Spin-Polarized Radical Pair in Photosystem I Reaction Center That

Faculty of Science, Kwansei Gakuin UniVersity, Nishinomiya 662-8501, Japan. ReceiVed: May 6, 1998; In Final Form: October 1, 1998. The spin-polarized ...
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10440

J. Phys. Chem. B 1998, 102, 10440-10445

Spin-Polarized Radical Pair in Photosystem I Reaction Center That Contains Different Quinones and Fluorenones as the Secondary Electron Acceptor Masayo Iwaki* and Shigeru Itoh National Institute for Basic Biology, Okazaki 444-8585, Japan

Hideyuki Hara and Asako Kawamori Faculty of Science, Kwansei Gakuin UniVersity, Nishinomiya 662-8501, Japan ReceiVed: May 6, 1998; In Final Form: October 1, 1998

The spin-polarized radical pair of the primary donor chlorophyll P700+ and the secondary acceptor Q- was studied by the electron spin echo envelope modulation (ESEEM) technique in the spinach photosystem I reaction center in which artificial quinones and fluorenones were reconstituted as Q. From the dipole interaction between the spins on P700+ and Q-, the distance between P700 and anilinochloronaphthoquinone or hydroxyanthraquinone was estimated to be 25.6 ( 0.3 Å or 25.7 ( 0.3 Å, respectively, suggesting their proper binding at the original phylloquinone binding site. Distances of 26.0 Å with larger heterogeneity were estimated for (NO2)3- and (NO2)4-fluorenone compounds that have only one carbonyl group, suggesting their multiple orientations in the binding pocket. The lifetimes of the radical pairs estimated by the time dependency of the two-pulse ESE signals were almost temperature independent (30-80 µs at 4-80 K). In the three-pulse ESE measurement, the lifetimes were lengthened by 10-103 times and showed strong temperature dependencies.

1. Introduction In the photosynthetic reaction centers, quinone molecules function as the secondary electron acceptors to stabilize the reducing power after the light-induced charge separation. The photosystem (PS) I reaction center of plants and cyanobacteria contains two molecules of phylloquinone (phyQ: 2-methyl-3phytyl-1,4-naphthoquinone).1 One of them functions as the secondary electron acceptor2,3 and was designated A1 historically4 and designated Q here. Upon photo-excitation, an electron is transferred from the special pair of chlorophyll a (P700; the primary electron donor) to A0 (chlorophyll a-690), phyQ, and then to a 4Fe4S cluster (FX) with reaction times of 3 ps, 23 ps, and 25-200 ns, respectively (see Figure 1 and reviews in ref 4). The two extra 4Fe4S clusters, FB and FA, function as the terminal acceptors. The acceptor phyQ is characterized by the extremely negative redox midpoint potential (Em) of around -0.78 V (NHE)5 (Figure 1). The locations of phyQs have not been determined yet in the X-ray crystallographic structure of the cyanobacterial PS I complex due to the limited resolution of 4 Å.6 A wide variety of artificial quinones and quinonoid molecules were shown to replace the function of the acceptor phyQ after its extraction with diethyl ether3,7-9 or hexane-methanol10 treatment. The reaction center protein seems to fix the phyQ molecule at the Q binding site by hydrophobic interactions, π-π interactions, and hydrogen bindings.9 The contribution of the one carbonyl group to the binding energy at the Q-site was estimated to be -3 kcal/mol.9 In the Q-reconstituted PS I complexes, analysis of the energy-gap dependence of the rate constants of the P700+A0-Q f P700+A0Q- reactions suggested a 7.8 Å edge-to-edge distance11 between A0 and Q according * Corresponding author. E-mail: [email protected].

