J. Phys. Chem. 1994,98, 10335-10342
10335
Primary Photochemical Processes in WOO-Enriched Photosystem I Particles: Trap-Limited Excitation Decay and Primary Charge Separation Shigeichi Kumazaki? Hideki Kandori,t?$Hrvoje Petek,ts Keitaro Yoshihara,*J and Isamu Ikegami" Institute f o r Molecular Science, Myodaiji, Okazaki 444, Japan, and Faculty of Pharmaceutical Sciences, Teikyo University, Sagamiko, Kanagawa 199-01, Japan Received: April 25, 1994; In Final Form: July 13, 1994@ The energy transfer and primary charge separation in photosystem I (PS I) reaction center (RC) particles with an antenna size of 12 chlorophyllsP700 were studied by subpicosecond transient absorption spectroscopy. Upon excitation of the particles at 638 nm under the donor chlorophyll (P700)-neutral conditions, the transition from the excited state of chlorophylls to the charge-separated state of P700'Ao- (Ao; acceptor chlorophyll a ) proceeded with a time constant of 6.5 ps at -278 K. This time constant was obtained from the absorption increase of P700'Ao- at 740 ( f 1 0 ) nm, where excited chlorophylls gave only a negligible absorbance change. The distribution of electronic excitation energy among different chlorophyll forms, which was monitored in the wavelength region 650-720 nm, did not show any significant change from 500 fs to 12 ps under both the P700-preoxidized and -neutral conditions. Comparison of the experimental distribution with simulated ones suggests that the excitation energy is distributed within 500 fs highly on P700 and also among other higherenergy chlorophyll forms. On assuming a Boltzmann distribution of excitation energy among all the chlorophyll forms in the particles at 278 K, the intrinsic time constant of the electron transfer from the excited state of P700 to A0 is estimated to be 3.0 ps.
1. Introduction The initial step in photosynthesis is light absorption, electronic excitation energy transfer among chlorophylls, and subsequent charge separation. The conversion of excitation energy into energy for the charge separation is highly efficient. However, there is a variety in designs of antenna pigments and electron carriers in the reaction centers (RCs) of various photosynthetic organisms. The isolated RC complex of purple bacteria contains only six pigment molecules.' At least four of them are directly involved in the primary charge separation2 This relative simplicity has enabled us to observe the photochemical processes by selective excitation of the so-called special air.^^^ The mechanism of the sequential electron transfer in the RC has been related to the three-dimensional structure clarified by the X-ray crystallographic ~ t u d i e s . ~ - ~ The RC core complex of photosystem I (PS I) includes more than 100 antenna chlorophylls. The absorption spectra of the different chlorophyll forms in the RC of PS are more highly overlapping than in that of purple bacteria. The absorption spectra of the primary donor P700 and acceptor chlorophyll A0 are included in the congested spectra as suggested by other timeresolved s t u d i e ~ . ' ~ - 'Their ~ spatial arrangement in the PS I complex has been proposed in a recent X-ray crystallographic study.I4 In addition, the amino acid sequence of PS I polypeptides shows almost no homology to that of purple bacteria nor to that of photosystem 11 (PS II).15J6 It is interesting to compare the mechanisms in the different types of RCs to extract some essential features in the optimized systems for energy collection and the following charge separation. An arrangement of the molecules in the electron relay system in the PS I RC was proposed on the basis of an X-ray Institute for Molecular Science.
* Present address: Department of Biophysics, Faculty of Science, Kyoto University, Kyoto 606, Japan. * Present address: Advanced Research Laboratory, Hitaxhi, Ltd., Hatoya+
ma, Saitama 350-03, Japan. 'I Tekyo University. Abstract published in Advance ACS Abstracts, September 1, 1994. @
0022-365419412098-10335$04.50/0
crystallographic study with a 6 8, res01ution.l~ The primary donor in the electron transfer chain seems to be a dimer of chlorophyll a molecules (P700). Two monomeric chlorophyll molecules are located close to P700. Their positions are analogous to those of the monomeric bacteriochlorophyll (BL and BM)in the RC of purple bacteria. The acceptor chlorophyll Ao, which is thought to transfer an electron to the phylloq ~ i n o n e , ~ is ~ Jtentatively ~J~ located to be 12 8, apart from one of the monomeric chlorophyll molecules close to WOO. l4 Selective excitation of P700 may be difficult due to the spectral congestion and to the presence of most red-shifted antenna chlorophylls (long-wavelengthabsorbing chlorophylls) with adsorption peaks even redder than that of WO0.18319 Nonselective excitation mainly induces absorbance changes due to excited antenna chlorophylls. Wasielewski et al. reported that the electron transfer from P700* (excited state) to A0 proceeds with a time constant of 13.7 ps upon nonspecific excitation at 610 This time constant seems to be relatively slow compared to the primary charge separation in the RCs of purple bacteria (tile = 2.8-4.1 ps)3,4,20and PS I1 (tile = 3.0 ps,21but see also ref 22 (21 ps)). The electron transfer from P700* may compete with back energy flow to other chlorophylls (detrapping). Energy flow from WOO to minor long-wavelength absorbing chlorophylls (downhill energy flow) seems to be significant due to the overlap of the P700 emission spectrum with the absorption spectra of the long-wavelength absorbing chlorophyll^.^^ Thermally-assisted excitation transfer from lower- (e.g. P700) to higher-energy chlorophyll forms (uphill energy flow) has been s u g g e ~ t e d .The ~ ~possible ~ ~ ~ ~distribution ~~ of excitation energy not only on P700 but also among other chlorophylls may slow down the overall charge separation rate.26 The intrinsic rate constant of the electron transfer from P700* to is important for a reliable kinetic modeling of the primary processes in the PS I core c o m p l e ~ . ~In~order . ~ ~ to know the intrinsic rate constant, previously estimated rate constants of overall primary charge separation should be reanalyzed with consideration of the distribution of excitation energy among 0 1994 American Chemical Society
10336 J. Phys. Chem., Vol. 98, No. 40, 1994 chlorophylls. Some recent estimations of the primary electron transfer were based on the assumption that transient absorbance changes due to excited chlorophylls are independent of the redox state of P700 (antenna subtraction m e t h ~ d ) . ' ~ , 'A~ direct measurement of the absorption of Ao- which is independent of the dynamics of excited chlorophylls is highly desired. The present investigation concerns the distribution of excitation and the electron transfer rate constant from P700* to AD. PS I RC particles with an antenna size of 12 (number of chlorophylls per P700 in one RC unit) are used to obtain large absorbance changes by the primary charge separation. Simultaneous detection of the bleach of A0 around 685-690 nm and the rise of the absorption of Ao- around 750 nmZ8are applied by probing a wide spectral region (650-800 nm). Lack of the minor long-wavelength absorbing chlorophylls in the present particle^^,^ may concentrate excitation energy mainly on WOO. The overall primary charge separation is expected to be faster than those in other s t ~ d i e s . ~Previous ~ , ~ ~ , Gaussian ~~ decomposition of the absorption spectra of these particles is used in order to know what and how many chlorophyll forms exist in the present s y ~ t e m .This ~ ~ ~information is used to simulate the spectral shape of the transient bleach of the particles. Delocalization of excitation energy in the system is studied by comparing simulated spectra with experimental ones. The intrinsic rate constant of the electron transfer from P700* to A0 is estimated on the basis of a distribution of excitation and of observed overall primary charge separation rate constants.
