Alteration of the Axial Met Ligand to Electron Acceptor A0 in Photosystem I: Effect on the Generation of P 700 ·+ A 1 ·− Radical Pairs as Studied by W-band Transient EPR

Photosystem I (PS I) mutants from the cyanobacterium Synechocystis sp. PCC 6803 bearing point mutations to the axial ligands of A_0A (M688N_PsaA) and A_0B (M668N_PsaB) were studied by high-field W-band electron paramagnetic resonance (EPR) spectroscopy. It was found that the EPR observables of PS I from the M668N_PsaB mutant were virtual identical to that of the wild type (WT), and are clearly distinct from the M688N_PsaA mutant. In particular, the P ₇₀₀ ^·+ decay kinetics in the M688N_PsaA mutant is significantly slower than in the WT or the M668N_PsaB mutant. The analysis of the out-of-phase electron–electron dipolar electron spin echo envelope modulation shows that in the M668N_PsaB mutant, the estimated distance of 26.0 ± 0.3 Å agrees well with the 25.8 Å distance for the P ₇₀₀ ^·+ A _1A ^·? radical pair measured in the X-ray crystal structure. In the M688N_PsaA mutant, two populations are found with estimated distances of 26.0 ± 0.3 and 25.0 ± 0.3 Å in a ratio of 0.7–0.3, which agree well with the 25.8 Å distance for the P ₇₀₀ ^·+ A _1A ^·? radical pair and the 24.6 Å distance for the P ₇₀₀ ^·+ A _1B ^·? radical pair measured in the X-ray crystal structure. The data confirm that under the experimental conditions employed in this work, which involve dark-adapted samples without the pre-reduction of the iron–sulfur clusters, electron transport in cyanobacterial PS I is asymmetrical at 100 K, with the majority of electron transfer taking place through the A-branch of cofactors.     

Pulse ENDOR studies on the radical pair P 700 +· A 1 ·− and the photoaccumulated quinone acceptor A 1 ·− of photosystem I

The secondary acceptor A₁ of the electron transport chain(s) of photosystem (PS) I is a phylloquinone (vitamin K₁, VK₁). Pulse electron paramagnetic resonance and electron nuclear double resonance (ENDOR) experiments at X-band frequencies were performed on the photoaccumulated acceptor radical A ₁ ^·? and the radical pair state P ₇₀₀ ^·+ A ₁ ^·? in PS I ofThermosynechococcus elongatus. The data obtained were compared with data from the respective radical anion of VK₁ in organic solvents. The unusualg tensor magnitude of A ₁ ^·? is explained by the hydrophobic binding pocket of this radical. The hyperfine couplings and the spin (and charge) density distribution is very different for A ₁ ^.? in PS I and VK ₁ ^·? in frozen alcoholic solution. This is attributed to a rather strong one-sided hydrogen bond to A ₁ ^·? . The presence of a hydrogen bond to A ₁ ^·? has only a minor effect ong. The hyperfine coupling constants of A ₁ ^·? determined from the radical pair spectra deviate only slightly from those derived from photoaccumulated A ₁ ^·? in PS I treated with dithionite at high pH. ENDOR resonances of the proton in a H bond were detected and an estimate of the strength and geometry of this bond to A ₁ ^·? was obtained. The significance of the hydrogen bond and other (hydrophobic) interactions of A₁ with the surrounding are briefly discussed.     

Investigation of the Stationary and Transient A 1 ·− Radical in Trp → Phe Mutants of Photosystem I

Photosystem I (PS I) contains two symmetric branches of electron transfer cofactors. In both the A- and B-branches, the phylloquinone in the A₁ site is π-stacked with a tryptophan residue and is H-bonded to the backbone nitrogen of a leucine residue. In this work, we use optical and electron paramagnetic resonance (EPR) spectroscopies to investigate cyanobacterial PS I complexes, where these tryptophan residues are changed to phenylalanine. The time-resolved optical data show that backward electron transfer from the terminal electron acceptors to P₇₀₀^·+ is affected in the A- and B-branch mutants, both at ambient and cryogenic temperatures. These results suggest that the quinones in both branches take part in electron transport at all temperatures. The electron-nuclear double resonance (ENDOR) spectra of the spin-correlated radical pair P₇₀₀^·+A₁^·− and the photoaccumulated radical anion A₁^·−, recorded at cryogenic temperature, allowed the identification of characteristic resonances belonging to protons of the methyl group, some of the ring protons and the proton hydrogen-bonded to phylloquinone in the wild type and both mutants. Significant changes in PS I isolated from the A-branch mutant are detected, while PS I isolated from the B-branch mutant shows the spectral characteristics of wild-type PS I. A possible short-lived B-branch radical pair cannot be detected by EPR due to the available time resolution; therefore, only the A-branch quinone is observed under conditions typically employed for EPR and ENDOR spectroscopies.     

