Electron spin resonance studies of the bound iron-sulfur centers in Photosystem I. Photoreduction of center A occurs in the absence of center B

The Photosystem I acceptor system of a subchloroplast particle from spinach was investigated by optical and electron spin resonance (ESR) spectroscopy following graduated inactivation of the bound iron-sulfur proteins by urea/ferricyanide solution. The chemical analysis of iron and sulfur and the ESR properties of centers A, B and X are consistent with the participation of three iron-sulfur centers in Photosystem I. A differential decrease in centers A, B and X is observed under conditions that induce S^2? →S⁰ conversion in the bound iron-sulfur proteins. Center B is shown to be the most susceptible, while center ‘X’ is the least susceptible component to oxidative denaturation. Stepwise inactivation experiments suggest that electron transport in Photosystem I does not occur sequentially from X→B→A, since there is quantitative photoreduction of center A in the absence of center B. We propose that center A is directly reduced by X; thus, X may serve as a branch point for parallel electron flow through centers A and B.     

Photosystem I photochemistry at low temperature. Heterogeneity in pathways for electron transfer to the secondary acceptors and for recombination processes

Electron transport has been studied by flash absorption and EPR spectroscopies at 10–30 K in Photosystem I particles prepared with digitonin under different redox conditions. In the presence of ascorbate, an irreversible charge separation is progressively induced at 10 K between P-700 and iron-sulfur center A by successive laser flashes, up to a maximum which corresponds to about two-thirds of the reaction centers. In these centers, heterogeneity of the rate for center A reduction is also shown. In the other third of reaction centers, the charge separation is reversible and relaxes with a t_1/2 ≈ 120 μs. When the iron-sulfur centers A and B are prereduced, the 120 μs relaxation becomes the dominant process (70–80% of the reaction centers), while a slow component (t_1/2 = 50–400 ms) reflecting the recombination between P-700⁺ and center X⁻ occurs in a minority of reaction centers (10–15%). Flash absorption and EPR experiments show that the partner of P-700⁺ in the 120 μs recombination is neither X nor a chlorophyll but more probably the acceptor A⁻₁ as defined by Bonnerjea and Evans (Bonnerjea, J. and Evans, M.C.W. (1982) FEBS Lett. 148, 313–316). The role of center X in low-temperature electron flow is also discussed.     

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.     

Regulation of Photosystem I electron flow activity by phosphatidylglycerol in thylakoid membranes as revealed by phospholipase treatment

Thylakoid membranes were treated by potato lipolytic acyl hydrolase, phospholipases A₂ from pancreas and snake venom, and by phospholipase C from Bacillus cereus under various conditions. The changes in the uncoupled rates of electron transport through Photosystem I (PS I) and in lipid composition were followed during these treatments. Pancreatic phospholipase A₂ which destroyed all phospholipids in thylakoid membranes stimulated the NADP⁺ reduction supported by reduced 2,6-dichlorophenolindophenol. This stimulation concerned only the dark but not the light reactions of this pathway. The main site of action of pancreatic phospholipase A₂ may be located on the donor side of PS I; the hydrolysis of phospholipids at this site caused an increased ability of reduced 2,6-dichlorophenolindophenol and ascorbate alone to feed electrons into PS I. A second site may be located on the acceptor side of PS I, probably between the primary acceptor and the ferredoxin system. When thylakoid membranes were first preincubated with or without lipolytic acyl hydrolase at 30°C (pH 8), the NADP⁺ photoreduction was inhibited whilst the methyl viologen-mediated O₂ uptake was stimulated. A subsequent addition of pancreatic phospholipase A₂ (which had the same hydrolysis rates for phosphatidylglycerol but not for phosphatidylcholine) further stimulated the O₂ uptake and restored NADP⁺ photoreduction. The extent of this stimulation, which depended on the presence of lipolytic acyl hydrolase, was ascribed partly to the hydrolysis of the phospholipids and partly to the generation of their lyso derivatives but not to the release of free fatty acids. On the contrary, phospholipase C which destroyed only phosphatidylcholine failed to restore this activity. It is suggested that phosphatidylglycerol is the only phospholipid associated with thylakoid membrane structures supporting PS I activities and that this lipid may play a physiological role in the regulation of these activities.     

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.     

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.     

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.     

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.     

