Investigation of the electron transfer site of p-benzoquinone in isolated photosystem II particles and thylakoid membranes using alpha- and beta-cyclodextrins

The electron transfer sites of p-benzoquinone (pBQ) and 2,6-dichloro-p-benzoquinone (DCBQ) were investigated in thylakoid membranes and isolated photosystem II (PSII) particles from barley (Hordeum vulgare) using alpha- and beta-cyclodextrins (CD) at concentrations up to 16 mM. In CD-treated thylakoid membranes incubated with DCBQ the electron transport through PSII, estimated as oxygen evolution (OE), is largely enhanced according to a S-shaped (sigmoidal) dose-response curve displaying a sharp inflection point, or transition. The maxima percent OE enhancement at cyclodextrin concentrations above 14 mM are about 100% (alpha-CD) and 190% (beta-CD). On the contrary, in thylakoid membrane preparations incubated with pBQ as electron acceptor one observes an OE inhibition of about 30% which might result from the depletion of the thylakoid membrane of its plastoquinone content. It was also found that in isolated PSII particles incubated with either pBQ or DCBQ the cyclodextrins induce only a small OE enhancement. Moreover, the observation in CD-treated thylakoid membranes incubated with pBQ of a residual, non-inhibited oxygen-evolving activity of about 70% puts a twofold question. That is, either the plastoquinone depletion was not complete, or, pBQ binds to electron acceptor sites of different nature. From this and data published in the literature, it is concluded that in the thylakoid membrane (i) DCBQ binds to Q(B), as is generally accepted, and (ii) pBQ binds to the plastoquinol molecules in the PQ pool and most likely also to Q(B), thereby in accord with Satoh et al.'s model [K. Satoh, M. Ohhashi, Y. Kashino, H. Koike, Plant Cell Physiol. 36 (1995) 597-605]. An attractive alternative hypothesis is the direct interaction of pBQ with the non-haem Fe(2+) between Q(A) and Q(B).     

Carotenoid triplet states associated with the long-wavelength-emitting chlorophyll forms of photosystem I in isolated thylakoid membranes

The carotenoid triplet populations associated with the long-wavelength-emitting chlorophyll forms of photosystem I (PS I)(dagger) have been investigated in isolated spinach thylakoids by means of fluorescence-detected magnetic resonance in zero field. The spectra collected in the 730-800 nm emission range can be globally fitted assuming the presence of four different carotenoid triplet states coupled to long-wavelength-emitting forms of PS I, having zero-field-splitting parameters /D/ = 0.0359 cm(-1) and /E/ = 0.00371 cm(-1), /D/ = 0.0382 cm(-1) and /E/ = 0.00388 cm(-1), /D/ = 0.0395 cm(-1) and /E/ = 0.00397 cm(-1), and /D/ = 0.0405 cm(-1) and /E/ = 0.00411 cm(-1). On the basis of the triplet-associated fluorescence emission profile, it is suggested that those triplets are associated with light-harvesting complex I, the peripheral antenna complex of PS I.     

Kinetic model for electron and proton transport in chloroplasts with a nonuniform distribution of protein complexes in thylakoid membranes

A mathematical model for describing electron and proton transport in chloroplasts of higher plants with nonuniform distribution of photosystem 1 (PS1) and photosystem 2 (PS2) between granal and stromal thylakoids was proposed. Along with the noncyclic (linear) electron transport from PS2 to terminal acceptor PS1, we considered cyclic electron transport around PS1, ATP synthesis coupled to transmembrane proton transfer through ATP synthase, and ATP consumption in the Calvin cycle. Numerical simulations of the kinetics of the photoinduced redox transformations of the primary PS1 donor (P₇₀₀) and the behavior of pH in the intrathylakoid space (pH_i) and interthylakoid space (pH₀), as well as the time evolution of the ATP concentration for various conditions of the functioning of the chloroplast were performed.     