High-frequency EPR approach to the electron spin-polarization effects observed in the photosynthetic reaction centers

Time-resolved high-frequency electron paramagnetic resonance (EPR) spectroscopy was applied to study the structure and dynamics of the electron transfer pathways in the photosynthetic RC proteins. When the spin-polarized EPR spectra are recorded at the high field, the singlet-triplet mixing in the radical pairs becomes faster due to the increase of Zeeman interaction, and a sequential electron transfer polarization model, which includes both the primary and secondary radical pairs, should be considered. Application of the sequential electron transfer polarization model for the interpretation of the bacterial RC proteins with a “slow” electron transfer rate reveals the importance of the protein dynamics. It was shown that the reorganization energy for the electron transfer process between P ₈₆₅ ⁺ H^?Q_A and P ₈₆₅ ⁺ HQ _A ^? , but not the change in the structure of the donor-acceptor complex, is a dominant factor that alters the electron transfer rate. The relaxation data, obtained in the delay after laser flash experiment, have been used to estimate the magnetic interaction in the weakly coupled radical pair. High-frequency spin-polarized EPR spectra allow the quantitative characterization of isotopically labeled quinone exchange in the PS I reaction center proteins.     

Interpretation of multifrequency transient EPR spectra of the P 700 + A0Q K − state in photosystem I complexes with a sequential correlated radical pair model: Wild type versus A0 mutants

Transient electron paramagnetic resonance (TR-EPR) spectra of the electron-hole pair state P ₇₀₀ ⁺ A₀Q _K ^? in photosystem I are numerically calculated. Parameter variation concerns mainly the exchange integralJ of the precursor spin pair state P ₇₀₀ ⁺ A ₀ ^? Q_K and its lifetime τ. A prominent emissive feature in the high-field region (P ₇₀₀ ⁺ part) of the EPR spectrum turns out to be diminished with increasing lifetime τ of the precursor pair state in the case of positive exchange couplingJ>0 (ferromagnetic type). Correspondingly, the emissive feature becomes more pronounced with increasing lifetime τ in the case of negative exchange couplingJ<0 (antiferromagnetic type). These results can be used to interpret the changes in the pattern observed in TR-EPR spectra comparing wild-type and specific A₀ mutants. The central ligating amino acid residue to the A₀ chlorophyll cofactor is mutated from native methionine (M) to leucine (L) in either the PsaA or the PsaB branch. Changes are observed only for the A-side mutant: M688L(PsaA). They are consistent with the following parameters in the precursor pair P ₇₀₀ ⁺ A ₀ ^? :J≈0.5÷1.0 mT and τ=1.5÷2 ns (as compared to τ～0.05 ns in the wild type).     

ESEEM study of the location of spin-polarized chlorophyll-quinone radical pair in membrane-oriented spinach photosystems I and II complexes

Light-induced spin-polarized radical pairs, P700⁺A₁ ^? in spinach photosystem (PS) I particles and P680⁺Q_A ^? in Zn-substituted PS II core complexes, in oriented membranes were studied by pulsed electron paramagnetic resonance (EPR). Based on the determined distance of 25.2 ± 0.2 Å between P700 and A₁, the angular dependence of the spin-polarized electron spin echo envelope modulation (ESEEM) spectra on the magnetic field suggests that the angle between $R_{P700 - {\rm A}_1 } $ , the radius-vector connecting P700 and A₁, and the membrane normaln was 24 ± 4° in PS I particles. Obtained angle and distance of P700-A₁ axis suggested Q_K side in the molecular geometry of cofactors presented in a recent X-ray crystallography of cyanobacterial PS I reaction center to be an active branch of electron transfer. The distance between P680 and Q_A was determined to be 27.4 ± 0.3 Å for a nonoriented PS II. The angle between $R_{P680 - Q_{\rm A} } $ , the radius-vector connecting P680 and Q_A, andn was determined to be 21 ± 5°. The angle of P680-Q_A axis was close to that of 20° of P870-Q_A axis reported in X-ray analysis of the purple bacterial reaction center crystal.     

