Excitation Dynamics in Eukaryotic PS I from - ACS Publications

EVents in Photosynthesis, Arizona State UniVersity, Tempe Arizona 85287-1601, USA and ...... Woodbury, N. W.; Blankenship, R. E.; Webber, A. N. J. Phy...
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J. Phys. Chem. B 2002, 106, 6322-6330

Excitation Dynamics in Eukaryotic PS I from Chlamydomonas reinhardtii CC 2696 at 10 K. Direct Detection of the Reaction Center Exciton States Krzysztof Gibasiewicz,†,§,| V. M. Ramesh,†,§ Su Lin,‡,§ Neal W. Woodbury,‡,§ and Andrew N. Webber*,†,§ Department of Plant Biology, Department of Chemistry and Biochemistry and Center for the Study of Early EVents in Photosynthesis, Arizona State UniVersity, Tempe Arizona 85287-1601, USA and Department of Physics, Adam Mickiewicz UniVersity, ul. Umultowska 85, 61-614 Poznan´ , Poland ReceiVed: December 19, 2001; In Final Form: April 8, 2002

Excitation energy transfer in PS I particles from the green alga Chlamydomonas reinhardtii CC 2696 was studied at 10 K by femtosecond transient absorption spectroscopy. Five-nm wide excitation pulses at 670, 680, 695, and 700 nm were applied to selectively excite different spectral forms contributing to the wide QY transition band of chlorophyll a. Absorbance changes between 630 and 770 nm, up to 100 ps after excitation, were collected with a time step of 54 fs during the first 5 ps. Excitation at 700 nm leads to a structured initial absorbance difference spectra with four positive bands clearly resolved at 634, 645, 652, and 661 nm, and four negative bands at 667, 675, 684, and 695 nm. These spectra are interpreted in terms of excitonic coupling between the six electron-transfer chlorophyll a molecules: a special pair, two accessory and two A0 chlorophylls. The negative bands were ascribed to photobleaching of the four one-exciton states in line with theoretical predictions (Beddard, G. S. J. Phys. Chem. B. 1998, 102, 10 966), and the positive ones to excited-state absorption. The significance of the broad absorbance changes is proposed to be the introduction of spectral overlap between the reaction center and different spectral forms of the antenna chlorophylls that is expected to increase the efficiency of energy flow to the reaction center. Excitation at different wavelengths shows indeed that trapping can occur from different spectral pools of chlorophylls with similar efficiency (trapping time 29-44 ps). Following the excitation at 670 and 680 nm, trapping was shown to occur from the same pool as at room temperature centered at 682-685 nm, containing apparently only a minority of antenna molecules located close to the reaction center. The trapping time was found to be only slightly longer compared to that at room temperature (20-23 ps at RT). At 10 K, a significant amount of chlorophylls cannot exchange excitation energy with their neighbors. Our results are consistent with previous reports that at cryogenic temperatures, charge separation is possible in ∼50% of PS I particles and that excitation quenching by the oxidized and reduced primary donor is equally effective. As was observed at room temperature, there is no indication of red chlorophylls absorbing above 700 nm. This lack of red chlorophylls makes it possible to directly excite reaction center chlorophylls and study interaction between them in wild type and, in future, mutant PS I from Chlamydomonas.

Introduction The availability of the PS I core antenna structure, first at 4 Å1,2 and recently at 2.5 Å resolution,3 raises many questions about the relationship between structure and function of this membrane protein. Of particular interest is the process of excitation energy migration within PS I and the dependence of this process on features of the structure. Crystallographic studies of PS I from the cyanobacterium Synechococcus elongatus have identified 90 antenna chlorophylls (Chls) that capture photons and then deliver their energy to the electron transport chain (ETC), where the excited primary donor easily gives an electron to the primary acceptor. The * To whom correspondence should be addressed. Mailing address: Department of Plant Biology, P.O. Box 871601, Arizona State University, Tempe, AZ 85284-1601. Phone: (480) 965-8725. Fax: (480) 965-6899. E-mail: [email protected]. † Department of Plant Biology, Arizona State University. ‡ Department of Chemistry and Biochemistry, Arizona State University. § Center for the Study of Early Events in Photosynthesis, Arizona State University. | Institute of Physics, Adam Mickiewicz University, Poznan ´.