Figure 1. Energy level and electron transfer pathways in the PS I reaction center. Charge recombination between P700+ and Q- is indicated by a dashed arrow. Estimated energy levels of the P700+Qstate in which Q is replaced by different compounds are also indicated.5,8 Chemical structures of compounds used in this study are shown in the right panel. From top to bottom, intrinsic phylloquinone (PhyQ), 3-anilino-2-chloro-1,4-napththoquinone (An-Cl-NQ), 1,2,5,8-tetrahydroxy-9,10-anthraquinone ((OH)4-AQ), 2,4,7-trinitro-9-fluorenone ((NO2)3Fl), and 2,4,5,7-tetranitro-9-fluorenone ((NO2)4-Fl).

to the empirical law of the distance dependency of the electron transfer rate.12 The fast reaction time of 23 ps from A0- to phyQ11,13 seems to be enabled by a strong electronic coupling between A0 and phyQ, the optimized energy gap and the small reorganization energy.11 PhyQ- rapidly reduces FX at room temperature and directly reduces P700+ below 200 K because of the decrease in the reduction rate of FX.14,15 Some reconsti-

10.1021/jp9821477 CCC: $15.00 © 1998 American Chemical Society Published on Web 11/13/1998

P700-Q Radical Pair in PS I

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TABLE 1: Estimated Parameters and the Distances between P700 and Different Q Speciesa Q intact (phylloquinone) An-Cl-NQ (OH)4-AQ (NO2)3-Fl (1 component) (NO2)3-Fl (3 component) (NO2)4-Fl (1 component) (NO2)4-Fl (3 component) Br2-NQ intact benzyl-NQ butyl-NQ NQ-d6 DQ-d12

D (µT) -164 ( 5 -171 ( 5a -166 ( 5 -164 ( 5 -158 ( 5 -158 ( 5 -200 ( 20 -100 ( 20 -158 ( 5 -158 ( 5 -200 ( 20 -100 ( 20 -175 ( 5c -170 ( 4 -167 ( 4 -173 ( 4 -176 ( 4 -164 ( 4

p Spinach PS I 1.0 1.0 1.0 1.0 0.1 0.4 0.5 1.0 0.2 0.4 0.4

r (Å)

T2 (µs)

Em (V, NHE)b

25.7 ( 0.3 25.3 ( 0.3c 25.6 ( 0.3 25.7 ( 0.3 26.0 ( 0.3 26.0 ( 0.3 24.1 ( 1.2 30.3 ( 1.2 26.0 ( 0.3 26.0 ( 0.3 24.1 ( 1.2 30.3 ( 1.2 25.3 ( 0.3c

1.0

-0.78

1.4 0.6 0.8 2.0 0.2 0.2 0.8 2.0 0.2 0.2

-0.62 -0.61 -0.34

-0.06

-0.37

Cyanobacterial PS Id 25.4 ( 0.3 25.6 ( 0.3 25.3 ( 0.3 25.1 ( 0.3 25.7 ( 0.3

a Abbreviations: An-Cl-NQ, 3-anilino-2-Cl-1,4-naphthoquinone (NQ); Fl, 9-fluorenone; NQ-d6, deuterated naphthoquinone; DQ-d12, deuterated duroquinone. p is the ratio of amplitude of the component used in the simulation. In the case of (NO2)n-Fl, parameters for a single component and three components are represented. b Estimated redox potential of Q-/Q couple in situ in the PS I reaction center. An E1/2 value of each Q-/Q couple measured in dimethylformamide was shifted by -0.316 V according to refs 5 and 8. c Data taken from ref 16. d Data taken from ref 19.

tuted quinones and fluorenones, on the other hand, formed the stable P700+Q- state and directly re-reduced P700+ with a time constant of 60-250 µs even at 280 K.5,7,8 The results were interpreted by their Em values in situ at the phyQ binding site that are more positive than the Em of FX.5,7,8 The distance between P700 and phyQ was determined to be 25.3-25.5 Å in spinach16 and cyanobacterial17 PS I by the electron spin echo envelope modulation (ESEEM) measurements. The angle between the line connecting the two carbonyls of phyQ and the membrane normal was estimated to be 27° by simulation of the electron paramagnetic resonance (EPR) spectrum in the cyanobacterial PS I.18 Possible locations of phyQ in PS I reaction center complexes have been proposed from these distances and angles.6,18 Similar P700-Q distances were estimated with some artificial quinones16,19 (seee also Table 1). A different arrangement of phyQ based on the estimated phyQFX distance of 25 Å was also proposed by Berry et al.20 Deuterated 1,4-naphthoquinone, on the other hand, was suggested to have an orientation different from that of phyQ.21 The relation between the molecular structures of reconstituted quinones and their localization in PS I has not been clearly established. We have analyzed the relation between the molecular structure of the Q species and their locations in the PS I reaction center based on the P700-Q distances estimated by the twopulse ESEEM measurements. The Q species were selected from a group of molecules that are known to tightly bind to PS I and not to reduce FX, i.e., to yield a stable P700+Q- radical pair5,8,9,11 (see Figure 1). They are different from intact phyQ molecules in the molecular shape and/or size, the possible number of hydrogen bonds, and the redox potential. The effects of an additional microwave pulse on the lifetimes of the spin-polarized radical pairs and their temperature dependencies were also examined in the three-pulse ESE measurements. 2. Electron Spin Echo (ESE) Measurements in the Radical Pair The electron spin echo envelope modulation (ESEEM) technique has been a powerful tool for the precise determination of the distance between the radical pair based on the dipolar