High-frequency EPR studies on cofactor radicals in photosystem I

Electron paramagnetic resonance (EPR) spectroscopy at W-band (94 GHz) is used to resolve theg-tensors of the radical ions of the primary chlorophyll donor P ₇₀₀ ^+? and the quinone acceptor A ₁ ^?? in photosystem I. The obtainedg-tensor principal values are compared with those of the isolated pigment radicals in organic solvents and the shifts are related to an impact of the protein environment. P ₇₀₀ ^+? has been investigated in photosystem I single crystals at 94 GHz. W-band EPR applied to the photoinduced radical pair P ₇₀₀ ^+? A ₁ ^?? is used to correctly assign theg-tensor axes of P ₇₀₀ ^+? to the molecular structure of the primary donor. Density functional theory calculations on a model of A ₁ ^?? in its binding pocket derived from the recent crystal structure of photosystem I were utilized to correlate experimental magnetic resonance parameters with structural elements of the protein.     

Transient and pulsed EPR spectroscopy on the radical pair state P 865 +. Q A −. to study light-induced changes in bacterial reaction centers

The radical pair state P ₈₆₅ ^+. Q _A ^?. (P₈₆₅: primary donor, Q_A: quinone acceptor) in Zn-substituted bacterial reaction centers is investigated using transient and pulsed EPR spectroscopy. For photoexcited samples not frozen in the dark but under continuous illumination, a prolonged lifetime of this radical pair state is observed in agreement with previous studies using time resolved optical spectroscopy. The transient EPR spectra revealed neither a different orientation of the quinone acceptor anion nor a change of itsg-anisotropy in the sample frozen in the charge separated state as compared with that frozen in the dark. The latter finding indicates a similar hydrogen bonding situation for Q _A ^?. in both samples. Changes observed in the transient EPR spectra are interpreted as result of contributions from spin-polarized Q _A ^?. which was generated in part of the sample while freezing under illumination. From the out-of-phase echo modulation pattern observed in the pulsed EPR measurements, it follows that the distance between P ₈₆₅ ^+. and Q _A ^?. is the same in dark frozen samples and in those frozen under continuous illuminaton. This is in contrast to the model suggested by Kleinfeld D., Okamura M.Y., Feher G.: Biochemistry23, 5780 (1984), in which an increased distance and a larger distribution of distances was suggested for samples frozen under illumination. The prolonged lifetime of the radical pair P ₈₆₅ ^+. Q _A ^?. is discussed in terms of differences in the relaxation behaviour of the protein.     

Magnetic-field-induced orientation of photosynthetic reaction centers as revealed by time-resolved W-band EPR of spin-correlated radical paris: Development of a molecular model

Spin-correlated radical pairs are the short-lived intermediates of the primary energy conversion steps of photosynthesis. In this paper, we develop a comprehensive model for the spin-polarized electron paramagnetic resonance (EPR) spectra of these systems. Particular emphasis is given to a proper treatment of the alignment of the photosynthetic bacteria by the field of the EPR spectormeter. The model is employed to analyze time-resolved W-band (94 GHz) EPR spectra of the secondary radical pair P ₇₀₀ ⁺ A ₁ ^? in photosystem I formed by photoexcitation of the deuterated and¹⁵N-substituted cyanobacteriumSynechococcus lividus. Computer simulations of the angular-dependent EPR spectra of P^700/+A_1/? provide values for the order parameter of the cyanobacterial cells and for the orientation of the membrane normal in a molecular reference system. The order parameter from EPR compares favorably with corresponding data from electron microscopy obtained for theS. lividus cells under similar experimental conditions. It is shown that high-field EPR of a magnetically aligned sample in combination with the study of quantum beat oscillations represents a powerful structural tool for the short-lived radical pair intermediates of photosynthesis.     

