Heathcote, P., Williams-Smith, D.L. and Evans, M.C.W. (1978) Biochem. J. 170, 373–378) and Mössbauer spectroscopy (Evans, E.H., Dickson, D.P.E., Johnson, C.E., Rush, J.D. and Evans, M.C.W. (1981) Eur. J. Biochem. 118, 81–84) did not show unequivocally whether Fe-S_X has one or two centres. Experiments are described which support the proposal that Fe-S_X has two centres. Fe-S_X can be photoreduced irreversibly by 210 K illumination of dithionite-reduced samples or reversibly by 7.5 K illumination of these samples. The amplitude of the Fe-S_X signal reversibly induced by illumination at 7.5 K is never more than 50% of the amplitude of the signal when Fe-S_X is prereduced by room temperature illumination or by 210 K illumination. Approx. half of the Fe-S_X is rapidly reduced by 210 K illumination, the remainder more slowly. The extent of reversible Fe-S_X reduction and P-700 photooxidation is little affected by the fast reduction of about half of the Fe-S_X. Subsequent reduction of the remaining Fe-S_X is paralleled by loss of the reversible photoreaction.     

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.     

Partial reconstitution of the photosynthetic oxygen evolution system by rebinding of the 33-kDa polypeptide

Treatment with 2.6 M urea of the Photosystem II particles depleted of two polypeptides of 24 kDa and 18 kDa completely released a polypeptide of 33 kDa and eliminated the oxygen-evolution activity. The 33-kDa polypeptide rebound to the urea-treated particles and partially reactivated the oxygen evolution. A quantitative analysis of the rebinding suggests tha there is a specific binding site for the 33-kDa polypeptide on the membrane surface.     

Recognition of interaction between the donor electron-transfer chains of Photosystem II under conditions of partial inhibition of oxygen evolution

The electron-transfer pathway on the donor side of Photosystem (PS) II has been examined using unfractionated and inside-out thylakoid membrane vesicles. A number of treatments are identified which result in the inhibition of light-dependent oxygen evolution. The differential capacities of the exogenous donors diphenylcarbazide and NH₂OH to restore the PS II-mediated reduction of 2,6-dichlorophenolindophenol (DCIP) in the inhibited membranes is discussed in terms of multiple donor sites for the electron-transfer pathway on the oxidising side of PS II. We also present data which indicate that the donor chains are not isolated from each other but that an individual PS II reaction centre may be able to interact with several oxygen-evolving complexes. The implication of such an interaction to the mechanism of oxygen evolution is discussed.     

The state of manganese in the photosynthetic apparatus. 3. Light-induced changes in X-ray absorption (K-edge) energies of manganese in photosynthetic membranes

Photosynthetic water oxidation by higher plants proceeds as though five intermediates, S₀-S₄, operate in a cyclic fashion. In this study of the manganese involvement in the process, a low temperature EPR signal is used as an indicator of S-state composition for manganese X-ray absorption K-edge measurements of a spinach Photosystem II preparation. A dramatic change is observed in the edge properties between samples prepared in states S₁ and either S₂ or S₃, establishing a direct relation between the local environment of Mn and the S-state composition. Samples in S₂ or S₃ exhibit a broadening of the principal absorption peak and a shift to higher energy by as much as 2.5 eV relative to S₁ samples. The magnitude of these changes is directly related to the EPR signal intensity induced by illumination. Models are discussed in which these data may be interpreted in terms of a conformation-induced change in Mn ligation and/or oxidation during the S₁ to S₂ transition.     

Kinetics of manganese redox transitions in the oxygen-evolving apparatus of photosynthesis

The kinetics of the S-state transitions of the oxygen-evolving complex were analyzed in dark-adapted, oxygen-evolving Photosystem-II preparations supplied with the electron acceptor 2,5-dichloro-p-benzoquinone. The kinetics of flash-induced absorbance changes at 350 nm, largely due to the successive S-state transitions S₀ → S₁, S₁ → S₂, S₂ → S₃ and S₃ →; S₀, confirm the +1, +1, +1, ?3 sequence of manganese oxidation reported earlier (Dekker, J.P., Van Gorkom, H.J., Wensink, J. and Ouwehand, L. (1984) Biochim. Biophys. Acta 767, 1–9), and reveal half-times of 30, 110, 350 and 1300 μs, respectively, for these transitions.     

Precursors to microsecond delayed luminescence in oxygenevolving and inhibited chloroplasts

We have investigated submillisecond delayed luminescence in spinach chloroplasts under a variety of conditions. In Tris-washed chloroplasts, which are inhibited on the oxidizing side of P-680, the delayed light emission in the 7–200 μs time-range decayed with biphasic behavior. In fully dark-adapted samples illuminated by a single saturating laser pulse, the fast phase of delayed luminescence followed a nearly identical pH-dependent time-course as that observed optically and by ESR for P⁺-680 reduction, thus verifying the recombination hypothesis for the origin of delayed light. The observed slower phase of delayed luminescence was also pH dependent, but unlike the fast phase, could not be ascribed to specific electron transfer events of PS II. This phase could be rationalized by a heterogeneity in the population of P-680. While kinetic parameters were found to be insensitive to changes in ionic strength, the overall luminescence intensity was quite sensitive to the electrical parameters, thus indicating the role of ionic strength and local charges in delayed luminescence modulation. A similar series of experiments was performed on untreated chloroplasts. The pH-dependent delayed luminescence behavior in both untreated chloroplasts and Tris-washed chloroplasts was similar despite significantly faster kinetics associated with the reduction of P⁺-680 by the secondary PS II electron donor, Z, in the former preparation (e.g., Van Best, J.A. and Mathis, P. (1978) Biochim. Biophys. Acta 503, 178–188). Thus, it was concluded that, in untreated samples, microsecond delayed luminescence emanates primarily from centers which are not competent in oxygen evolution. The nearly identical delayed luminescence intensity in untreated chloroplasts and in Tris-washed chloroplasts was rationalized by a model which predicts modulations in delayed luminescence yield by the exciton-quenching effect of P⁺-680. Computer simulations demonstrate the feasibility of this model. The previously documented flash oscillations in microsecond delayed luminescence intensity in untreated chloroplasts (Bowes, J.M. and Crofts, A.R. (1979) Biochim. Biophys. Acta 547, 336–346), which we readily observed, were attributed to alterations in delayed luminescence yield (in nonfunctional centers) by variations in charge density stored at the oxygen-evolving complex of functional centers. Taken together, our results emphasize the dependence of delayed luminescence kinetics upon electron-transfer kinetics and the dependence of delayed luminescence amplitude upon the photochemical parameters, the exciton yield and the emission yield.