coupling between polarized electron spins on the radical pair. The polarized spins on the radical pair state can be precisely detected by the transient out-of-phase ESE, which shows a deep envelope modulation.22,23 The basic spin functions for the spin Hamiltonian of the radical pair are given by |S〉 for the singlet state and |T+〉, |T-〉 and |T0〉 for the triplet state. |S〉 and |T0〉 are mixed into two eigenstates |Φa〉 and |Φb〉 as,

|Φa〉 ) cos φ|S〉 + sin φ|T0〉

(1)

|Φb〉 ) -sin φ|S〉 + cos φ|T0〉

(2)

where φ represents the coupling angle. It was suggested that the light-induced radical pair P700+Q- is initially created in the singlet state, showing the zero-quantum coherent oscillation between the |Φa〉 and |Φb〉 states, which was reported to relax within a few hundred nanoseconds in the deuterated PS I.24 In a two-microwave pulse experiment of ESEEM (laser-T-P1-τP2-τ-echo), the transient intensity of the ESE signal, V(τ), is expressed as23,25,26

V(τ) ) (∆ω2B2/R4) sin Aτ[1 - cos(Rτ)] exp(-2τ/T2)

(3)

A ) -2D(3 cos2 θ - 1)/3 + 2J

(4)

B ) D(3 cos2 θ - 1)/3 + 2J

(5)

R ) ∆ω + B

(6)

2

2

2

where D is the dipolar coupling, J is the value of the spin exchange interaction, θ is the angle between the line connecting the radical pair and the direction of the external magnetic field, and ∆ω is the difference in reosnance frequencies for the two spins in the absence of dipolar and exchange interactions. T2 is a phase memory time constant for four single-quantum transitions, from |Φa〉 to |T+〉 or |T-〉 and from |Φb〉 to |T+〉 or |T-〉.23,25,26 For randomly oriented smaples, eqs 3-6 are averaged over the angle θ. The spin-spin distance (r) is obtained from the relation D ) -3gβ/2r3. The charge recombination (P700+Q- f P700Q) occurs only from the singlet state. The application of the extra microwave