Kumazaki et al.
-80
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Wavelength (nm) Figure 1. Series of transient absorption spectra at representative delay
2. Experimental Section The P700-enriched particles was prepared as described p r e v i o ~ s l y . In ~ ~brief, the lyophilized spinach PS I particles obtained by digitonin treatment were extracted twice with diethyl ether that contained water at 70% saturation. They were solubilized with 20 mM phosphate buffer (pH 8) containing 0.2% Triton X-100 by incubation for 30 min. The insoluble, grayish-white material was removed by centrifugation, and the blue-green supernatant, which had a Chl (chlorophyll a)/P700 ratio of 12, was used in the subsequent experiments. Transient absorption spectra were measured by using a double-beam spectrometer and an amplified subpicosecond dye laser, as was described p r e v i o ~ s l y . ' The ~ ~ ~dye ~ ~laser ~ ~ was replaced by a subpicosecond system (Coherent SATORI, Model 774). The pulses at 638 nm with a full width at half maximum of 200 fs were used at a repetition rate of 10 Hz. This beam was used to excite the sample and to generate a white light continuum for probing. The pump and probe beams overlapped with a 5" angle in a flow cell with a 4 mm path length through the sample. The polarization angles of the two beams were set parallel. The excitation light at the sample had a beam diameter of about 2 mm, while the probe beam was mildly focused into this excited area to give a beam diameter of about 0.5 mm. The absorbance at the 676 nm peak of the sample was adjusted to 1.3. The intensity of the pump beam was adjusted to between 0.2 and 1.3 photons absorbed per RC (=photons/RC). This estimation is based on the cross section of the P700-enriched particles at 638 nm and excitation light i n t e n ~ i t i e s . ~The ~ excitation intensity was varied by inserting neutral density filters in the pump beam branch. The filters which adjusted the shape and intensity of the white light continuum in the probe beam branch were not changed in all experiments. The instrument response function was measured by observing the instantaneous bleach of Oxazine 750 in 1-chloronaphthalenein the same cell. The 10-90% rise time of the bleach between 660 and 720 nm was 500 fs. Since the excitation intensity was adjusted by neutral density filters, the zero delay was set at the time for the
times: solid lines, under the P700-neutral conditions; broken lines, under the P700-preoxidized conditions. Excitation intensity was set at 0.8 photons absorbedlRC.
half rise of the bleach at 680 nm under the WOO-preoxidized conditions at each intensity level. Because of the chirp of the white light continuum, the time zero at each probing wavelength was corrected by measuring the cross correlations between the pump and the probe pulses at several wavelengths in a BBO crystal with a thickness of 0.5 mm. The full width at half maximum of the cross correlations was 250-280 fs between 650 and 800 nm. The sample was kept in a reservoir and was circulated by a peristaltic pump to avoid denaturation. The reservoir was cooled in an ethylene glycol bath with temperatures in the -3 to 0 "C range. The temperature of the sample during the measurement was kept below 278 K. In each set of measurements, the sample in the presence of 0.2 mM femcyanide (P700-preoxidized conditions) was measured first. P700+ was then reduced by addition of 10 mM ascorbate and 50 pM 2,6-dichlorophenolindophenol,and the measurements were repeated (WOO-neutral conditions). When quinone was added to the sample, an aliquot of 2-methyl-1,Cnaphthoquinone (vitamin K3) in ethanol was added directly to the suspension of the particles to give a final concentration of 40 pM quinone. 3. Results
3.1. Transient Absorption Spectra under the WOONeutral and -Preoxidized Conditions. Figure 1 shows a series of the transient absorption spectra under the P700-neutral (solid lines) and -preoxidized (P700+) (broken lines) conditions. The excitation intensity was set at 0.8 photonsiRC. At 500 fs, a main bleach (negative absorbance change) between 650 and 710 nm and a positive absorbance change in the wavelength region longer than 750 nm are observed (Figure l a and a'). Under the P700-preoxidized conditions, the bleach is mainly due to superposition of the ground state (SO)depletion and stimulated
WOO-Enriched Photosystem I Particles emission from the singlet excited state (SI) of chlorophylls. The positive signal is due to the absorption from SI. A slight negative absorbance change between 730 and 750 nm probably represents a stimulated emission from the vibronic bottom in S1 to a vibrationally excited state in The transient spectrum at 500 fs under the P700-neutral conditions had an extra bleach at 685-710 nm and an extra absorption at 710-800 nm compared to that under the WOO-preoxidized conditions (Figure l a and a’). They are attributed to the absorbance changes due to excitation of WOO. From 500 fs to 36 ps, the bleach and positive absorption under the P700-preoxidized conditions decay rapidly (Figure lb-d and b’-d’). These represent the ground state recovery of the chlorophylls. Under the P700-neutral conditions, however, the bleach remains much larger than that under the WOO-preoxidized conditions (Figure lb-d). Simultaneously, a broad positive absorption appears in the longer-wavelength region (710-800 nm) from 500 fs to 36 ps (Figure 1c’-e’). From 36 to 230 ps, the change of the spectral shape is quite slow in both the redox states of P700 (Figure Id-e and d’-e’). The persistent broad extra bleach at 670-700 nm under the P700-neutral conditions reflects the ground state depletion of both P700 and A0.11-13 The charge-separated state of WOO+&- is also reported to show a broad absorption in the near infrared region.28 In the 720800 nm wavelength region, the positive absorption remained under the P700-neutral conditions. The charge-separated state has a lifetime of 47-53 ns in the quinone-depleted particles under the P700-neutral condition^.