Backward electron transport in photosystem 2 reaction center and temperature dependence of delayed luminescence characteristics

The temperature dependence of parameters of light-induced changes in millisecond delayed luminescence (half-width of the maximum, maximal and steady-state luminescence intensity) is studied within the temperature range from -23 to 45 degrees C in leaf segments of Chinese rose (Hibiscus rosa sinensis). Delayed luminescence (DL) is induced and registered by a homemade setup based on a Lewis-Kasha-type phosphoroscope. The temperature dependence of steady-state luminescence intensity is shown to have two maxima, at -10 and 35 degrees C. At room temperatures, the steady-state value of luminescence intensity is minimal, and its value correlates with the temperature tolerance of the plant. Depending on cooling and heating regimes, the DL steady-state value vs. temperature curves is found to be different. We suppose this effect to be caused by temperature-induced destructive changes in the structure of photosystem 2 reaction centre and probably by salting out.     

Coupling of electron and proton transport in photosynthetic membranes: molecular mechanism

Using the method of Modified Neglect of Diatomic Overlap (MNDO), the electronic structure of plastoquinol (PQH(2)) and plastoquinone (PQ) in neutral, singly (PQ(-)) and doubly (PQ(2-)) reduced states is studied. The conformational analysis performed on these molecules shows that in the lowest energy conformation, the angle between the first link of the tail backbone and the ring plane of neutral and singly reduced PQ and plastoquinol is nearly the same and differs by 15 degrees from that of doubly reduced PQ. Nevertheless, for all states of plastoquinone and for plastoquinol, the total energy changes by less than 0.2 eV when the studied angle is varied from 0 degrees to 180 degrees. As in Rhodobacter sphaeroides, the oxygen of the PQ ring is reported to form a hydrogen bond with a nitrogen in the ring of Histidine (His) L 190. The energy of the PQ-His complex was calculated for different redox states of PQ and for several values of the distance between the molecules (N-O distance from 0.2 to 0.5 nm). For every considered complexes, the total energy dependence on the proton position on the line connecting the N and O atoms was determined, to see if the hydrogen bond is formed. It is shown that for only singly reduced PQ this dependence has a symmetric two-well form, i.e. the hydrogen bond is formed. For neutral and doubly reduced PQ, the curve is also two-well but asymmetric, so that the proton is bound to His or to PQ, correspondingly.On the basis of these results, we propose the following scheme of electron-proton coupling. Negatively charged oxygens of PQ form H-bonds with proton donor groups of the surrounding protein and fix PQ in its pocket. While the negative charges of oxygens increase after quinone reduction, protons shift to PQ oxygens and form strong hydrogen bonds with them. Upon second PQ reduction, protons are torn away from surrounding amino acids and form covalent bonds with the quinol. Resulting PQH(2) detaches from its binding place and is replaced by a neutral PQ. The lacking protons on amino acids in the Q(B) pocket are replaced by a step-by-step transfer from the stroma bulk through the proton channels.     

Inhibition of photosynthetic electron transport by UV-A radiation targets the photosystem II complex

We have studied the inhibition of photosynthetic electron transport by UV-A (320-400 nm) radiation in isolated spinach thylakoids. Measurements of Photosystem II (PSII) and Photosystem I activity by Clark-type oxygen electrode demonstrated that electron flow is impaired primarily in PSII. The site and mechanism of UV-A induced damage within PSII was assessed by flash-induced oxygen and thermoluminescence (TL) measurements. The flash pattern of oxygen evolution showed an increased amount of the S0 state in the dark, which indicate a direct effect of UV-A in the water-oxidizing complex. TL measurements revealed the UV-A induced loss of PSII centers in which charge recombination between the S2 state of the water oxidizing complex and the semireduced Q(A)- and Q(B)- quinone electron acceptors occur. Flash-induced oscillation of the B TL band, originating from the S2Q(B)- recombination, showed a decreased amplitude after the second flash relative to that after the first one, which is consistent with a decrease in the amount of Q(B)- relative to Q(B) in dark adapted samples. The efficiency of UV-A light in inhibiting PSII electron transport exceeds that of visible light 45-fold on the basis of equal energy and 60-fold on the basis of equal photon number, respectively. In conclusion, our data show that UV-A radiation is highly damaging for PSII, whose electron transport is affected both at the water oxidizing complex, and the binding site of the Q(B) quinone electron acceptor in a similar way to that caused by UV-B radiation.     