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pulse is expected to change the lifetime of the radical pair through modification of the population in the |T+〉/|T-〉 and |Φa〉/ |Φb〉 states. This phenomenon had been observed by the pulsed ESR technique in the purple bacterial reaction centers27 and the intact PS I reaction center16 and recently analyzed by a semiclassical quantum theory.28 3. Materials and Methods 3.1. Quinone Reconstitution. PS I particles were prepared from spinach chloroplasts by the treatment with digitonin and lyophilized after washing in distilled water.3 The lyophilized PS I particles were extracted twice with diethyl ether at 0-5 °C.3 The extracted PS I particles are depleted of phylloquinone and about 60% of antenna chlorophylls but still contain the photoactive primary donor P700, the acceptors A0, and three FeS clusters.3 The Q-depleted PS I particles were dispersed in a 50 mM Tris-Cl buffer (pH 7.5). An-Cl-NQ, (OH)4-AQ, (NO2)4-Fl (Aldrich, Milwaukee, WI), and (NO2)3-Fl (Tokyokasei, Tokyo, Japan) were used for reconstitution. To reconstitute the Q species, aliquots of dimethyl sulfoxide solutions were added to the buffer suspension of Q-depleted PS I particles and incubated overnight at 0 °C. Dissociation constants between (NO2)3-Fl or (NO2)4-Fl and PS I were reported to be 0.3 or 0.6 µM, respectively,8 and the values for An-Cl-NQ and (OH)4AQ were determined to be 1 and 3 µM, respectively, according to the methods in ref 9. Before the EPR measurements, glycerol was added to the sample to give a final concentration of 60% (v/v). The final concentrations of P700 and the Q species were adjusted to 10-20 and 100-150 µM, respectively. 3.2. Pulsed EPR. Pulsed EPR measurements were performed as previously described16,25,26 with an FT-EPR spectrometer (Bruker ESP 380) employing a dielectric cavity (Bruker ER 4117 DHQ-H) inside a liquid-helium cyrostat (Oxford Inst. CF 935). To detect transient states, the out-of-phase ESE signal was observed with a pulse sequence of laser-T-P1-τ-P2-τ-echo. Two 32-ns microwave pulses (P1, P2) with spin rotation angles of 45°-τ-180°, which gave the maximum intensity of ESE signals were used.26 The interval time T was fixed at 1 µs to avoid the effects of zero quantum beats, which might be significant in a shorter time range,24 and to apply the first microwave pulse (P1) to the radical pair within its lifetime (see Figure 4). The time (τ)-dependent echo intensity of the P700+Qradical pair was recorded at 3440 G (g ) 2.0040), where the yield of the out-of-phase ESE signal became maximum. The time τ was varied from 96 ns with a 16-ns step. To generate theP700+Q- radical pair state, the samples were excited by a 4-ns (FWHM), 700-nm laser flash created by an optical parametric oscillator pumped by a Nd:YAG laser (Continuum Sure lite I) at a repetition rate of 10 Hz or lower to accumulate signals with adequate S/N ratio. The values of dipolar coupling (D), spin exchange interaction (J), and phase memory time (T2) were determined by the simulation of the ESEEM signal as described in the former section. The time (T) dependency of the ESE intensity was measured with the two-pulse sequence, laser-T-P1-τ-P2-τ-echo, with a fixed interval τ ) 120 ns. To study the effect of an additional microwave pulse (P0) on the time (T) dependency, the echo intensity was measured with a three-pulse sequence, laser-t0P0-T-P1-τ-P2-τ-echo with fixed intervals of t0 ) 1 µs and τ ) 120 ns. Pulse angles of P0, P1, and P2 were 180°, 45°, and 180°, respectively. In both measurements, the time T was scanned from 1 µs to 200 ms and the decay rate constants (k) of the ESE intensities were determined. All phases of the

Figure 2. Two-pulse ESEEM signal measured at 80 K as a function of the time separation τ between microwave pulses P1 and P2 in the PS I reaction centers with different Q species. From top to bottom: intact PS I, PS I reaction centers reconstituted with An-Cl-NQ, (OH)4AQ, (NO2)3-Fl, and (NO2)4-Fl. Solid lines indicate experimental data. In the cases of intact PS I, An-Cl-NQ-PS I, and (OH)4-AQ-PS I, dotted lines indicate simulated curves with a single component. In the cases of (NO2)n-Fls, dotted lines were obtained from calculations with three components and dashed-dotted lines with a single component. The parameters used for the simulation are given in Table 1. Inset: time sequence used.

microwave pulses were the same, and the pulse lengths were 32 ns. The signal was accumulated 100 times in each case. 4. Results and Discussion 4.1. ESEEM Signals of P700+Q- Radical Pair States. The flash-induced ESEEM signals of the P700+Q- radical pair in the intact and the Q-reconstituted PS I reaction centers were measured with a two-pulse sequence at 80 K. The Q molecules used were 3-anilino-2-chloro-1,4-naphthoquinone (An-Cl-NQ), 1,2,5,8-tetrahydroxy-9,10-anthraquinone ((OH)4-AQ), 2,4,7trinitro-9-fluorenone ((NO2)3-Fl), and 2,4,5,7-tetranitro-9-fluorenone ((NO2)4-Fl), as shown in Figure 1. They are known to oxidize A0- rapidly but cannot reduce FX.5,8,11 These quinones and fluorenones re-reduce P700+ directly with a time constant of 150-250 and 60-80 µs, respectively, estimated by optical measurements at room or cryogenic temperature.8,14 Their in situ Em values were estimated to be more positive (-0.62 V to -0.06 V) than that of phyQ (-0.78 V), as indicated in Figure 1 (see also Table 1).5,8 All the PS I preparations containing different Q species produced a remarkable out-of-phase ESE signal. The timedependent ESE intensity was recorded at 3440 G (g ) 2.0040), where the yield of the signal became maximum. The signal had a specific feature of the spin-polarized radical pairs expected from theories.23 The ESEEM profiles obtained in the PS I reaction centers reconstituted with An-Cl-NQ and (OH)4-AQ were almost the same as that of intact PS I (solid lines in Figure 2A-C). On the other hand, the ESEEM obtained in the reaction centers reconstituted with (NO2)3-Fl and (NO2)4-Fl were somewhat different, showing faster spin dephasing (solid lines in Figure 2D,E). Similar fast dephasing was also reported in the