^^^^^ Therefore, the extra absorbance changes observed under the WOO-neutral conditions can mostly be attributed to the formation of the charge-separated state (P700Ao P700+Ao-). During the preparation of P700-enriched particles, the acceptor phylloquinone is almost completely removed.’ 1~13,36 The addition of quinones with appropriate redox potentials accelerates the recovery of Ao, which has a reported absorption peak at around 685-690 nm.10311$13317 Transient absorption spectra were measured at 230 ps with vitamin K3 (2-methyl-1,4naphthoquinone)-added samples (Figure 2a). The bleach around 675-690 nm decayed within 230 ps on addition of vitamin K3 under the WOO-neutral conditions (Figure 2a). Thus, subtraction of the spectrum of WOO-neutral with quinone from that of WOOneutral without quinone gives the difference spectrum of Aoand A0 (solid line in Figure 2b) (hereafter abbreviated as Ao-/ Ao). Long-lived bleach of chlorophylls under the P700-neutral conditions was shown to be well-approximated by that under the P700-preoxidized condition^.'^*^' Subtraction of the spectrum of P700+ without quinone3*from that of P700-neutral with quinone gives the difference spectrum of P700+ and P700 (broken line in Figure 2b) (likewise denoted as P700+P700). The Ao-/Ao is in good agreement with the previously reported except that the peak position of the bleach (683 nm) is blue-shifted by about 3 nm.13 Such a blue shift of the peak position of the difference spectrum has been observed with decreasing number of antenna chlorophylls (data not shown). Note the broad positive absorbance change of Ao-/Ao in the near infrared wavelength region (720-800 nm) which is more prominent than that of P700+/P700. 3.2. Transient Absorption Spectra and Kinetics at Different Excitation Light Intensities. When excitation light induces multiple excitation (two or more excitations) of a single RC, interactions of two or more excited states may take place followed by anomalously fast decay of excited states (annihilat i ~ n ) .The ~ ~ contribution of the multiple excitation in the transient signals was estimated. The transient absorption spectra of WOO-neutral particles at 500 fs under different excitation
-
J. Phys. Chem., Vol. 98, No. 40, 1994 10337
P700+, without quinone -
2 -20 m
e
20 -40 P700-neutra1, without quinone
-60 I
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,
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7 0-
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0
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720 760 Wavelength (nm)
Figure 2. (a) Transient absorption spectra at 230 ps for particles with three different preparations. Excitation intensity was set at 1.3 photons absorbed/RC: P700+ without quinone, P700-preoxidized particles without any addition of quinone; P700-neutral without quinone, P700neutral particles without addition of quinone; €700-neutral with quinone, P700-neutral particles reconstituted with 2-methyl- 1.4-naphthoquinone. (b) Solid line, difference spectra of &-/&, which is given as the difference between €700-neutral with quinone and €700-neutral without quinone spectra; broken line, difference spectra of P7OO+P700, which is given as the difference between P700-neutral with quinone and WOO+ without quinone spectra. 0.2 ,I
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I 650
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1
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-0.2 photons / RC 660
680 690 700 Wavelength (nm)
6jO
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Figure 3. Transient absorption spectra at 500 fs on excitation of 0.2 and 1.3 photons absorbed/RC under the P700-neutral conditions.
light intensities are normalized and shown in Figure 3. At the stronger excitation intensity, the spectrum was slightly shifted to the blue. This is also the case under the P700-preoxidized conditions (data not shown). The time profile of absorbance changes induced by excitation pulses of different intensities is shown in Figures 4-6. The excitation intensities were 0.4,0.8, and 1.3 photons per RC. At 740 nm, the positive absorbance changes due to P700+ and Aowere monitored (Figure 2b).28 Under the P700-preoxidized conditions, a slow absorbance increase was observed at the highest excitation intensity (Figure 4a; the spectrum of the remaining positive absorbance changes is shown in Figure 2a), while a small negative absorbance change in the time domain of less than 10 ps was observed at lower excitation intensities (Figure 4b and c). This reflects the decay of stimulated emission. The kinetics under the P700-neutral conditions are fitted by a single-exponential curve with time constants of 6.4,
10338 J. Phys. Chem., Vol. 98, No. 40, 1994 154
Kumazaki et al.
c
8.6 (7.6 9.8) ps
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Figure 4. Time dependence of averaged difference absorbance at 740 (f10) nm on excitation of 1.3,0.8, and 0.4 photons absorbedRC.The open circles represent the data under the WOO-preoxidized conditions, and closed circles represent the data under the WOO-neutral conditions. The solid lines are single-exponential decay curves fitted to the data points. The open squares represent the difference between the two fitted curves (WOO-preoxidizedminus WOO-neutral). The open squares approximate the absorbance increase mainly due to &- (see text). The broken lines are single-exponential decay curves fitted to the open squares. The time constant and error ranges were estimated from the fitting and are shown in the figure.