P680 (P(D1)P(D2)) and Chl(D1) as alternative electron donors in photosystem II core complexes and isolated reaction centers

Low temperature (77-90 K) measurements of absorption spectral changes induced by red light illumination in isolated photosystem II (PSII) reaction centers (RCs, D1/D2/Cyt b559 complex) with different external acceptors and in PSII core complexes have shown that two different electron donors can alternatively function in PSII: chlorophyll (Chl) dimer P(680) absorbing at 684 nm and Chl monomer Chl(D1) absorbing at 674 nm. Under physiological conditions (278 K) transient absorption difference spectroscopy with 20-fs resolution was applied to study primary charge separation in spinach PSII core complexes excited at 710 nm. It was shown that the initial electron transfer reaction takes place with a time constant of ~0.9 ps. This kinetics was ascribed to charge separation between P(680)* and Chl(D1) absorbing at 670 nm accompanied by the formation of the primary charge-separated state P(680)(+)Chl(DI)(-), as indicated by 0.9-ps transient bleaching at 670 nm. The subsequent electron transfer from Chl(D1)(-) occurred within 13-14 ps and was accompanied by relaxation of the 670-nm band, bleaching of the Pheo(D1) Q(x) absorption band at 545 nm, and development of the anion-radical band of Pheo(D1)(-) at 450-460 nm, the latter two attributable to formation of the secondary radical pair P(680)(+)Pheo(D1)(-). The 14-ps relaxation of the 670-nm band was previously assigned to the Chl(D1) absorption in isolated PSII RCs [Shelaev, Gostev, Nadtochenko, Shkuropatov, Zabelin, Mamedov, Semenov, Sarkisov and Shuvalov, Photosynth. Res. 98 (2008) 95-103]. We suggest that the longer wavelength position of P(680) (near 680 nm) as a primary electron donor and the shorter wavelength position of Chl(D1) (near 670 nm) as a primary acceptor within the Q(y) transitions in RC allow an effective competition with an energy transfer and stabilization of separated charges. Although an alternative mechanism of charge separation with Chl(D1)* as the primary electron donor and Pheo(D1) as the primary acceptor cannot be ruled out, the 20-fs excitation at the far-red tail of the PSII core complex absorption spectrum at 710 nm appears to induce a transition to a low-energy state P(680)* with charge-transfer character (probably P(D1)(δ+)P(D2)(δ-)) which results in an effective electron transfer from P(680)* (the primary electron donor) to Chl(D1) as the intermediary acceptor.     

Electrokinetic parameters of ion transport across isolated pepper cuticular membranes

The transport of NaCl and CaCl₂ solutions across isolated pepper cuticular membranes was studied by means of conductivity, membrane potential and diffusion experiments. Some characteristic membrane parameters such as the electrical resistance, ionic and salt permeabilities were obtained as a function of the electrolyte concentrations. Cuticle morphological asymmetry accounts for differences in membrane potential values under external reverse gradients. The influence of temperature on the membrane structure was also considered, but only small changes in the electrokinetic parameters were obtained. From the NaCl diffusion experiments two activation energies were determined (54.8 kJ/mol for temperature ranging between 15 and 35°C, and 20.6 kJ/mol for the interval of temperature between 40 and 60°C), which could be associated to thermal transitions in the molecular structure of the cuticle for the interval 30–40°C.     