P700-Q Radical Pair in PS I case of QA quinone in the quinone-reconstituted or Znsubstituted PS II core reaction center complex.25 In all the PS I preparations, the signal amplitudes did not change significantly over a temperature range from 4 to 200 K (data not shown). This indicates no significant contribution of the other electron transfer pathway, such as the one from A0to FX, that competes with the recombination reaction between P700+ and Q- at low temperature. The results are consistent with the optical measurement of P700+ re-reduction.8,14 These artificial Q compounds were estimated to have Em values in situ at the binding site more positive than that of phyQ and FX, as shown in Figure 15,7,8 and seem to reduce P700+ preferentially at room temperature as well as at low temperature. The ESE signals were simulated to obtain D, J, and T2 values using eqs 3-6. The curves were calculated to fit the experimental data (solid lines in Figure 2). The parameters used in the calculations are listed in Table 1 together with those reported in the literature. Theoretical fitting indicates that the signal amplitude at τ ) 0 is zero. The J value was determined to be almost zero (0 ( 10 µT) in every case. For the intact PS I, the values of D ) -164 µT and T2 ) 1.0 µs were obtained (dotted lines in Figure 2A). The similar values of D ) -166 µT and T2 ) 1.4 µs were determined in the case of An-Cl-NQ and D ) -164 µT and T2 ) 0.6 µs in the case of (OH)4-AQ (dotted lines in Figure 2B,C). This suggests that the three quinones are located at the same site and that the binding features are similar. The ESEEM profiles were somewhat different in the cases of (NO2)3-Fl and (NO2)4-Fl. Although a single component with D ) -158 µT and T2 ) 0.8 µs well simulated the curves for τ > 300 ns (dashed-dotted lines in Figure 2D,E), in both cases better fits to the fast damping portion in the experimental data were obtained with three components; a slow component (D ) -158 µT, T2 ) 2.0 µs) and two faster (T2 ) 0.2 µs) components with D ) -200 and -100 µT (dotted lines in Figure 2D,E). The multiple T2 and D values may reflect the heterogeneity in the locations or mobility of the fluorenones inside the reaction center protein. In every case, the T2 value was 1-2 orders shorter than the lifetime (charge recombination time) of the P700+Q- state measured by the optical measurements (150250 µs in the case of quinones and 60-80 µs in the case of fluorenones8,14). 4.2. Distance between P700 and Different Q Species. The distance between the spins on P700 and each quinone/fluorenone was determined from the estimated D value, as summarized in Table 1. The center-to-center distances between P700 and Q were determined to be 25.6 ( 0.3 and 25.7 ( 0.3 Å in the An-Cl-NQ- and (OH)4-AQ-reconstituted PS I preparations, respectively. The P700-phyQ distance in the intact PS I was estimated to be 25.7 ( 0.3 Å. This is consistent with the estimation of previous studies on intact and quinone-reconstituted PS 1 reaction centers.16,17,19 The results suggest that the reconstituted quinone binds at the original phyQ site even with an anilino group, three aromatic rings, or hydroxy groups that are possible to have extra interactions with the surrounding protein or to form tautomers.29 It is also indicated that the locations of the Q sites in the PS I reaction center protein isolated from spinach and cyanobacteria are probably similar, judging from the D values obtained in the two reaction centers (see Table 1 and ref 19). The distance between P700 and (NO2)3Fl or (NO2)4-Fl was also estimated to be 26.0 ( 0.3 Å either from the D value in the one-component fitting (T2 ) 0.8 µs) or from that of slow T2 ) 2.0 µs component in the three-component fitting (Table 1). (NO2)n-Fl molecules, therefore, seem to bind at the original phyQ site with a hydrogen bond through their