6.5, and 8.6 ps for the excitation conditions at 0.4,0.8, and 1.3 photons/RC, respectively. When the excitation light intensity is weaker than 0.8 photons/RC (namely, 0.4 and 0.8 photons/ RC), the fittings for the kinetics give almost the same time constant (6.4 and 6.5 ps). The time constant of the overall primary charge separation is concluded to be 6.5 ps. The absorbance change at around 740 nm in P700+/P700 is only about 20% of that in Ao-/Ao (Figure 2b).28 The time constant of the rise of Ao- thus seems to be quite close to 6.5 ps. Interestingly,the difference between the kinetics under the WOOpreoxidized and -neutral conditions (antenna subtraction method) gives almost the same time constant of 6.0-6.5 ps for all the excitation conditions employed (Figure 4a-c). At 680 nm, the ground state recovery of antenna chlorophylls and the disappearance of A0 absorption were monitored (Figure 5a-c). The contribution of the absorbance changes by P700 is negligible at this wavelength (Figure 2b). The bleach under the WOO-preoxidized conditions (open circles) always recovers faster (ground state recovery) than that under the P700-neutral conditions (closed circles). After about 50 ps, the decays in the particles under both the redox conditions reached a plateau which persisted for up to 400 ps (the maximum delay examined). In other s t u d i e ~ ,the ~ ~difference ,~~ of kinetics at 685 or 690 nm between the P700-neutral and -preoxidized states was used to estimate the time constant for the bleaching of A0 (antenna subtraction method). The same method was applied to the kinetics at 680 nm in Figure 5a-c (open squares), which gave
(C) -0.4 photons / RC I
0
20
,
40
I
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Time (ps)
80
c
100
Figure 5. Time dependence of averaged difference absorbance at 680 ( f 2 ) nm on excitation with 1.3, 0.8, and 0.4photons absorbed/RC. Two types of symbols (open and closed circles) are used in the same
manner as in Figure 4. The solid lines are double-exponential decay curves fitted to the data points. The open squares represent the difference between the two fitted curves (WOO-preoxidized minus WOOneutral). This approximates the amount of bleach of & due to its reduction (antenna subtraction method). The broken lines are singleexponential decay curves fitted to the open squares. a time constant of 5.3 ps for the range of excitation intensities employed. This time constant is quite close to that for the overall primary charge separation (6.5 ps). At 700 nm, which is an isosbestic point of the difference spectrum of Ao-/Ao (Figure 2b), the bleach of P700 was monitored (Figure 6a-c). The initial amplitude of the bleach is much greater under the P700-neutral conditions than under the WOO-preoxidized conditions. A part of the initial signal under the P700-neutral conditions (solid circles) decays rapidly at the highest excitation intensity (Figure 6a), while it grows slightly at the lowest excitation intensity (Figure 6c). The initial rapid decay on the highest excitation intensiJy can be explained by the contribution of annihilation. A gradual increase of the bleach by P700 (tile = 15 ps) upon an excitation of relatively low intensity has been observed previously in the PS I RC complex with a larger antenna size.37 In the upper half of Figure 6c, the approximate amount of the bleach by P700 (open squares) is decomposed into two components. On an assumption that there is no kinetically-resolvable intermediary electron carrier between P700 and Ao, P700+ (thick, solid line) is expected to increase exponentially with the time constant of 6.5 ps (overall primary charge%eparation). The remaining bleach of P700 indicates the population of excited WOO (WOO*) (broken line). P700* rose with a time constant of less than 500 fs and decayed with the time constant of 6.5 ps on the assumption. 3.3. Spectral Change of the Transient Bleach Due Mainly to Excitation of Chlorophylls. The bleach in the wavelength region between 650 and 720 nm reflects the distribution of
P700-Enriched Photosystem I Particles
J. Phys. Chem., Vol. 98, No. 40, 1994 10339
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Figure 6. Time dependence of averaged difference absorbance at 700
(f2) nm on excitation of 1.3.0.8, and 0.4 photons absorbed/RC. Three types of symbols (open circles, closed circles, and open squares) are used in the same manner as in Figure 5 . The solid lines are fitted curves to the data. The open squares approximate the amount of bleach of P700 due to its excitation and subsequent oxidation. In the upper half of part c, the open squares are decomposed into two components. The broken line and thick, solid line represent the bleach by excitation and oxidation of P700, respectively. See text for details. I
-
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Wavelength (nm) Figure 7. Normalized transient spectra compared at 500 fs (solid line), at 12.4 ps (open squares),and at 37 ps (closed circles) under the P700preoxidized conditions. excitation among the different chlorophyll forms. In order to avoid the contribution of annihilation, the transient spectra with an excitation intensity at 0.4 photons/RC are analyzed. The normalized transient spectra at 500 fs, 12.4 ps, and 37 ps under the P700-preoxidized conditions are shown in Figure 7. The spectrum at 500 fs coincides well with the one at 12.4 ps. The spectrum at 37 ps is broader to the red than the one at 500 fs. Such a red shift of the bleach has been observed previously with the same type of particles." We tentatively estimated the distribution of excitation energy under the P700-neutral conditions as follows. The transient
40
50
Figure 8. (a) Series of calculated transient absorption spectra only due to excited chlorophylls under the P700-neutral conditions. The absorbance changes due to the charge separation were subtracted by the method explained in the text. The spectrum at 500 fs was reduced to that at 13.2 ps in order to compare the spectral shapes (dots). (b) Decay profile of excited chlorophylls under the P700-neutral conditions.