antenna function of Chls is supported by at least 22 identified carotenoids. The ETC is arranged in two quasi-symmetrical branches, composed of the primary donor, P700, and acceptors termed A0, A1, FX, and FAB. There are two A0 and two A1 molecules. P700 is a Chl a/Chl a′ heterodimer, each A0 is a Chl a monomer, each A1 is a phylloquinone, and FX is an iron sulfur center. There are two additional iron sulfur centers termed FAB, or FA and FB. According to recent papers, an electron residing on the excited primary donor, P700*, may flow either along one branch comprising one pair of A0 and A1 molecules or along the other,4,5 although electron-transfer activity of both branches is not yet well established. Between P700 and both A0 molecules, two additional monomeric Chls termed A or accessory Chls are located, but their role is not clear. The centerto-center distances between P700 and A is 11-13 Å, and between A and A0 9-10 Å in each branch.2,6,7 The edge-toedge distance between neighboring ETC Chls is 4-5 Å. There are only two antenna Chl molecules that are close to the ETC. They are located symmetrically at both sides of the two branches in the vicinity of the A0 molecules. The center-to-center distance

10.1021/jp014608l CCC: $22.00 © 2002 American Chemical Society Published on Web 05/25/2002

Excitation Dynamics in Eukaryotic PS I between these “connecting Chls” and respective A0 molecules is 12.8 and 10.9 Å. The distance between any of the other antenna Chls and ETC is >18 Å. Thus, the connecting Chls are thought to bridge structurally and possibly functionally the rest of the antenna with ETC. It has been hypothesized, that the energy from the antenna flows to the primary donor successively via connecting Chls, A0 and A molecules.2,3 However, this hypothesis has been questioned based on theoretical modeling of excitation energy transfer in PS I.8 The 41 innermost antenna Chls are located in the elliptically distorted cylindrical region. The ETC molecules are placed inside this cylinder, along its axis. There are two additional sets of more peripherally located antenna Chls forming two layers parallel to the plane of membrane and perpendicular to the axis of the cylindrical region, one close to the stromal and the other to the lumenal side of the membrane. Each of these sets contain 18 Chl molecules. The remaining 13 antenna Chls are two connecting Chls and 11 Chls bound by more peripheral protein subunits. The orientations of all 96 Chl headgroups and their Qx and Qy transition dipolar moments were determined very recently.3 The intrinsic rate of electron transport from P700 to A0 is on the order of 1 ps.9,10 However, the experimentally measured trapping time necessary for excitation decay in the PS I core antenna due to the charge separation in the ETC is as long as 20-40 ps.11-14 This decay is believed to occur from the energy equilibrated state of the antenna system.15 The equilibration takes 1.5-12 ps and is thought to originate from energy migration between the main pool of Chl molecules and red Chls absorbing at wavelengths about and longer than 700 nm.11-13,16,17 Fluorescence depolarization measurements have allowed detection of an exceptionally fast phase of 150-300 fs attributed to a single photon hop between two Chl molecules.18 Recent studies on excitation dynamics in PS I with subpicosecond resolution have been focused on particles isolated from cyanobacteria: Synechocystis sp. PCC 680314,19-21 and S. elongatus.22 These studies demonstrated an additional, subpicosecond phase of energy equilibration occurring within the main Chl pool. Both downhill14,19,20 and uphill14 subpicosecond and picosecond energy transfer was shown after selective excitation of different spectral forms of Chl molecules. Similar studies were performed at 77 K where the efficiency of the uphill energy transfer is decreased.21 On the basis of these studies, two dimers of Chls located close to ETC have been proposed to be red Chls (14, 21; see also 23). The central vs peripheral24-26 location of red Chls is however still a matter of debate. There is much less detailed information available on subpicosecond and picosecond excitation dynamics in eukaryotic PS I.27 Although PS I is believed to have similar structure and function in all organisms containing it,28-30 it was pointed out that, for e.g., the number and spectroscopic properties of low energy Chls (red Chls) is species dependent.22,31 In particular, PS I particles from C. reinhardtii CC 2696, a mutant that lacks the peripheral Chla/b-binding protein, were suggested to be completely devoid of Chl molecules absorbing above 700 nm.27 The presence of red Chls in the PS I core antenna absorbing clearly above 700 nm is well documented only in cyanobacteria32-34 and different hypothesis about their function have been put forward: they may increase the efficiency of energy trapping,11,35 increase the absorption of far red light36 or be for photoprotection.24 A possible lack of red Chls in eukaryotic PS I core complexes may implicate some differences in the structure compared to cyanobacterial PS I. An additional impulse to study excitation dynamics in C. reinhardtii at 10 K with subpicosecond