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Figure 3. Proposed arrangements of phylloquinone (left) and (NO2)4Fl (right) in the PS I reaction center. Possible heterogeneity of the binding of fluorenones in the phyQ binding pocket is shown. Positions of chlorophylls, which are represented by rectangles, are based on the X-ray crystallography.6 The edge-to-edge distance of P700-A0 was estimated from analysis of the electron transfer rate.11

one carbonyl group and/or π-π interactions of the aromatic rings, as proposed previously.9 A carbonyl group of (NO2)n-Fl may bind at one of the hydrogen-bonding sites of the two carbonyls of phyQ, as proposed by the study of affinity9 (see Figure 3). When the carbonyl group binds at a site more distant from P700, an edge-to-edge distance between one of the nitro groups and P700 might become even shorter. This mechanism may interpret their faster electron transfer to P700+ observed by the optical measurements.8,14 In contrast to PS I, the reaction centers of purple bacteria and PS II have secondary acceptor quinones (QA) that have more positive Em’s of -0.1 to -0.2 V. The reduction rate of QA by pheophytins and the charge recombination between the oxidized primary donor (P) and QA- are slower than the reactions of Q of PS I. The estimated distances between P and QA by using the ESEEM technique in the purple bacterial (28.4 Å)17 and PS II (27 Å)25 reaction center complexes are slightly longer than the distance between P700 and Q in PS I. The multiple D and T2 values were estimated with (NO2)nF1, suggesting wider distributions in their localization, orientation, and/or mobility in the Q-binding pocket (Figure 3). The D values of the two fast components (T2 ) 0.2 µs) estimated in the three-component fittings gave apparent distances of 24.1 ( 1.2 and 30.3 ( 1.2 Å with relative contributions of about 40% each (Table 1). The distance distribution could cause a multiple recombination kinetics. However, the optically measured rereduction kinetics of P700+ was composed of two decay components with relative contributions of 80% and 20%, and with t1/e values of 60 µs and >10 ms, respectively, in the (NO2)3Fl-reconstituted-PS I reaction centers.8,14 The small contribution of the t1/e > 10 ms phase apparently disagrees with a larger contribution of the 30-Å distance component. The calculated multiple distances, therefore, might either be an artifact in the fitting procedure or not be related to the recombination kinetics straightforwardly. In the purple bacterial reaction center, preillumination induced a significant change of the recombination kinetics of P+QA- with a slight shift (0.4 Å) of P-QA distance.30 It was also proposed that the change in the distribution of the P-QA distance on cooling may be due to a global change of the protein matrix, which affects the recombination rate.31 Structural changes in the PS I protein induced by the reconstitution of the fluorenones might be responsible for the observed distance distribution. The present study indicated that the artificial quinones and fluorenones bind to the PS I reaction center at almost the same distance from P700 as that of intrinsic phyQ. The inabilities to

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Figure 4. Temperature dependence of the decay rate constant (k ) (t1/e)-1, s-1) of the spin-polarized P700+Q- radical pair state detected in a two-pulse sequence ESE in the PS I with different Q species: (open circle), intact PS I; (open triangle), An-Cl-NQ; (open square), (OH)4AQ; (solid circle), (NO2)3-Fl; (solid triangle), (NO2)4-Fl. The k values were obtained from the time (T) dependency of the amplitude of ESE signals measured with the time sequence drawn in the inset.