Efficient energy transfer from the long-wavelength antenna chlorophylls to P700 in photosystem I complexes from Spirulina platensis

To study the role of the long-wavelength chlorophylls (Chl) in photosystem I (PSI), the action spectra of P700 photooxidation at 293 and 77 K have been measured for PSI trimeric and monomeric complexes isolated from Spirulina platensis. The long-wavelength Chls which absorb in the region 710dash740 nm transfer excitation energy to the reduced P700 with the same efficiency as bulk antenna Chls, causing the oxidation of P700. The relative quantum yield of P700 photooxidation is about unity (293-77 K) even under the direct excitation of Chl absorbing at 735 nm (Chl735). At 77 K Chl735 exhibits a fluorescence band at 760 nm (F760) whose intensity is quenched under illumination of the PSI trimeric complexes from Spirulina. The relative quantum yield of F760 quenching is not dependent on the wavelength of excitation in the region 620–750 nm. Since the value of the overlap integral between the band of F760 and the absorption band of the cation radical of P700 (P700⁺) is higher than that of the P700 band, it is suggested that Chl735 transfers energy to P700⁺ more efficiently than to reduced P700; energy transfer to P700⁺ causes the quenching of F760. A linear relationship between the photooxidation rate of P700 and the fraction of P700⁺ at 293 K indicates that the energy exchange between PSI subunits of the trimer is negligible. Thus, the antenna of PSI trimers of Spirulina is organized in separate photosynthetic units.     

Effect of pH on methylene blue transient states and kinetics and bacteria photoinactivation

We have measured directly by time-resolved spectroscopy the transient spectra and kinetics of the methylene blue (MB) excited singlet and triplet state as a function of pH from a few picoseconds to several microseconds. The data show that the acidic triplet state (3)MBH(2+) is the protonated analogue of the basic (3)MB(+). It is also shown that the singlet oxygen formation quantum yield is much higher in basic than in acidic media. The transient spectra and their kinetics suggest that because pH exerts a large influence in singlet oxygen and radical formation, it may also be important in bacteria inactivation. Therefore, we performed experiments, which showed that the rate of gram-positive and gram-negative bacteria inactivation at pH 9 is 3-25 times higher than the rate at pH 5.     

A comparison between chemically and photochemically driven electron transport reactions in immobilized liquid membranes

Electron transport in immobilized liquid membranes using a microporous polypropylene film as the support was studied in the reagent concentration independent regime and was kinetically controlled under the conditions employed in this study. The velocities depended on the concentration of the carrier (Vitamin K₃) in the membrane and varied exponentially with the reciprocal of the absolute temperature. Neither the membrane thickness, concentration of the oxidant (Fe(ophen)₃³⁺) nor the concentration of reductant (S₂O₄^2- or MV⁺ generated photochemically) affect the electron transport rate. Maximum velocities at 25°C (7.8 μmol-cm^-2-hr^-1 and 2.5 μmol-cm^-2-hr^- for the S₂O₄^2- and MV⁺ driven reactions, respectively) were obtained in the pH range of 6-7 for the reductant compartment and in the 0 to - 1 pH range in the oxidant compartment. The respective turnover rates were 2.1 hr^-1 and 0.65 hr^-1 based on 2e^-/Vitamin K₃ for the S₂O₄^2- and MV⁺ driven reactions, respectively. The mechanism of electron transport is best interpreted to involve formation of the hydroquinone in the membrane which then reacts with Fe(o-phen)₃³⁺ in the rate-limiting electron transfer step.     

Stabilization of oxygen evolution and primary electron transport reactions in photosystem II against heat stress with glycinebetaine and sucrose