J. Phys. Chem. B, Vol. 106, No. 24, 2002 6323 resolution was the previous finding at RT,27 that excitation at g700 nm leads to a very broad and structured band disappearing within ∼200 fs, which was hypothesized to originate from strong excitonic interaction, likely between the six ETC Chls. A similar effect of coupling between two transient absorption bands at 698 nm (or 708 nm) and 683 nm appearing after excitation at 700 nm (or 710 nm), has been reported for Synechocystis sp. PCC 6803 at 77 K.21 In that case, the resonant photobleaching at 683 nm was ascribed to the upper excitonic level of one of the dimers (red pigment) located close to the ETC. The absence of red Chls in PS I from C. reinhardtii would help understand this interesting phenomenon more clearly. On the basis of the 4 Å PS I structure, it has been calculated by Beddard37 that exciton coupling between the nearest neighbors within the group of the six ETC Chls is similar and strong (above 300 cm-1). In particular, the coupling between the two molecules of the special pair is not stronger than, for example, between them and respective accessory Chls. Thus, in contrast to the purple bacteria where the coupling between the special pair components is much stronger than between any other electron-transfer cofactors, the electronic identity of a “special pair” is not obvious. Instead, one can speak about a six-molecule structure. Exciton coupling within this structure leads to splitting of the QY transition band of the Chl a molecules into six bands corresponding to six one-exciton states. A similar splitting due to interaction between the 7 BChl a molecules in the FMO protein of green sulfur bacteria, and also between bacteriochlorophylls forming two rings in purple bacterial LH2, has been observed (see ref 38 for review). The aim of this study is to characterize excitation dynamics in the eukaryotic PS I core antenna at low temperature and compare it to similar studies performed at RT27 and on the cyanobacterium Synechocystis sp. PCC 6803 at 77 K.21 In particular, we wanted to (1) test the hypothesis put forward in the RT paper about the lack of red Chls absorbing above 700 nm and (2) examine further the nature of the very broad, structured and short-lived absorption changes caused by excitation at g700 nm. More detailed objects were to (3) study energy equilibration processes at 10 K, in particular uphill vs downhill energy transfer; (4) check the connection between different antenna Chls, and between them and the ETC at 10 K; (5) examine the quenching efficiency and dynamics by the ETC at 10 K; and (6) identify Chl pools over which equilibration takes place after selective excitation of different spectral pools of Chls. Materials and Methods Preparation of PS I Particles. PS I particles containing ∼85 chlorophyll molecules per RC were prepared from thylakoid membranes using a mild detergent procedure as described in ref 27, 39. Femtosecond Transient Absorption Measurements. To rereduce oxidized primary donor in PS I particles (OD674 nm,1 cm ) 30), 40 mM sodium ascorbate and 20 µM phenazine methosulfate were added. Then, the sample was mixed with glycerol (1:2 v/v), centrifuged in a vacuum to remove any dissolved air which disturbs homogeneous freezing of the sample, and placed in a thin cuvette (∼1 mm thick). The results obtained from cuvettes made of glass and plexiglass were checked to be essentially the same, so the majority of experiments were done with plexiglass cuvettes ensuring that no cracks in the sample appeared during the freezing. The final OD of the sample was between 1 and 1.2 at 674 nm. The cuvette was placed in the cryostat and frozen to 10 K in darkness. To avoid annihilation effects, the excitation pulse intensity was kept at a

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Figure 1. Room temperature and 10 K absorption spectra of PS I core antenna from C. reinhardtii CC 2696. Inset: 665-710 nm region of the spectra with excitation wavelengths indicated by arrows.