reduce FX by those Q compounds used in this study, thus, can be interpreted by their inappropriate redox potential values and not by their bindings at the nonfunctional sites. The reported large variation in the rate of forward electron transfer from A0 to various Q species (from 23 ps to 60 ns11) is therefore concluded to be mainly determined by the variation in the energy gap. The effects of slight changes in their geometries seem to be obscured by the larger effects of the energy gap, as assumed previously.11 On the other hand, the rate of the charge recombination from Q- to P700+ was only weakly dependent on the energy gap, presumably due to the large contribution of high-frequency vibrational modes.14 In this case, a slight change in the edge-to-edge distance between the cofactors might modify the electron transfer rate. 4.3. Temperature Dependency of the Lifetime of the P700+Q- Radical Pair Measured by Two- and Three-Pulse ESE. The lifetimes of different P700+Q- radical pair states with different Q species were studied by the two-pulse ESE method (laser-T-P1-τ-P2-τ-echo). The amplitudes of ESE signals were measured with the varied interval time (T) between the laser excitation (hν) and the first microwave pulse (P1) (see Figure 4, inset). The interval τ value was fixed to be 120 ns. The amplitudes of the signals decayed essentially mono-exponentially in the Q-reconstituted PS I reaction centers, as well as in the intact PS I, as seen in the previous work.16 The decay time t1/e of the ESE signal in intact PS I slightly increased from 30 µs (log k ) 4.5) to 60 µs (log k ) 4.2) with the decrease in temperature from 80 to 4 K (Figure 4). However, almost constant t1/e values of 60-80 µs (log k ) 4.2-4.1) were obtained in the PS I reaction centers reconstituted with An-Cl-NQ and (OH)4-AQ. In the cases of (NO2)3-Fl and (NO2)4-Fl, shorter t1/e values of 30-50 µs (log k ) 4.5-4.3) that are also almost temperature insensitive were obtained. The t1/e values obtained in the cases of quinones and fluorenones are shorter than the corresponding lifetimes of 150-250 and 60-80 µs, respectively, that were optically determined as the re-reduction time of lightinduced P700+.7,13 The reason for the discrepancy is not clear yet. A similar discrepancy was also observed in the radical pair of P680+QA- of the PS II reaction center,25 but not of the P+QAof the purple bacterial reaction center.27 The time (T) dependency of the amplitude of the ESE signal was also measured with the application of an extra microwave pulse (P0) at a fixed time t0 (at 1 µs) after the laser excitation (three-pulse ESE, laser-t0-P0-T-P1-τ-P2-τ-echo; see Figure 5,

Iwaki et al.

Figure 5. Temperature dependence of the decay rate constant (k ) (t1/e)-1, s-1) of the ESE signal of the spin-polarized P700+Q- radical pair state detected in a three-pulse sequence ESE in the PS I with different Q species: (open circles), intact PS I; (open triangle), AnCl-NQ; (open square), (OH)4-AQ; (solid circle), (NO2)3-Fl; (solid triangle), (NO2)4-Fl. The k values were obtained from the time (T) dependency of the amplitude of ESE signals measured with the time sequence drawn in the inset.

inset). The signal amplitudes were measured with the varied interval time (T) between the P0 and P1 pulses with the τ value fixed at 120 ns. The application of the P0 pulse produced a long-lived negative echo signal followed by a fast decay phase in the Q-reconstituted PS I reaction centers (not shown), as seen in the intact spinach PS I reaction center in the previous work. 16 In contrast to the two-pulse measurement (without P0 pulse), the lifetimes of the radical pair were significantly elongated, showing temperature-dependent variation in t1/e values over 3 orders of magnitude upon cooling from 50 to 4 K in each case (Figure 5). There seemed to be no apparent correlation between the molecular structure of the Q species and the temperature dependency of the t1/e values. The experimental results indicate that the application of a P0 pulse switched the rate-limiting step from the activationless simple charge recombination reaction to a slower process with an apparent activation energy of about 6 meV in both intact and Q-reconstituted PS I reaction centers. In the purple bacterial reaction center, the decay of the ESE signal of P+QA- radical pair became slower with the application of an additional 90° microwave pulse shortly after the laser excitation.27,28 This effect has been explained by the semiclassical quantum theory.28 If the P0 pulse could populate the |T+〉 and |T-〉 triplet sublevels that were initially unoccupied, the slow decay can represent the spin-lattice relaxation from the |T+〉 and |T-〉 states to the |Φa〉 and |Φb〉 states. The angle of the P0 pulse used in the present study (Figure 5), however, was 180°, and the 90° pulse gave similar results (not shown), although only the 90° pulse can convert the population in the |Φa〉/|Φb〉 states to the |T+〉/|T-〉 states theoretically.28 The pulse angle effect that is apparently inconsistent with the theory will be studied in detail elsewhere. 5. Conclusions The distances between P700 and the secondary acceptor Q were determined to be 25.6-25.7 Å in the intact and An-ClNq- or (OH)4-AQ-reconstituted PS I reaction centers by ESEEM methods. This indicates that these artificial quinones can bind at the original phyQ site, though their molecular structure and physicochemical nature are different from those of intrinsic phyQ. Themultiple distances of 24.1, 26.0, and 30.3 Å and T2 values of 2.0 and 0.2 µs estimated in the cases of (NO2)3-F1 and (NO2)4-F1 seem to indicate their heterogeneous orientations