The protective action of co-solutes, such as sucrose and glycinebetaine, against the thermal inactivation of photosystem II function was studied in untreated and Mn-depleted photosystem II preparations. It was shown that, in addition to the reactions that depend on the oxygen evolving activity of the photosystem, those that implicate more intimately the reaction center itself are protected by high concentrations of osmolytes. However, the temperature required to inhibit oxygen evolution totally in the presence of osmolytes is lower than that required to eliminate reactions, such as P680 (primary electron donor in photosystem II) photo-oxidation and pheophytin photo reduetion, which only involve charge separation and primary electron transport processes. The energy storage measured from the thermal dissipation yield during photoacoustic experiments and the yield of variable fluorescence are also protected to a significant degree (up to 30%) at temperatures at which oxygen evolution is totally inhibited. It is suggested that a cyclic electron transport reaction around photosystem II may be preserved under these conditions and may be responsible for the energy storage measured at relatively high temperatures. This interpretation is also supported by thermoluminescence data involving the recombination between reduced electron acceptors and oxidized electron donors at - 30 and - 55 °C. The data also imply that a high concentration of osmolyte allows the stabilization of the photosystem core complex together with the oxygen-evolving complex. The stabilization effect is understood in terms of the minimization of protein-water interactions as proposed by the theory of Arakawa and Timasheff (Biophys. J., 47 (1985) 411--414).     

Polypeptide patterns obtained by polyacrylamide gel electrophoresis of thylakoid membranes isolated from light and dark grown strains of Chlorogloea fritschii

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis has been used to determine the difference between thylakoid polypeptide patterns of light and dark grown strains of the cyanobacterium Chlorogloea fritschii. There were only 2 prominent bands present in the dark grown strains, polypeptide Mr50,000 and polypeptide Mr90,000, also five fainter bands in the Mr range 45,000-66,200 corresponding to photosystem one, compared with the 32 bands present in the light grown strains. There was no obvious indication of the Mr 33,000 3-(3,4-chlorophenyl)-1,1-dimethyl-urea binding protein. In addition the progressive daily development of the various photosystem components in the light and their relationship in photosynthesis was determined. It was observed that the increase of the relative concentrations of the photosystem two and phycocyanin components indicated their developments are mutually synchronized. The effect of light to dark and dark to light transfer on established strains was investigated. Appreciable loss of photosystem two components and the presence of an additional band Mr22,500 of unknown function in the light to dark transfer, and little reactivation of the photosynthetic capabilities in the dark to light transfer was observed.     

Comparative modulation analysis of electron spin echo signals from oxidized chlorophyll a in vitro and P 700 centres in chloroplasts

A comparative analysis of electron spin echo modulation effects from ¹⁴N nuclei of pyrrole rings for Chla⁺ and P 700⁺ has been performed, yielding parameters of quadrupole and isotropic hyperfine couplings with nitrogen nuclei in these paramagnetic centres.     

Bilirubin-sensitized photoinactivation of enzymes in the isolated membrane of the human erythrocyte.

Abstract— Bilirubin has been found to sensitize the photodynamic inactivation of several enzymes in the isolated membrane (ghost) of the human red cell. When ghosts (pH 8.0, 10°C) + bilirubin (0.1 mM) were irradiated with blue light (350 Wm^-2), the activity of glyceraldehyde 3-phosphate dehydrogenase decayed with t_1/2? 15 min. No effect was observed in the absence of pigment or with incident yellow light. Diazabicyclo-octane (DABCO) sharply reduced the inactivation rate, suggesting that ¹O₂ is involved. Sodium dodecyl sulfate-gel electrophoresis of ghosts containing fully inactivated glyceraldehyde 3-phosphate dehydrogenase revealed no change in the polypeptide band corresponding to the subunit of the enzyme. Solubilized enzyme, which was similarly photosensitive, could be partially protected by nicotinamide adenine dinucleotide or glyceraldehyde 3-phosphate. The integral enzymes Mg²⁺-ATPase, Na⁺, K⁺-ATPase, and acetylcholinesterase were also affected. Under the above conditions and bilirubin = 0.37 mM, these enzymes were photoinactivated in first-order fashion, k? 2, 1.2 and 0.2 h^-1, respectively. The rate of decay of total ATPase was found to vary as the square root of the bilirubin concentration over the range 7–370 μM. At a fixed bilirubin concentration (0.37 mM), this rate was also shown to be directly proportional to light intensity. Inasmuch as the —SH content of bilirubin-containing ghosts diminished during irradiation, oxidation of essential cysteine residues could be responsible for the inactivation of some of the enzymes studied.     