level of e1 photon per RC, as monitored by the maximal transient absorbance signal (∆A below 0.01). The experimental pump/probe setup was described earlier.27,40 Spectrally narrow (fwhm of ∼5 nm) laser pulses with a ∼150 fs duration at 670, 680, 695, and 700 nm, were used to excite the sample with a repetition rate of 1 kHz. The polarization of the pump and white probe beams was set at the magic angle with respect to each other. Transient absorbance spectra in the region between 630 and 750 nm were collected on two time scales: from -1 ps to 5 ps with a step size of 54 fs and from 5 to 100 ps with a step size of 2 ps. Time zero was assigned to the spectrum recorded at the longest pump-probe delay time at which any signal was not detected yet. The spectral resolution of the spectrometer was about 0.14 nm per channel. Data were averaged over 2 nm intervals for all spectra recorded. Decay associated spectra (DAS) were calculated from global fitting accounting for deconvolution of the recorded signals with the instrument response function using locally written software (ASUFIT, web address: www.public.asu.edu/∼laserweb/asufit/ asufit.html). The instrument response function was modeled by a Gaussian function with a width of ∼0.3-0.4 ps. In addition, the time vs wavelength absorbance change surfaces were corrected for the spectral dispersion of the probe beam. Results Absorption Spectra. The absorption spectra of PS I particles from C. reinhardtii CC 2696 used in this work are shown in Figure 1. In contrast to the RT spectrum, at 10 K, two shoulders on the red slope of QY transition band of the Chl molecules are seen at ∼683 nm and ∼695 nm. Absorbance Difference Spectra. Absorbance difference spectra recorded at different pump-probe delay times at 670, 680, 695, and 700 nm excitation wavelengths are shown in Figure 2A-D, respectively. Excitation at 670 nm results in the immediate appearance of a narrow, 5 nm wide (fwhm) negative signal (Figure 2A; trace 0.17 ps) assigned to photobleaching and stimulated emission of initially excited Chl molecules. The signal is centered exactly at the peak of the excitation wavelength. After 0.60 ps, the amplitude at ∼670 nm reaches its maximal value but the initial band is already accompanied by a ∼683 nm band seen as a shoulder on the red slope of the main band. In the trace marked 2.1 ps, the ∼683 nm shoulder is already seen as a distinct band