P700-Q Radical Pair in PS I and/or larger mobility in the phyQ-binding pocket, probably due to their one-carbonyl structure. The inability of the quinones and fluorenones used in this study to reduce FX at room temperature cannot be interpreted by their bindings at the nonfunctional sites, and it is probably due to their inappropriate redox potential at the Q site. The lifetimes of the spin-polarized radical pair states were only weakly dependent on temperature in all the Q-reconstituted PS I reaction centers. The application of an extra microwave pulse (P0) significantly elongated the lifetimes of the radical pair states and induced strong temperature dependency of the lifetime. This indicates a switch of the ratelimiting step from the charge recombination process itself to a slower phonon-bottleneck spin-lattice relaxation process of interconversion between triplet sublevels. Abbreviations: An-Cl-NQ, 3-anilino-2-chloro-1,4-naphthoquinone; ESE, electron spin echo; ESEEM, electron spin echo envelope modulation; P700, the primary donor chlorophyll a in PS I; phyQ, phylloquinone ) 2-methyl-3-phytyl-1,4-naphthoquinone; PS, photosystem; (NO2)3-Fl, 2,4,7-trinitro-9-fluorenone; (NO2)4-Fl, 2,4,5,7-tetranitro-9-fluorenone; (OH)4-AQ, 1,2,5,8-tetrahydroxy-9,10-anthraquinone. Acknowledgment. We are very grateful to Drs. Jau Tang of Argonne National Laboratory and Tatsuhisa Kato of Institute for Molecular Science (Japan) for their helpful discussions. This work was supported by Grants-in-Aid on Priority-AreaResearch, Decoding Earth Evolution Program (09214220), Molecular Biometallics (09235235), Single Electron Devices (09233222) to S. I. from the Ministry of Education, Science, Sports, and Culture, Japan, and by Hyogo Science and Technology Association to A.K. References and Notes (1) Takahashi, Y.; Hirota, K.; Katoh, S. Photosyn. Res. 1985, 6, 183. Schoeder, H.-U.; Locau, W. FEBS Lett. 1986, 199, 23. (2) Brettel, K.; Setif, P.; Mathis, P. FEBS Lett. 1986, 203, 220. Rustandi, R. R.; Snyder, S. W.; Feezel, L. L.; Michalski, T. J.; Norris, J. R.; Thurnauer, M. C.; Biggins, J. Biochemistry 1990, 29, 8030. (3) Itoh, I.; Iwaki, M.; Ikegami, I. Biochim. Biophys. Acta 1987, 893, 508. Itoh, S.; Iwaki, M. FEBS Lett. 1989, 243, 47. (4) Brettel, K. Biochim. Biophys. Acta 1997, 1318, 322. Malkin, R. AdVances in Photosynthesis; Ort, D. R., Yocum, C. F., Eds.; Kluwer Academic Publishers: Dordrecht, The Netherlands; 1996, Vol. 4, pp 313332. Golbeck, J. H. The molecular Biology of Cyanobacteria; Bryant, D. A., Ed.; Kluwer Academic Publishers: Dordrecht, The Netherlands; 1994, pp 319-360. (5) Iwaki, M.; Itoh, S. Plant Cell Physiol. 1994, 35, 983. (6) Schubert, W.-D.; Klukas, O.; Krauss, N.; Saenger, W.; Fromme, P.; Witt, H. T. J. Mol. Biol. 1997, 272, 741. Fromme, P.; Witt, H. T. Biochim. Biophys. Acta 1998, 1365, 175.

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