Closed circles are the averaged absorbance changes at 680 ( f 2 ) nm in the series of spectra shown in part a. The solid line is a singleexponential decay curve fitted to the data points. The optimum time constant was 9.1 (8.1-10.4) ps. signals under the P700-neutral conditions are the sum of absorbance changes due to excited chlorophylls and to the primary charge separation between WOO and &. The contribution of the former decreases while that of the latter increases with time. The contribution of P700Ao WOO+&- at a certain delay time can be measured by the amount of the positive absorbance changes at around 740 nm, where excited chlorophylls have little absorption (cf. Figure 4b and c). The difference spectrum for P700+Ao-/P700& (Figure 2b) was normalized and subtracted from the transient spectrum by minimizing the absorbance changes between 720 and 750 nm. The resultant spectra which now contain only the absorbance changes due to excited chlorophylls (including P700*) are shown in Figure 8a. The spectral shape did not change from 500 fs up to 13.2 ps (Figure Sa). The bandwidth of the bleach for WOO-neutral particles is broader to the red side than that for WOO-preoxidized particles (Figures 7 and 8a). Spectral broadening to the red side with time was also observed at 120 ps under the P700-neutral conditions (Figure 8a). The kinetics at 680 (3~2)nm calculated from the series of spectra in Figure 8a was fitted to a single exponential function with a time constant of 9.1 (8.1-10.4) ps (Figure 8b). This time constant was in good agreement with the fast component of the fluorescence decay (9 ps), which was previously measured by Kamogawa et al. in PS I particles with a ChlP700 ratio of 8-10 under the P700-neutral condition^.^^ In summary, the decay rate constant of excited chlorophylls was slightly larger in the P700-neutral particles ((9.1 ps)-l in Figure 8b) than in the P700-preoxidized ones ((11.0 ps)-' in Figure 5c40) with an excitation intensity of 0.4 photons/RC. In both the redox states of P700, the distribution of excitation
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10340 J. Phys. Chem., Vol. 98, No. 40, 1994
Kumazaki et al.
among different chlorophyll forms did not change from 500 fs to 12 ps and spectral broadening to the red side becomes noticeable at later delay times.
4. Discussion The overall transition rate constant from the excited state of chlorophylls to the charge-separated state of P700+Ao- in the WOO-enriched PS I RC was determined to be (6.5 ps)-' without interference of the excited state of chlorophylls. The rate is deduced from the simultaneous measurements of the depletion of A0 and rise of Ao-. The contribution of excitation migration to the overall transition rate is discussed below. 4.1. Is Excitation Decay in WOO-Enriched Particles TrapLimited or Diffusion-Limited? In the diffusion-limited model of excitation decay, irreversible energy flow from antenna chlorophylls to P700 is the rate-determining step for the decay. In the trap-limited electronic excitation energy is rapidly exchanged among chlorophylls and the decay rate is determined by the electron transfer from the primary donor to the acceptor. In the present experiments, the shape of the bleach due to excited chlorophylls remained unchanged from 500 fs to 12 ps (Figures 7 and 8a). The distribution of excitation energy among chlorophylls seems to be equilibrated within 500 fs, even though the chlorophylls are not selectively excited at 638 nm. Under the P700-neutral conditions, the decay rates of the excited states of different chlorophyll forms are almost constant (tile = 9.1 ps) in the wavelength region 660-705 nm (Figure 8a and b). The decay time constant is almost comparable to the rise time constant of P700+Ao- (6.5 ps). These results suggest that the transient signals at 500 fs consist mainly of the bleach of chlorophylls which are rapidly exchanging excitation energy with P700 in the subpicosecond time scale and that excitation is quenched at P700 in a few picoseconds. Although the broadening of the bleach due to excited chlorophylls at 37 ps or later (Figures 7 and 8a) may suggest an energy flow from high- to low-energy chlorophylls, such a slow energy transfer is not associated with the primary charge separation which was substantially finished within 12 ps (Figure 4b and c). Therefore, the trap-limited model of excitation decay seems to be preferred to the diffusion-limited one in the P700-enriched PS I particles. 4.2. Estimation of the Intrinsic Rate Constant of the Electron Transfer from WOO* to Ao. In the trap-limited model, the intrinsic rate constant of the electron transfer from P700* to A0 can be estimated if the distribution of excitation energy among chlorophylls is known.26341 The bleach at 500 fs under the P700-neutral conditions with an excitation intensity at 0.2 photonsRC (Figures 9c) had a wider bandwidth (710 (h30) cm-I in full width at e-' of the maximum ( W e - ' M)) than that for the bleach of a monomeric chlorophyll a in ether (-410 cm-' ( W e - ' M)).22 In both the redox states of P700, the distribution of excitation among different chlorophyll forms did not change from 500 fs to 12 ps. The observation indicates a redistribution of excitation energy among several chlorophyll forms within 500 fs. We simulated transient absorption spectra by using the ground state absorption spectrum which has previously been decomposed into several chlorophyll form^.^,^ First, the ground state absorption spectrum was fitted by the sum of Gaussian spectra Ai (v,wi, vi) for the ith chlorophyll form) multiplied with the assumed numbers of each chlorophyll Ni, where v is the frequency of the light and vi and wi are the peak position and the bandwidth ( W e - ' M). Figure 9a shows the ground state absorption spectrum of the particles with neutral P700 at -278 K and a simulated spectrum using the parameters in the figure c a p t i ~ n . ~For . ~ a simulation of transient absorbance
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c
15.5h O3
Figure 9. (a) Simulated ground state absorption spectra of the RC particles under the P700-neutral conditions (solid line) and an experimental one (broken line). The solid line is a weighted sum of chlorophyll spectra with the following parameters. Six chlorophyll forms (1-6) were assumed with absorption peak position (nm), bandwidth (cm-I), and number in one RC unit as follows: (1) 689, 598, 2.0 (P700); ( 2 ) 685, 456, 1.2 (A& (3) 681, 377, 2.0; (4) 674, 430, 2.0; ( 5 ) 669, 459, 3.1; (6) 659, 485, 1.1. These parameters are taken from ref 9 (Gaussian decomposition of the spectra of 11 chlorophylldP700 particle at 90 K) after the following modifications. On increasing the temperature from 90 to 278 K, the absorption peak positions were assumed to show blue shifts by 5 nm for P700 and 1 nm for the other chlorophylls." The bandwidths were increased by 35-50%. (b and c) Experimental (dots) and simulated (solid line) transient absorption spectra at 500 fs under the P700-neutral conditions. The dots represent the experimental transient absorbance changes with excitation intensity at 0.8 (b) and 0.2 (c) photons absorbed/RC. The solid lines are weighted sums of the chlorophyll bleaches given by eqs 1-3 in the text. In part c, the area fiiled with vertical lines indicates the contribution by WOO in the simulated spectrum (solid line). change, the bleach of chlorophyll form i is approximated by the sum of ground state depletion and the stimulated emission from SI as in eq 1.