Energy transfer and quantum yield in Photosystem II

The photoreduction and dark reoxidation of Q_α and Q_β, the primary electron acceptors of Photosystems (PS) IIα and IIβ, respectively, in the presence of 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea (DCMU) were studied in tobacco chloroplasts by means of fluorescence and absorbance measurements. The magnitude of a correction for an absorbance change by the oxidizing side of PS II needed in our previous study of the quantum yield of Q reduction (Biochim. Biophys. Acta 635 (1981), 111–120) has been determined. The absorbance change occurs in PS IIα mainly. The maximum fluorescence yield was found to be the same as in the mutant Su/su, which has a 3-fold higher reaction center concentration and a lower PS IIα to PS IIβ ratio. The kinetics of the light-induced fluorescence increase were measured after various pretreatments and the corresponding kinetics of the integrated fluorescence deficit were analyzed into their α and β components. From the results the contribution to the minimum fluorescence level, the degree of energy transfer between units, and the quantum efficiency of Q reduction were calculated for both types of PS II. This led to the following conclusions. The absence of energy between PS IIβ antennae is confirmed. Fluorescence quenching in PS IIα was adequately described by the matrix model, except for a decrease in the energy transfer between units during photoreduction of Q_α, possibly due to the formation of ‘islets’ of closed centers. PS II reaction centers in which Q is reduced do not significantly quench fluorescence. The ratio of variable to maximum fluorescence, 0.77 in PS IIα and 0.92 in PS IIβ, multiplied by the fraction of Q remaining in the reduced state after one saturating flash, 0.88 in PS IIα and greater than 0.95 in PS IIβ, leads to a net quantum efficiency of Q reduction in the presence of DCMU and NH₂OH of 0.68 in PS IIα and about 0.90 in PS IIβ. These values are in good agreement with the measured overall quantum efficiency of Q reduction.     

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.     

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.     

The detection,isolation and characterization of a light-harvesting complex which is specifically associated with Photosystem I

10% of the chlorophyll associated with a ‘native’ Photosystem (PS) I complex (110 chlorophylls/P-700) is chlorophyll (Chl) b. The Chl b is associated with a specific PS I antenna complex which we designate as LHC-I (i.e., a light-harvesting complex serving PS I). When the native PS I complex is degraded to the core complex by LHC-I extraction, there is a parallel loss of Chl b, fluorescence at 735 nm, together with 647 and 686 nm circular dichroism spectral properties, as well as a group of polypeptides of 24-19 kDa. In this paper we present a method by which the LHC-I complex can be dissociated from the native PS I. The isolated LHC-I contains significant amounts of Chl b (Chl $\text{a}\text{b ? 3.7}$). The long-wavelength fluorescence at 730 nm and circular dichroism signal at 686 nm observed in native PS I are maintained in this isolated complex. This isolated fraction also contains the low molecular weight polypeptides lost in the preparation of PS I core complex. We conclude that we have isolated the PS I antenna in an intact state and discuss its in vivo function.     

Potentiometric titration of Photosystem II fluorescence decay kinetics in spinach chloroplasts

The fluorescence yield of chloroplasts reflects the redox state of the electron acceptor of the Photosystem II reaction center, with increasing yield as the acceptor is reduced. Chemical reductive titrations of fluorescence yield in chloroplasts at room temperature indicate two distinct midpoint potentials, suggesting the possibility of Photosystem II electron acceptor heterogeneity. We have carried out a potentiometric titration of the fluorescence decay kinetics in spinach chloroplasts using a continuous mode-locked dye laser with low-intensity excitation pulses and a picosecond-resolution single-photon timing system. At all potentials the fluorescence decay is best described by three exponential components. As the potential is lowered, the slow phase changes 30-fold in yield with two distinct midpoint potentials, accompanied by a modest (3-fold) increase in the lifetime. The titration curve for the slow component of the fluorescence decay of spinach chloroplasts is best characterized by two single-electron redox reactions with midpoint potentials at pH 8.0 of +119 and ?350 mV, with corresponding relative contributions to the fluorescence yield of 49 and 51%, respectively. There is little change in the fast and middle components of the fluorescence decay. We found that the oxidized form of the redox mediator 2-hydroxy-1,4-naphthoquinone preferentially quenches the fluorescence, causing an anomalous decrease in the apparent midpoint of the high-potential transition. This effect accounts for a significant difference between the midpoint potentials that we observe and some of those previously reported. The selective effect of reduction potentials on particular fluorescence decay components provides useful information about the organization and distribution of the Photosystem II electron acceptor.     