Gibasiewicz et al. with an amplitude higher than that at 0.60 ps. Comparison of the 0.60 and 2.1 ps traces leads to the observation that simultaneously with build-up of the amplitude at ∼683 nm, decreasing of the band ∼670 nm occurs. This suggests downhill energy transfer from chlorophylls absorbing at ∼670 nm to those absorbing at ∼683 nm. Further temporal evolution of the spectrum leads to a decrease of the amplitudes of both these bands along with appearance of another shoulder at ∼693 nm (see trace 11 ps). Finally, at 97 ps, the ∼683 nm band disappears completely, and only two bands are still observed: the bigger one at ∼670 nm and the small one at 693 nm. Spectral evolution following excitation at 680 nm resembles that initiated by the 670 nm excitation, except that it occurs in a narrower spectral region that makes the overall picture less clear (Figure 2B). The initially narrow signal (0.16 ps) gradually becomes asymmetric with the red slope less steep than the blue one (0.70 ps). With time, the asymmetry increases (2.1 ps). It is easy to see that the signal amplitude at ∼680 nm decreases and that at >683 nm increases with time when comparing the traces at 0.70 and 2.1 ps. This again suggests downhill energy transfer. The trace marked 11 ps peaking still at ∼680 nm is largely asymmetric, the asymmetry being increased by the appearance of another shoulder at ∼693 nm similar to that seen at the respective delay time after excitation at 670 nm. At a delay time of 99 ps, only a peak at ∼677 nm and a wide shoulder in the 685-695 nm region remain. Excitation at 695 nm, similarly to shorter wavelengths, causes a very narrow initial absorbance difference signal (Figure 2C; trace 0.17 ps). However the maximum of the signal is slightly blue shifted relative to the excitation wavelength, similar to what was observed at room temperature at this excitation wavelength.27 Due to the finite duration of the excitation pulse, the initial signal increases and reaches its maximal amplitude at 0.71 ps. At this time, a small additional band at ∼677 nm is also seen. Further temporal evolution of the spectrum depends, essentially, on a gradual decrease of the main band at ∼693 nm and of a small band at ∼677 nm (compare traces 2.1, 11, and 97 ps). Unlike at shorter wavelengths, exciting at 700 nm immediately causes absorbance changes that cover a very broad spectral region between 630 and 710 nm (Figure 2D). These absorbance changes are positive between 630 and 665 nm and negative between 665 and 710 nm (trace 0.16 ps; see also traces 0.37 and 0.48 ps). Moreover, several bands can be distinguished that contribute to these changes peaking at: 633-635 nm, 645 nm, 651-653 nm, and 661 nm in the positive part of the transient spectra, and at 667 nm, 675 nm, 683-685 nm, and 695 nm in the negative part of transient spectra. It should be noticed that all of these peaks do not result from the noise as confirmed by two independent experiments. Comparing the traces marked 0.16 and 0.37 ps, one can see that the band peaking at 695-697 nm is growing faster than the other bands. At the delay time of 0.48 ps, the amplitudes of all bands, except for that peaking at 697 nm, have started to decrease. After 2.1 ps, no positive bands in the 640-665 nm region are observed and the negative bands at ∼675 nm and ∼683 nm can no longer be distinguished. Instead, the peak at 697 nm dominates the spectrum and its amplitude is about 3 times higher than the amplitudes in the region between 675 and 683 nm. At the delay time of 103 ps, the band 697 nm is still dominant and accompanied by a smaller band with a flat maximum at 675680 nm. Comparison of the very initial absorbance changes following the excitation at four wavelengths, 670, 680, 695, and 700 nm,

Excitation Dynamics in Eukaryotic PS I

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Figure 2. Time-resolved transient absorption spectra (A-D) and decay associated spectra (E-H) at 10 K of PS I core antenna from Chlamydomonas reinhardtii CC 2696 using four different excitation wavelengths.

in the region 630-720 nm, normalized to the same maximal amplitude (Figure 3), demonstrates qualitative differences between the nature of species excited at 700 nm and at shorter wavelengths. Excitation at above 700 nm results in very weak signals that cannot be reliably analyzed due to the low signalto-noise ratio. This was not the case at RT where difference spectra of good quality were measured at excitation wavelength up to 710 nm.27 This is not surprising as PS I absorbs stronger at RT than at 10 K above 700 nm (Figure 1).

Decay Associated Spectra. Decay Associated Spectra (DAS) obtained from global fitting (Figure 2E-2H) allows the identification of spectra, characterized by different lifetimes, that contribute to the overall absorbance dynamics shown in Figure 2A-2D. In line with the similarities between absorbance dynamics triggered by excitation at 670 and 680 nm, respective sets of DAS are also largely similar (compare Figure 2E and 2F). The fastest components of 0.40 and 0.45 ps clearly demonstrate downhill energy transfer from the initially excited

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Figure 3. Comparison of the initial time-resolved spectra recorded after a pump-probe delay time of ∼160 fs at excitation wavelengths of 670, 680, and 695 nm (A), and 700 nm (B). Traces A and B in panel B come from two independent experiments. Excitation wavelengths are indicated by arrows.

pools of Chls, centered at 670 and 680 nm, respectively, to another pool of Chls absorbing at about 685 nm. In addition to these subpicosecond components, additional ∼2 ps phases have been resolved. The shapes of these latter components, with negative amplitudes at lower wavelengths and positive at higher, indicate further downhill energy transfer. It is worth noting that the width of the negative parts of the ∼2 ps DASs, especially after excitation at 670 nm, are broader and red-shifted, compared to the respective negative parts of subpicosecond DASs. This indicates energy transfer from a partly equilibrated pool of Chl molecules different from those initially excited. On the other hand, the positive parts of the ∼2 ps DASs are peaking at 693695 nm, 10 nm further toward the red compared to the positive parts of the subpicosecond DASs, thus indicating energy transfer to lower energy Chls. The next kinetic component was found to decay in 29-30 ps and is peaking at 685 nm. At the 670nm-excitation, an additional small band at 670 nm contributes to the 29 ps DAS. The 29-30 ps component is attributed to excitation trapping by the neutral or oxidized primary donor. Finally, a nondecaying (ND) spectra have been found with shapes similar to those of the respective time-resolved spectra recorded at a pump-probe delay time of 97 and 99 ps (Figure 2A and 2B). In both cases, a band peaking close to the excitation wavelength dominates and is accompanied by another band centered at 693 nm. All four kinetic components found at 695 nm excitation (Figure 2G), 0.14 ps, 1.3 ps, 44 ps and ND, have similar