In eq 1 the stimulated emission is assumed to be exactly the mirror image of the absorption spectrum with a peak shift of Avi. A Stokes shift of 105 cm-' (-5 nm) for WOO was assumed on the basis of the excitation and emission spectrum in ref 8. The same Stokes shift was applied to the other chlorophyll In the wavelength region between 650 and 720 nm, the shape of the absorption spectrum of S1 of chlorophyll a seems to be quite broad compared to that of S0?2,42 Since, however, only the spectral shape is concerned, it is not necessary to include the absorption spectrum of SI in eq 1. We also assumed an infinitely fast excitation redistribution to the
J. Phys. Chem., Vol. 98, No. 40, 1994 10341
P700-Enriched Photosystem I Particles Boltzmann distribution at a temperature of 278 K. A finite time scale of energy exchange will be reconsidered later in this section. The probability for the electronic excitation energy to be on the chlorophyll form i (Pi) is proportional to the amount of the chlorophyll form in one RC unit (Ni) and the Boltzmann factor as
P i = Niexp(-(Ei - Ep)lkBT) where Ei and E p are the vibrationally relaxed energy levels of the excited states of i and P700*, respectively, k~ is the Boltzmann constant, and T is the absolute temperature. The energy level E , is assumed to be vi - Avil2. The transient spectrum is now given by U ( V ) = ~PiAAi(v,wi,vi,Avi)
(3)
1
The calculated spectral shape based on eq 3, in which all the chlorophyll forms in the figure caption participate, is shown in Figure 9b and c (solid lines). It is normalized and fitted to the experimental spectra (at 500 fs, under the P700-neutral conditions) on the lower-energy side. The mismatch (difference) between the experimental spectra and the simulated ones on the higher-energy side has a peak at -676 nm (-14 800 cm-l) and decreases with decreasing the excitation light intensity. This is calculated to be about 49, 39, 29, and 18% of the total experimental signal upon excitation of 1.3, 0.8, 0.4, and 0.2 photons/RC, respectively (cf. Figure 3). Thus, the transient spectrum at 500 fs induced by an excitation pulse of even lower intensity (even lower than 0.2 photons/RC) seems to be wellsimulated by eq 3, in which all the chlorophyll forms in the present particles participate. The simulated bleach of P700 is indicated by the vertical lines in Figure 9c. It amounts to 46% of the total simulated spectrum (broken line in Figure 9c), indicating that the probability for excitations of residing on WOO ( P p 7 ~is) 0.46.26.43 This delocalization of excitation energy slows down the overall primary charge separation. We now estimate the intrinsic rate constant of the electron transfer from P700* to Ao. The rate constant of the overall primary charge separation measured from the absorbance change at around 740 nm (k740) (Figure 4b and c) is given by the product of P p 7 ~and the intrinsic rate constant of the electron transfer from P700* to Ao (kcs). (4) Using eq 4, kcs is calculated to be (3.0 ps)-'. The calculated rate constant is as large as those reported for the purple bacterial RCs ((2.8-4.1 P S ) - ' ) . ~ ~ ~The ~ ~ similarity O may reflect common features of the RCs. A structural analogy between the RCs of purple bacteria and PS I was implied by recent X-ray crystallographic ~tudies.~,'J~ The energy gap for the reaction P7OO*Ao P700'Ao- (0.2-0.3 eV)la$u is also similar to that for the corresponding step (P*H P+H-: P, special pair; H, bacteriopheophytin) in purple bacterial RCs (0.16-0.26 eV).45-47 A finite time scale of energy exchange between P700 and other chlorophylls is now reconsidered. A simple model for the energy exchange and the primary charge separation at P700 is given as
-
-
Chl*P700&
k,
km
ChlP700*Ao
-ChlP700+A0kcs
where Chl represents a pool of antenna chlorophylls and km and ~ D are T the rate constants for energy transfer from the pool
to WOO and from WOO to the pool, respectively. In the limit of km kDT >> ks,a fast redistribution of excitation proceeds with a time constant of 1I(km ~ D T )until equilibration of the excitation energy between WOO and the pool is established. This limit corresponds to the trap-limited For the time scale of single-step energy transfer in the PS I RC complex, values of 150-300 fs48 or 220 fs49have been proposed. These time constants probably give a reasonable estimation of both l / k ~ ~ and l k m , since the present measurements have shown a rapid equilibration within 500 fs (Figures 7 and 8a). The estimated ilks of 3.0 ps is much longer than lI(km ~ D T ) ,which is estimated to be '0.5 ps. The estimation also suggests that the excitation decay in P700-enriched RC particles is essentially trap-limited. Thus, eqs 2-4 seem to be applicable to the present study. 4.3. Antenna Size Dependence of the Rate Constant for Overall Primary Charge Separation. According to eqs 2 and 3, the probability of excitation energy localizing on P700 ( P p 7 ~ ) becomes smaller with the increase of number of chlorophylls which are excited together with P700. This leads to a slower overall charge separation (eq 4). The rate constant for the bleaching of A0 has been investigated in PS I RC particles with a larger number of antenna chlorophylls (16- 100 chlorophyllsl P700).12J3,37 The rate constants obtained by the antenna subtraction method are (5.3 ps)-' in 12 ChW700 particles in the present study, (11 ps)-' in 16 ChW700 particle^,'^^^^ (8 ps)-' in 30 Chvp700 particle^,'^^^^ and (13.7 ps)-' in 30-40 ChW700 particles.'2 The relationship between the rate constants and antenna size roughly reflects a larger delocalization of excitation energy in the particles with more antenna chlorophylls. Fluorescence decay measurements may be more suitable to study the effect of antenna size on the overall primary