Reactions of hydrogen and oxygen with Photosystem I of isolated heterocysts from Anabaena variabilis (ATCC 29413)

Isolated heterocysts from Anabaena variabilis show high rates of light- and hydrogen-dependent acetylene reduction. This heterocyst preparation also shows light-induced redox reactions of cytochromes f-556 and b-564. Both cytochromes are reversibly oxidized by light or by oxygen (in the dark). Oxygen-oxidized cytochromes assume their initially reduced state by addition of dithionite or flushing the reaction vessel with hydrogen or argon. All three agents remove oxygen from solutions more or less efficiently, thereby creating reducing conditions; hydrogen-induced reduction is slow and generally completed after about 10 min. No evidence has been obtained for a direct electron donation of hydrogen to either of the cytochromes measured. Photoreduction of nitrogen in the presence of hydrogen is explained by a combined operation of properly poised cyclic photophosphorylation providing ATP, and a light-independent hydrogenase reaction providing reductant.     

Characteristics of the Photosystem II reaction centre. I. Electron acceptors

The EPR characteristics of Photosystem II electron acceptors are described, in membrane and detergent-treated preparations from a mutant of Chlamydomonas reinhardii lacking Photosystem I and photosynthetic ATPase. The relationship between the quinone-iron and pheophytin acceptors is discussed and a heterogeneity of reaction centres is demonstrated such that only a minority of reaction centres were capable of secondary electron donation at temperatures below 100 K. Only these centres were therefore able to stabilise a reduced acceptor below 100 K. Parallel experiments using a barley mutant (viridis zb63) which also lacks Photosystem I, provide similar results indicating that the C. reinhardii system can provide a general model for the Photosystem II electron acceptor complex. The similarity of the system to that of the purple photosynthetic bacteria is discussed.     

The dependence of reaction center and antenna triplets on the redox state of Photosystem I

The formation of chlorophyll triplet states during illumination of Photosystem I reaction center samples depends upon the redox state of P-700, X and ferredoxin Centers A and B. When the reaction centers are in the states P-700⁺A₁XFd_BFd^?_A and P-700 A₁XFd^?_BFd^?_A prior to illumination, we observe electron paramagnetic resonance (EPR) spectra from a triplet species which has zero-field splitting parameters (|D| and |E|) larger than those of either the chlorophyll a or chlorophyll b monomer triplet, and a polarization which results from population of the triplet spin sublevels by an intersystem crossing mechanism. We interpret this triplet as arising from photoexcited chlorophyll antenna species associated with reaction centers in the states P-700⁺Fd^?_A and P-700⁺X^?, respectively, which undergo de-excitation via intersystem crossing. When the reaction centers are in the states P-700A₁XFd^?_BFd^?_A and P-700A₁X^?Fd^?_BFd^?_A prior to illumination, we observe a triplet EPR signal with a polarization which results from population of the triplet spin sublevels by radical pair recombination, and which has a |D| value similar to that of chlorophyll a monomer. We interpret this triplet (the radical pair-polarized triplet) as arising from ³P-700 which has been populated by the process $\text{P-700}{}^{\mathrm{+}}\text{A}{}^{\mathrm{?}}{}_1\text{→}{}^3\text{P-700}\text{A}{}_1$. We observe both the radical pair-polarized triplet and the chlorophyll antenna triplet when the reaction centers are in the state P-700 A₁XFd^?_BFd^?_A, presumably because the processes P-700⁺A^?₁X → P-700⁺A₁X^? and $\text{P-700}{}^{\mathrm{+}}\text{A}{}^{\mathrm{?}}{}_1\text{X}\text{→}{}^3\text{P-700}\text{A}{}_1\text{X}$ have similar rate constants when Centers A and B are reduced, i.e., the forward electron transfer time from A^?₁ to X is apparently much slower in the redox state P-700 A₁XFd^?_BFd^?_A than it is in state P-700 A₁XFd_BFd_A. The amplitude of the radical pair-polarized triplet EPR signal does not decrease in the presence of a 13.5-G-wide EPR signal centered at g 2.0 which was recorded in the dark prior to triplet measurements in samples previously frozen under intense illumination. This g 2.0 signal, which has been attributed to phototrapped A^?₁ (Heathcote, P., Timofeev, K.N. and Evans, M.C.W. (1979) FEBS Lett. 101, 105–109), corresponds to as many as 12 spins per P-700 and can be photogenerated during freezing without causing any apparent attenuation of the radical pair-polarized triplet amplitude. We conclude that species other than A^?₁ contribute to the g 2.0 signal.     