Comparison of electron spin polarization of P 870 + Qa a − observed in Zn2+-containing and Fe2+-depleted bacterial reaction centers

Recently, a general model has been developed to explain electron spin polarized (ESP) electron paramagnetic resonance (EPR) signals found in systems where radical pairs are formed sequentially. The photosynthetic bacterial reaction center (RC) is such a system in which we can experimentally vary parameters (lifetime, structure, and magnetic interactions in the sequentially formed radical pairs) that affect ESP development in order to test this model. In Fe²⁺-depleted transfer step from intermediate radical pair, P ₈₇₀ ⁺ Q _a ^? which is produced in an electron transfer step from intermediate radical pair, P ₈₇₀ ⁺ I^?. (P ₈₇₀ ⁺ is the oxidized primary donor, a special pair of bacteriochlorophyll molecules, I^? is the reduced bacteriopheophytin acceptor, and Q _a ^? is the reduced primary quinone acceptor.) The lifetime of P ₈₇₀ ⁺ I^? can be shortened relative to the lifetime of P ₈₇₀ ⁺ I^? in Fe²⁺-depleted RCs by substitution of Zn²⁺. We report the first observation of X-band and Q-band ESP EPR signals due to P ₈₇₀ ⁺ Q^? from bacterial reaction centers that contain Zn²⁺. Comparison of these signals to those observed from Fe²⁺-depleted bacterial reaction centers shows intensity differences and g-factor shifts. The results are discussed in terms of the general sequential radical pair model.     

Light-induced EPR spectra of reaction centers fromRhodobacter sphaeroides at 80 K: Evidence for reduction of QB by B branch electron transfer in native reaction centers

Photosynthetic reaction centers (RCs) fromRhodobacter sphaeroides capture solar energy by electron transfer from primary donor D to quinone acceptor Q_B through the active A branch of electron acceptors. The light-induced electron paramagnetic resonance (EPR) spectrum from native RCs that had Fe²⁺ replaced by Zn²⁺ was investigated at cryogenic temperature (80 K, 35 GHz). In addition to the light-induced signal due to the formation of D^+.Q _A ^?. observed previously, a small fraction (ca. 5%) of the signal displayed very different characteristics. The signal was absent in RCs in which the Q_B was displaced by the inhibitor stigmatellin. Its decay time (τ=6 s) was the same as observed for D^+.Q _B ^?. in mutant RCs lacking Q_A, which is significantly slower than for D^+.Q _A ^?. (τ=30 ms). Its EPR spectrum was identical to that of D^+.Q _B ^?. . The quantum efficiency for forming the major component of the signal was the same as that found for mutant RCs lacking Q_A (?=0.2%) and was temperature independent. These results are explained by direct photochemical reduction of Q_B via B branch electron transfer in a small fraction of native RCs.     

Structural analysis of three-spin systems of photosystem II by PELDOR

The pulsed electron electron double resonance (PELDOR) pulse sequence is applied to a three-spin system consisting of three radicals (Y _D ^· , Y _Z ^· and Q _A ^? ) generated in spinach PS II. The distance between Y_Z and Q_A has been determined to be 3.4 nm with the previously derived distances of the other radical pairs, 2.9 nm for Y _D ^· -Y _Z ^· and 3.9 nm for Y _D ^· -Q _A ^? . This distance has been derived from the Y _Z ^· -Q _A ^? radical pair trapped in Y_D-less mutants ofChlamydomonas reinhardtii. Furthermore the method was applied to the Y _D ^· -Q _A ^? -Chl _Z ⁺ system to find the unknown distance between Q_A and Chl_Z. The derived distance was 3.4 nm. A triangular configuration was found in the membrane system that gives the relative positions of the electron transfer components.     

Theoretical investigation of Q A −· -ligand interactions in bacterial reaction centers ofRhodobacter sphaeroides

Density functional theory was used to calculate magnetic resonance parameters for the primary stable electron acceptor anion radical (Q _A ^?· ) in its binding site in the bacterial reaction center (bRC) ofRhodobacter sphaeroides. The models used for the calculations of the Q _A ^?· binding pocket included all short-range interactions of the ubiquinone with the protein surroundings in a gradual manner and thus allowed a decomposition and detailed analysis of the different specific interactions. Comparison of the obtained hyperfine and quadrupole couplings with experimental data demonstrates the feasibility and reliability of calculations on such complex biologically relevant systems. With these results, the interpretation of previously published 3-pulse electron spin echo envelope modulation data could be extended and an assignment of the observed double quantum peak to a specific amino acid is proposed. The computations provide evidence for a slightly altered binding site geometry for the Q_A ground state as investigated by X-ray crystallography with respect to the Q _A ^t-· anion radical state as accessible via EPR spectroscopy. This new geometry leads to improved fits of the W-band correlated-coupled radical pair spectra of Q _A ^?· -P ₈₆₅ ^+· compared to orientation data from the crystal structure. Finally, a correlation of the¹⁴N quadrupole parameters of His219 with the hydrogen bond geometry and a comparison with previous systematic studies on the influence of hydrogen bond geometry on quadrupole coupling parameters (J. Fritscher: Phys. Chem. Chem. Phys. 6, 4950–4956, 2004) is presented.     