charge separation rate. The lifetimes of fluorescence decay measured in the previous ~ t u d i e sshow ~ ~ , an ~ ~approximate linear relationship between lifetime and the antenna size. Owens et al. estimated the intrinsic rate constant for the electron transfer from WOO* on the basis of the results of the antenna size dependence of fluorescence lifetime and a lattice model of antenna chlorophylls for excitation migration and trapping.49 The predicted intrinsic rate constant for the electron transfer from P700* was (3.4 P S ) - ' . ~ ~Trissl et al. suggested a rapid equilibrium of the excitation energy among many antenna chlorophylls according to their excited state energy levels and abundances in the particles with an antenna size of 213.43 On the basis of the trap-limited model and the trapping time (tile = 70 ps), they predicted an intrinsic rate constant of (2-3 ps)-' for the electron transfer from P700*.43 The trap-limited model for the excitation decay seems to be applicable to particles with a smaller antenna size. 4.4. Excitation Decay in P700-Enriched Particles Which Is Not Associated with the Primary Charge Separation. The excitation decay under the WOO-neutral conditions (tile = 9.1 ps) is slightly slower than the rise of the charge-separated state (tile = 6.5 ps). The lifetime of the excited states of chlorophylls would be increased in the presence of chlorophylls isolated from WOO in terms of energy e ~ c h a n g e and/or ~ ~ ~ ~of~particles ~* which are photochemically inactive to the charge separation. The longlived bleach at 120 ps (Figure 8a) may also indicate the presence of such isolated chlorophylls and/or photochemically inactive particles.11-13,37,53 Under the WOO-preoxidized conditions, quenching of excited chlorophylls was observed to be quite fast (tile = 11.0 ps). The decay kinetics of the excited state was reported to be almost independent of the redox state of P700 in the particles with a
+
+
+
10342 J. Phys. Chem., Vol. 98, No. 40, I994 ChllP700 ratio of 8-100.33,37v54955The distribution of the excitation energy is also similar in both redox states of P700. These similarities seem to support the previously used antenna subtraction method (analysis of electron transfers based on the differences of transient spectra between P700-neutral and -preoxidized conditions).12.13 Interestingly, the lifetime of excited chlorophylls under the P700-preoxidized conditions seems to be increased when the antenna size is larger. The lifetimes in the particles with different antenna sizes are 11.O ps (12 ChW700) in this study, 15 ps (25-31 ChW700),13.37 and 19.5 ps (42 Chl/P700).54 These results may suggest the presence of a quencher as efficient as neutral P700 even under the P700-preoxidized conditions.
5. Concluding Remarks The present study has shown a direct measurement of the rise of &- (tile = 6.5ps), which does not depend on the kinetics of excited chlorophylls. Ao- was also shown to appear simultaneously with the depletion of A0 (tile = 5.3 ps). The intrinsic rate constant of the electron transfer from P700* to A0 was estimated to be (3.0 ps)-'. This estimation is based on the trap-limited model, in which excitation energy is distributed among all the chlorophyll forms in one RC unit. A recent X-ray crystallographic study for the PS I RC14 suggested the presence of two monomeric chlorophylls (C) close to P700 which are analogous to the accessory chlorophylls in purple bacterial R C S . ~ -However, ~ we did not obtain any clear evidence for an intermediate state WOO+C-&, possibly because of the present spectral and time resolution.
Acknowledgment. We are grateful to Prof. S. Itoh and Drs. M. Iwaki and M. Mimuro of the National Institute for Basic Biology for their invaluable discussions on the present work and to Mr. Y. Jia of the University of Chicago for his helpful communication. This work was supported in part by a Cooperative Research Program in the Institute for Molecular Science and by the New Energy and Industrial Technology Development Organization. References and Notes (1) Straley, S. C.; Parson, W. W.; Mauzerall, D. C.; Clayton, R. K. Biochim. Biophys. Acta 1973, 305, 597. (2) Kirmaier, C.; Holten, D. Photosynth. Res. 1987, 13, 225. (3) Holzapfel, W.; Finkele, U.; Kaiser, W.; Oesterhelt, D.; Scheer, H.; Stilz, H. U.; Zinth, W. Proc. Natl. Acad. Sci. USA. 1990, 87, 5168. (4) Martin, J.-L.; Breton, J.; Hoff, A. J.; Migus, A,; Antonetti, A. Proc. Natl. Acad. Sci. USA. 1986, 83, 957. ( 5 ) Deisenhofer, J.; Epp, 0.;Miki, K.; Huber, R.; Michel, H. Nature 1985, 318, 618. (6) Deisenhofer, J.; Michel, H. EMBO J. 1989, 8, 2149. (7) Allen, J. P.; Feher, G.; Yeates, T. 0.; Komiya, H.; Rees, D. C. Proc. Natl. Acad. Sci. U S A . 1987, 84, 5730. (8) Iwaki, M.; Mimuro, M.; Itoh, S. Biochim. Biophys. Acta 1992,1100, 278. (9) Ikegami, I.; Itoh, S.Biochim. Biophys. Acta 1988, 934, 39. (10) Mathis, P.; Ikegami, I.; SCtif, P. Photosynth. Res. 1988, 16, 203. (1 1) Kim, D.; Yoshihara, K.; Ikegami, I. Plant Cell Physiol. 1989, 30, 679. (12) Wasielewski, M. R.; Fenton, J. M.; Govindjee. Photosynth. Res. 1987, 12, 181. (13) Kumazaki, S.; Iwaki, M.; Ikegami, I.; Kandori, H.; Yoshihara, K.; Itoh, S. J. Phys. Chem., submitted. (14) Krauss, N.; Hinrichs, W.; Witt, I.; Fromme, P.; Pritzkow, W.; Dauter, Z.; Betzel, C.; Wilson, K. S.; Witt, H. T.; Saenger, W. Nature 1993, 361, 326. (15) Kirsch, W.; Seyer, P.; Hermann, R. G. Curr. Genet. 1986,10, 843. (16) Golbeck, J. H.; Bryant, D. A. Light-Driven Reactions in Bioenergetics; Current Topics in Bioenergetics Series, Vol. 16; Academic Press: New York, 1991; p 83. (17) Itoh, S.; Iwaki, M. In Dynamics and Mechanisms of Photoinduced Transfer and Related Phenomena; Mataga, N., Okada, T., Masuhara, H., Eds.; Elsevier Science Publishers: Amsterdam, 1992; p 527.