Oxygen-evolution patterns from spinach Photosystem II preparations

Patterns of O₂ evolution resulting from sequences of short flashes are reported for Photosystem (PS) II preparations isolated from spinach and containing an active, O₂-evolving system. The results can be interpreted in terms of the S-state model developed to explain the process of photosynthetic water splitting in chloroplasts and algae. The PS II samples display damped, oscillating patterns of O₂ evolution with a period of four flashes. Unlike chloroplasts, the flash yields of the preparations decay with increasing flash number due to the limited plastoquinone acceptor pool on the reducing side of PS II. The optimal pH for O₂ evolution in this system (pH 5.5–6.5) is more acidic than in chloroplasts (pH 6.5–8.0). The O₂-evolution, inactivation half-time of dark-adapted preparations was 91 min (on the rate electrode) at room temperature. Dark-inactivation half-times of 14 h were observed if the samples were aged off the electrode at room temperature. Under our conditions (experimental conditions can influence flash-sequence results), deactivation of S₃ was first order with a half-time of 105 s while that of S₂ was biphasic. The half-times for the first-order rapid phase were 17 s (one preflash) and 23 s (two preflashes). The longer S₂ phase deactivated very slowly (the minimum half-time observed was 265 s). These results indicate that deactivation from S₃ → S₂ → S₁, thought to be the dominant pathway in chloroplasts, is not the case for PS II preparations. Finally, it was demonstrated that the ratio of S₁ to S₀ can be set by previously developed techniques, that S₀ is formed mostly from activated S₃ (S₄), and that both S₀ and S₁ are stable in the dark.     

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     

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 $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 1.1}\text{ms}$.     

Studies on well coupled Photosystem I-enriched subchloroplast vesicles. Content and redox properties of electron-transfer components

Stable and well coupled Photosystem (PS) I-enriched vesicles, mainly derived from the chloroplast stroma lamellae, have been obtained by mild digitonin treatment of spinach chloroplasts. Optimal conditions for chloroplast solubilization are established at a digitonin/chlorophyll ratio of 1 ($\text{w}\text{w}$) and a chlorophyll concentration of 0.2 mM, resulting in little loss of native components. In particular, plastocyanin is easily released at higher digitonin/chlorophyll ratios. On the basis of chlorophyll content, the vesicles show a 2-fold enrichment in ATPase, chlorophyll-protein Complex I, P-700, plastocyanin and ribulose-1,5-bisphosphate carboxylase as compared to chloroplasts, in line with the increased activities of cyclic photophosphorylation and PS I-associated electron transfer as shown previously (Peters, A.L.J., Dokter, P., Kooij, T. and Kraayenhof, R. (1981) in Photosynthesis I (Akoyunoglou, G., ed.), pp. 691–700, Balaban International Science Services, Philadelphia). The vesicles have a low content of the light-harvesting chlorophyll-protein complex and show no PS II-associated electron transfer. Characterization of cytochromes in PS I-enriched vesicles and chloroplasts at 25°C and 77 K is performed using an analytical method combining potentiometric analysis and spectrum deconvolution. In PS I-enriched vesicles three cytochromes are distinguished: c-554 (E′₀ = 335 mV), b-559_LP (E′₀ = 32 mV) and b-563 (E′₀ = ? 123 mV); no b-559_HP is present (LP, low-potential; HP, high-potential). Comparative data from PS I vesicles and chloroplasts are consistent with an even distribution of the cytochrome b-563- cytochrome c-554 redox complex in the lateral plane of exposed and appressed thylakoid membranes, an exclusive location of plastocyanin in the exposed membranes and a dominant location of plastoquinone in the appressed membranes. The results are discussed in view of the lateral heterogeneity of redox components in chloroplast membranes.