Structure of the P700(+ )A1(-) radical pair intermediate in photosystem I by high time resolution multifrequency electron paramagnetic resonance: analysis of quantum beat oscillations.

The geometry of the secondary radical pair P700(+)A1(-), in photosystem I (PSI) from the deuterated and 15N-substituted cyanobacterium Synechococcus lividus, has been determined by high time resolution electron paramagnetic resonance (EPR), performed at three different microwave frequencies. Structural information is extracted from light-induced quantum beats observed in the transverse magnetization of P700(+)A1(-) at early times after laser excitation. A computer analysis of the two-dimensional Q-band experiment provides the orientation of the various magnetic tensors of with respect to a magnetic reference frame. The orientation of the cofactors of the primary donor in the g-tensor system of is then evaluated by analyzing time-dependent X-band EPR spectra, extracted from a two-dimensional data set. Finally, the cofactor arrangement of P700(+)A1(-) in the photosynthetic membrane is deduced from angular-dependent W-band spectra, observed for a magnetically aligned sample. Thus, the orientation of the g-tensor of P700(+) with respect to a chlorophyll based reference system could be determined. The angle between the g1(z) axis and the chlorophyll plane normal is found to be 29 +/- 7 degrees, while the g1(y) axis lies in the chlorophyll plane. In addition, a complete structural model for the reduced quinone acceptor, A1(-), is evaluated. In this model, the quinone plane of is found to be inclined by 68 +/- 7 degrees relative to the membrane plane, while the P700(+)-A1(-) axis makes an angle of 35 +/- 6 degrees with the membrane normal. All of these values refer to the charge separated state, observed at low temperatures, where forward electron transfer to the iron-sulfur centers is partially blocked. Preliminary room temperature studies of P700(+)A1(-), employing X-band quantum beat oscillations, indicate a different orientation of A1(-) in its binding pocket. A comparison with crystallographic data provides information on the electron-transfer pathway in PSI. It appears that quantum beats represent excellent structural probes for the short-lived intermediates in the primary energy conversion steps of photosynthesis.     

Proton-coupled electron transfer and tyrosine D of photosystem II

Photosystem II (PSII) is a photosynthetic reaction center that oxidizes water and reduces bound plastoquinone. PSII electron transfer is mediated by two redox-active tyrosine residues. One of these residues, tyrosine D (YD), has been assigned as Tyr160 of the D2 polypeptide by site-directed mutagenesis and isotopic labeling. Previous spectroscopic evidence has established that His189 in the D2 subunit forms a hydrogen bond with YD* and donates a proton to YD* when the radical is reduced. However, the mechanism of this reaction has not been elucidated. In this report, EPR spectroscopy and 2H2O solvent exchange were used to investigate the pL dependence of the YD* reduction rate. The kinetic isotope effect (KIE), induced by solvent exchange, was also measured as a function of pL. Under the conditions employed, the reduction of YD* is attributed to recombination with the QA- plastoquinone acceptor of PSII. The kinetic data were fit with a biexponential function. The majority, slow phase exhibited a pL-dependent rate constant, with a minimum at pL 7.5. Solvent exchange gave significant KIE at values between pL 5.5 and 8.0. In particular, at high pL (> or =7.5), the values of the KIE were determined to be 2.1 +/- 0.6 and 2.4 +/- 0.5. These values are consistent with a coupled electron and proton reaction, which occurs with a single kinetic step at pL values > or =7.5. The lower KIE values and the rate acceleration observed at low pL may be consistent with a change of mechanism in which the protonation of YD* occurs first, followed by rate-limiting electron transfer. The more modest acceleration in rate at high pL values is attributed to a small, pL-induced change in the distance between YD* and QA-.     