A selective hole burning method applied to determine distances between paramagnetic species in photosystems

A method of selective hole burning in EPR spectra was applied to determine the distances from a radical to the acceptor quinone-iron in bacterial and plant photosystems. A low amplitude hole burning 180° pulse and high amplitude 90° and 90° pulses applied to detect ESE of P870⁺ inRb. Sphaeroides and the distance from the primary electron donor P870⁺ to the acceptor Q _A ^? Fe²⁺ was determined to be 26±2 Å from the dipolar broadening of the burned hole in P870⁺ EPR. This result is consistent with that given by X-ray analysis and susceptibility measurement. In plant photosystem II the same method was applied to the EPR spectrum of tyrosine D⁺, but the effect of crystalline field splitting of Fe²⁺ ion was taken into consideration. The effective spin value for the ferrous iron in PS II was found to be 0.8 and the distance between the radical and the non-heme iron was obtained to be 42±2 Å.     

Temperature dependence of Mn2+ EPR in Sn2P2S6 near the phase transition

The X-band EPR spectrum of Mn²⁺ in Sn₂P₂S₆ was studied in the temperature rangeT=223–363 K. At room temperature the spin-Hamiltonian constants areg=2.00±0.01,B ₂ ⁰ =(163±3)·10^?4 cm^?1,B ₂ ² =(159±3)·10^?4 cm^?1,A=?(75±1)·10^?4 cm^?1. The effect of the invariance in temperature of the resonance magnetic fields in the narrow temperature rangeT=337–340 K and the model of the paramagnetic centre are discussed. According to EPR data a phase transition occurs atT=337 K. This transition from the paraelectric phase to the ferroelectric one is accompanied by a dramatic change in value of the spin-Hamiltonian constantB ₂ ⁰ .     

The magnetic field dependence of the electron spin polarization in consecutive spin correlated radical pairs in type I photosynthetic reaction centres

The magnetic field/microwave frequency dependence of the spin polarized EPR spectra of the sequential spin correlated radical pairs P⁺A^? ₁ and P⁺F^? _x in type I photosynthetic reaction centres is investigated. Experimental data are presented for photosystem (PS) I and reaction centres of heliobacteria at × band (9.7 GHz) and K band (24 GHz). In photosystem I at ambient temperatures the lifetime of A ^? ₁ is ~290 ns and both states are observable by transient EPR. In heliobacteria, electron transfer to F_x occurs within ~600 ps and only the state P⁺F^? _x is observed. The experimental data show a net polarization of P⁺ in the state P⁺F^? _x, which displays a clear dependence on the strength of the external field. The net polarization generated in sequential radical pairs is expected to pass through a maximum as a function of the Zeeman energy when the characteristic time of singlet-triplet mixing is comparable with the lifetime of the precursor. In PS I, the precursor lifetime (290ns) is much longer than the characteristic time of singlet-triplet mixing at × band (9 GHz, 3 kG) and K band (24 GHz, 8 kG). As a result, the observable net polarization decreases with the field strength in this region. In contrast, in heliobacteria, the precursor lifetime (600 ps) is much shorter than the characteristic time of singlet-triplet mixing, and the net polarization increases in the same range of Zeeman energies. The polarization patterns in these two systems can be described using the specific limiting cases of a short lived and long lived precursor radical pair and written as a sum of several contributions. The spectra are simulated on this basis using parameters derived entirely from independent experimental data, and good agreement between the experimental polarization patterns is obtained. The calculated polarization patterns are sensitive to spin dynamics on a timescale much shorter than the spectrometer response time, and the expected influence of a 10 ns component in the electron transfer, as observed optically in some PS I, preparations is discussed. No significant influence from such a component is found in the spin polarization patterns of PS I from the cyanobacterium Synechocystis 6803.     

EPR study of the ferroelectric phase transition in chromium-doped DMAAS

X-band electron paramagnetic resonance (EPR) investigations of single crystals of Cr³⁺-doped dimethylammonium aluminium sulphate hexahydrate are presented from 100 K to room temperature. The crystal undergoes a phase transition at 152 K from the ferroelastic to the ferroelectric phase. The spin-Hamiltonian parameters have been determined for both phases. The spin-Hamiltonian parameters in the ferroelectric phase are:g=1.980±0.003,b ₂ ⁰ =(1140±15)·10^?4 cm^?1,b ₂ ² =(214±10)·10^?4 cm^?1. Remarkable EPR line width changes confirm the order-disorder character of the ferroelectric phase transition on a microscopic level and demonstrate that the dimethylammonium reorientation freezing-out is the prime reason for this transition.     