Kumazaki et al. (18) Mimuro, M. Plant Cell Physiol. 1993, 34, 321. (19) Trinkunas, G.; Holzwarth, A. R. Biophys. J. 1994, 66, 415. (20) Woodbury, N. W.; Becker, M.; Middendorf, D.; Parson, W. W. Biochemistry 1985, 24, 7516. (21) Wasielewski, M. R.; Johnson, D. G.; Seibert, M.; Govindjee. Proc. Natl. Acad. Sci. U S A . 1989, 86, 524. (22) Durrant, J. R.; Hastings, G.; Joseph, D. M.; Barber, J.; Porter, G.; Klug, D. R. Biochemistry 1993, 32, 8259. (23) Forster, Th. In Modem Quantum Chemistry, Part III; Sinanoglu, O., Ed.; Academic Press, Inc.: New York, 1965; p 93. (24) Lyle, P. A.; Struve, W. S. J. Phys. Chem. 1991, 95, 4152. (25) Werst, M.; Jia, Y.; Mets, L.; Fleming, G. R. Biophys. J. 1992, 61, 868. (26) Schatz, G. H.; Brock, H.; Holzwarth, A. R. Biophys. J. 1988, 54, 397. (27) Jia, Y.; Jean, J. M.; Werst, M. M.; Chan, C.-K.; Fleming, G. R. Biophys. J. 1992, 63, 259. (28) Warren, P. V.; Golbeck, J. H.; Warden, J. T. Biochemistry 1993, 32, 849. (29) Holtzwarth, A. R.; Schatz, G.; Brock, H.; Bittersmann, E. Biophys. J. 1993, 64, 1813. (30) Ikegami, I.; Katoh, S. Biochim. Biophys. Acta 1975, 376, 588. (31) Petek, H.; Yoshihara, K.; Fujiwara, Y.; Frey, J. G. J. Opt. SOC. Am. E 1990, 7, 1540. (32) Kandori, H.; Kemnitz, K.; Yoshihara, K. J. Phys. Chem. 1992,96, 8042. (33) Kamogawa, K.; Moms, J. M.; Takagi, Y.; Nakashima, N.; Yoshihara, K.; Ikegami, I. Photochem. Photobiol. 1983, 37, 207. (34) Itoh, S.; Iwaki, M. Biochim. Biophys. Acta 1988, 934, 32. (35) Ikegami, I.; SCtif, P.; Mathis, P. Biochim. Biophys. Acta 1987,894, 414. (36) Itoh, S.; Iwaki, M.; Ikegami, I. Biochim. Biophys. Acta 1987,893, 508. (37) Klug, D. R.; Giorgi, L. B.; Crystall, B.; Barber, J.; Porter, G. Photosynth. Res. 1989, 22, 277. (38) Positive absorbance changes in the region above 720 nm under the WOO-preoxidized conditions were observed upon excitation of 1.3 photons/RC (Figures 2a and 4a) and was not observed with weaker excitation intensities (Figures la'-e' and 4b and c). (39) van Grondelle, R. Biochim. Biophys. Acta 1985, 811, 147. (40) A single-exponential curve was used here for simple comparison with the kinetics for the €700-neutral particles, even though a doubleexponential curve gives a better fitting to the open circles in Figure 5c. (41) Durrant, J. R.; Hastings, G.; Joseph, D. M.; Barber, J.; Porter, G.; Klug, D. R. Proc. Natl. Acad. Sci. USA. 1992, 89, 11632. (42) Shepanski, J. F.; Anderson, R. W. Chem. Phys. Lett. 1981, 78, 165. (43) Trissl, H.-W.; Hecks, B.; Wulf, K. Photochem. Photobiol. 1993, 57, 108. (44) Iwaki, M.; Itoh, S. In Electron Transfer in Inorganic, Organic and Biological Systems; Bolton, J. R., Mataga, N., McLendon, G., Eds.; Advances in Chemistry Series 228; American Chemical Society: Washington, DC, 1991; p 163. (45) Woodbury, N. W. T.; Parson, W. W. Biochim. Biophys. Acta 1984, 767, 345. (46) Woodbury, N. W.; Parson, W. W. Biochim. Biophys. Acta 1986, 850, 197. (47) Horber, J. K. H.; Gobel, W.; Ogrodnik, A.; Michel-Beyerle, M. E.; Cogdell, R. J. FEES Lett. 1986, 198, 273. (48) Du, M.; Xie, X.; Jia, Y.; Mets, L.; Fleming, G. R. Chem. Phys. Lett. 1993, 201, 535. (49) Owens, T. G.; Webb, S. P.; Mets, L.; Alberte, R. S.; Fleming, G. R. Proc. Natl. Acad. Sci. U S A . 1987, 84, 1532. (50) This time constant was obtained with excitation wavelength and intensity of 605 nm and 0.8 photonsRC. (51) This time constant was obtained with excitation wavelength and intensity of 605 nm and 1.5 photonsRC. (52) Breton, J.; Ikegami, I. Photosynth. Res. 1989, 21, 27. (53) This long-lived bleach was observed under all the excitation conditions used. (54) Owens, T. G.; Webb, S. P.; Alberte, R. S.; Mets, L.; Fleming, G. R. Biophys. J. 1988, 53, 733. ( 5 5 ) Turconi, S.; Schweitzer, G.; Holzwarth, A. R. Photochem. Photobiol. 1993, 57, 113.