Dipyridamole and its derivatives modify the kinetics of the electron transport in reaction centers from Rhodobacter sphaeroides

A well known vasodilator dipyridamole (DIP), 2,6-bis(diethanolamino)-4,8-dipiperidinopyrimido[5,4-d]pyrim idine, and its derivatives have recently been shown as potential co-activators (modulators) in the phenomenon of multidrug resistance (MDR) in cancer therapy. They inhibit the specific function of a transmembrane P-glycoprotein responsible for the ex-flux of anti-cancer drugs from tumor cells. To clarify molecular mechanisms of the anti-MDR activity of DIP and its two derivatives, RA25 and RA47, we have studied their effects on electron transport in reaction centers (RC) from purple photosynthetic bacteria Rb. sphaeroides, using RC as a model system. Increasing concentrations of DIP and RA47 progressively accelerate the back electron transfer from the primary quinone acceptor QA to the bacteriochlorophyll dimer Bchl2 (Bchl2+ -QA- recombination). In the absence of o-phenantroline, when both quinone acceptors QA and QB are involved in the electron transport, RA47 is more effective than DIP. DIP stabilizes the electron on the secondary quinone acceptor QB, the effect manifested as the retardation of Bchl2+ -QB- recombination. Effects of RA25 are negligible in all cases. The drugs are proposed to change the electron transport affecting the RC structural dynamics and the stabilization of the electron on quinone acceptors through modification of H-bonds in the system.     

Exploring the electron transfer pathways in photosystem I by high-time-resolution electron paramagnetic resonance: observation of the B-side radical pair P700(+)A1B(-) in whole cells of the deuterated green alga Chlamydomonas reinhardtii at cryogenic temperatures

Crystallographic models of photosystem I (PS I) highlight a symmetrical arrangement of the electron transfer cofactors which are organized in two parallel branches (A, B) relative to a pseudo-C2 symmetry axis that is perpendicular to the membrane plane. Here, we explore the electron transfer pathways of PS I in whole cells of the deuterated green alga Chlamydomonas reinhardtii using high-time-resolution electron paramagnetic resonance (EPR) at cryogenic temperatures. Particular emphasis is given to quantum oscillations detectable in the tertiary radical pairs P700(+)A1A(-) and P700(+)A1B(-) of the electron transfer chain. Results are presented first for the deuterated site-directed mutant PsaA-M684H in which electron transfer beyond the primary electron acceptor A0A on the PsaA branch of electron transfer is impaired. Analysis of the quantum oscillations, observed in a two-dimensional Q-band (34 GHz) EPR experiment, provides the geometry of the B-side radical pair. The orientation of the g tensor of P700(+) in an external reference system is adapted from a time-resolved multifrequency EPR study of deuterated and 15N-substituted cyanobacteria (Link, G.; Berthold, T.; Bechtold, M.; Weidner, J.-U.; Ohmes, E.; Tang, J.; Poluektov, O.; Utschig, L.; Schlesselman, S. L.; Thurnauer, M. C.; Kothe, G. J. Am. Chem. Soc. 2001, 123, 4211-4222). Thus, we obtain the three-dimensional structure of the B-side radical pair following photoexcitation of PS I in its native membrane. The new structure describes the position and orientation of the reduced B-side quinone A1B(-) on a nanosecond time scale after light-induced charge separation. Furthermore, we present results for deuterated wild-type cells of C. reinhardtii demonstrating that both radical pairs P700(+)A1A(-) and P700(+)A1B(-) participate in the electron transfer process according to a mole ratio of 0.71/0.29 in favor of P700(+)A1A(-). A detailed comparison reveals different orientations of A1A(-) and A1B(-) in their respective binding sites such that formation of a strong hydrogen bond from A1(-) to the protein backbone is possible only in the case of A1A(-). We suggest that this is relevant to the rates of forward electron transfer from A1A(-) or A1B(-) to the iron-sulfur center F(X), which differ by a factor of 10. Thus, the present study sheds new light on the orientation of the phylloquinone acceptors in their binding pockets in PS I and the effect this has on function.