Alternative pathways of photoinduced electron transport in chloroplasts

The influence of the alternative pathways of electron transport in photosynthetic systems of the oxygen type on the kinetics of the photoinduced redox transitions of P₇₀₀, ferredoxin, NADP, pH of the intrathylakoid space or lumen, and relative concentration of ATP was studied. The oxygen effect on the kinetics of photooxidation of P₇₀₀ was analyzed. The retardation of the photooxidation of P₇₀₀ at low oxygen concentrations can be explained by the “over-reduction” of the acceptor side of PS1 as a result of a decrease in the electron outflow from PS1 to oxygen during hypoxia. The results of numerical experiments are in good agreement with known experimental data that the withdrawal of electrons from PS1 (on the ferredoxin-NADP segment of the chain) can be the limiting stage in the noncyclic electron transport chain. The functioning of the cyclic electron transport chain provides additional synthesis of ATP molecules and weakens the excess reduction of the acceptor segment of PS1. The alternative pathway of electron transport, namely, electron outflow from PS1 to oxygen also favors the optimum conditions for the functioning of the photosynthetic electron transport chain.     

Mechanism by which a single water molecule affects primary charge separation kinetics in a bacterial photosynthetic reaction center of Rhodobacter sphaeroides

Using quantum-chemical methods, we have studied the role played by water molecules W-A and W-B that are bound by hydrogen bonds to accessory bacteriochlorophyll molecules B _A and B _B in the process of primary charge separation in the reaction center of Rhodobacter Sphaeroides. We have found that the occurrence of a rotational mode of the W-A molecule at 32 cm^?1 and/or its harmonics in stimulated emission of an electron donor P* and the dynamics of population of the states P⁺B _A ^? and P⁺H _A ^? may be related to the structural heterogeneity of the reaction center and the existence of a conformation in which the W-A molecule is predominantly involved in one hydrogen bond (with BA). Based on the calculated redox potentials B _A and P, it has been shown that the appearance of the W-A molecule in the reaction center reduces the energy of the P⁺B _A ^? state by ??600 cm^?1. This is somewhat smaller than the influence of the amino-acid residue Tyr^M210 (??870 cm^?1) and correlates well with a substantial decrease in the electron transfer rate in mutant forms of reaction centers GM203L (which do not contain W-A molecules) and YM210F (in which Tyr^M210 is replaced with Phe). The data obtained allow us to suggest that rotation of the water molecule with a fixed position of its H atom that is involved in a hydrogen bond with the keto carbonyl group of B _A is initiated due to the charge separation between the halves of special pair P and the formation of the state P _A ⁺ P _B ^? . The large effect of this rotation on the kinetics of population of the states P⁺B _A ^? and P⁺H _A ^? after the excitation of P is quite consistent with its influence on the energy of the state P⁺B _A ^? .     

Anomalous pulse-angle and phase dependence of Hahn’s electron spin echo and multiple-quantum echoes of the spin correlated radical pair P700+A 1 − in photosystem I

In a spin-correlated radical pair system, anomalous pulse-angle and phase dependence of electron spin echo and multiple-quantum echoes were theoretically calculated by Tanget al. (J. Chem. Phys.106, 7471 (1997)). The maximum intensity of the out of phase signal at 45 degree of spin rotation angle was experimentally verified in two-pulse echoes of the light-induced P700⁺A ₁ ^? radical pair in Photosystem I. The values,D = 1.64 G andJ = 0.00 G, fit well with the experimental ESEEM spectra. Single and double quantum echoes were observed at the value oft = τ andT = 2τ with the laser flash-t-P1_70,ζ1-τ-P2_{140, ζ2}-T pulse sequence, which led to determination of the relaxation time T₂₃ between the singlet and triplet ¦T₀〉 states. The relaxation times of the zero and single quantum transitions were determinedT ₂₃ ≈ 100 ns andT ₂ = 1000 ns, respectively. The field sweep ESE signal shape can be fitted with the hyperfine inhomogeneities of 7 G for P700⁺ and of 10 G for A ₁ ^? .