Gibasiewicz et al.

Figure 4. Comparison of the 29-44 ps trapping components (A) and the nondecaying components (B) of decay associated spectra at four different excitation wavelengths.

shapes: they have only negative bands, are relatively narrow and are centered at 693 nm. In all cases, except for 1.3 ps component, a very small additional band peaking at 675-677 nm occurs. As little as a 5 nm red shift of excitation wavelength to 700 nm causes a dramatic change in the shape of the DASs (Figure 2H). The fastest component, 0.14 ps, resembles the very initial time resolved spectra (Figure 2D; 0.16 ps trace) with broad and clearly structured bands. The second component has a lifetime of 35 ps and three bands with negative amplitudes: the dominant one centered at 697 nm, a smaller one at 685 nm and the smallest one at 675 nm. The third component is nondecaying (ND) and has two bands with negative amplitudes: the dominant one at 697 nm and a smaller one with a flat maximum at 675-680 nm. The trapping and ND components from Figure 2E-2H have been normalized to the same maximal amplitudes in order to compare their shapes and maximum positions (Figure 4A and 4B, respectively). When exciting at the two shorter wavelengths, the main bands in the trapping components both peak at 685 nm, indicating that the equilibration has occurred essentially over the same pool of Chls (different from that initially excited). However, excitation at 670 nm leads to the appearance of the additional small band at ∼670 nm as well as a slight blue shift of the main band, suggesting a minor difference in the composition of Chl pools over which the equilibration is established. Excitation at longer wavelengths results in a red shifting of the DAS of the trapping components indicating energy equilibration over different pools of Chl molecules,

Excitation Dynamics in Eukaryotic PS I depending on the excitation wavelength. The main common feature of all of the ND spectra (Figure 4B) is that they are each dominated by a band centered close to the excitation wavelength. Discussion Steady-State Spectra. In contrast to RT, the low-temperature absorption spectrum of the PS I core antenna from C. reinhardtii CC 2696 reveals additional structure in the QY transition region (Figure 1). On the red slope of the main band peaking at 674 nm, two shoulders are seen: one between 680 and 685 nm and the other centered at about 695 nm. The former is probably due to a fraction of Chls contributing to the main Chl pool, whereas the latter may originate from a pool of clearly redshifted, lower energy Chls. This spectrum should be compared to the 4 K spectra of PS I from the cyanobacteria Synechocystis PCC 6803 (Figure 10 in ref 32) and S. elongatus (Figures 1 and 2 in ref 33). In both these species, a more red-shifted pool of Chls, absorbing at 708 nm, has been found after Gaussian decomposition of the spectra. In addition, a second pool of Chls absorbing at 714 nm in Synechocystis PCC 680326 and at 719 nm in S. elongatus was found. The number of red Chl molecules absorbing at 708 nm was estimated to be 2-3 in Synechocystis PCC 680326,31,32 and 4-5 in S. elongatus.31,33 Comparison of cyanobacterial spectra with that in Figure 1 indicates that there are no Chls in C. reinhardtii with absorption maximum above 700 nm. This is consistent with previous observations at RT.27 Excitation Energy Equilibration. Direct excitation of the main pool of Chls at 670 and 680 nm results in a biphasic energy equilibration process (Figure 2A, B, E, F). In 0.40-0.45 ps, the energy from the initially excited spectrally narrow pools of Chls is transferred to another pool absorbing at ∼685 nm. The latter pool remains excited for about 30 ps as seen from the respective DASs centered at 685 nm. During that time, excitation decays. The pool absorbing at ∼685 nm may correspond to the shoulder at ∼685 nm seen in the 10 K spectrum (Figure 1). This indicates that the observed subpicosecond downhill energy transfer leads to concentration of excitons in a smaller pool that absorbs about 10 nm more to the red compared to the maximum absorption at 674 nm ascribed to the main pool of Chls. This concentration is not only an effect of lowering the temperature to 10 K resulting in only downhill energy transfer with final equilibration over lower energy forms of Chls. Even at RT a similar, although less pronounced, red shift of the absorbance changes in the subpicosecond time scale was observed.27 Thus, RT and 10 K measurements consistently show that equilibration of excitation energy is at least 1 order of magnitude faster than trapping and that it takes place not over the whole antenna system but over a minor pool of Chls. An alternative or additional explanation of the subpicosecond red shift of the transient spectra could in principle be vibrational relaxation, assuming a high contribution of stimulated emission to the observed transient signals. However, it would be unusual to find a Stokes shift of as much as 15 nm for Chls absorbing at 670 nm and of only 5 nm for Chls absorbing at 680 nm. The second, 2-3 ps phase of energy equilibration was observed at RT, but no clear direction of energy transfer was found because of a lack of a positive phase in the DAS. At 10 K, further downhill energy transfer to the Chl pool absorbing at ∼695 nm is demonstrated by the positive amplitudes of the 2.4 and 2.3 ps components of the DASs in Figure 2E and 2F, respectively. This phase has a similar lifetime to ones found in cyanobacterial PS I that were ascribed to energy equilibration between the main pool of Chls and red Chls absorbing at about 710 nm.14,19,20

J. Phys. Chem. B, Vol. 106, No. 24, 2002 6327 This suggests that “red Chls” in C. reinhardtii CC 2696 are 10-15 nm blue shifted compared to those in cyanobacteria and, in consequence, their nature may be quite different. For example, instead of being Chl dimers, as proposed for cyanobacteria,14,21,32 their absorption maximum shift to the red may be caused by a strong interaction with the protein environment, similar to how the protein modulates spectral properties of Chls absorbing at shorter wavelengths. Alternatively, “red Chls” in C. reinhardtii may be weakly coupled dimers. The area under the positive parts of 2.3-2.4 ps DAS components is significantly smaller relative to the respective areas above the negative parts. This may be caused by an excited state absorption of “red Chls” which partly compensate photobleaching in the ∼695 nm region. Excitation at 695 nm results in DASs ascribed to energy equilibration (0.14 and 1.3 ps in Figure 2G) that do not show any positive amplitudes. The same excitation wavelength at RT caused clear subpicosecond uphill energy transfer27 that is not possible at 10 K. Comparison of all four DASs, all being centered at 693 nm, demonstrates that excitation remains on the same spectral pool of Chls. However, the presence of two equilibration components suggests some heterogeneity in this pool. For example, initially excited molecules with bigger oscillator strength may transfer their energy to neighbors absorbing less strongly at ∼693 nm or to the Chls with photobleaching at ∼693 nm being partly compensated by excited-state absorption. Exciton Coupling. Both the shape and the amplitudes of the very initial absorbance changes caused by 700 nm excitation (trace 0.16 ps in Figure 2D and Figure 3) are unusual when compared to those at the 670, 680, and 695-nm excitations. In contrast to the latter, apart from a very broad and structured negative part due to photobleaching and stimulated emission, there are wide positive changes between 630 and 665 nm, which can be attributed to the excited state absorption. Also, the amplitudes of the maximum at 675, 685, and 695 nm are about twice as big as the amplitudes of the signals denoted 0.17 ps and/or 0.16 ps in Figure 2A-2C. Another difference is that absorbance differences are relatively far from the excitation wavelength in contrast to shorter wavelength excitations. All these features reveal the very different nature of the molecules excited at 700 nm from those excited at wavelengths shorter than 700 nm. Similar differences, although less pronounced, were observed at RT. The DAS (0.14 ps in Figure 2H) demonstrates that in overall dynamics of excitation, the decay of these very initial absorbance changes dominates. Excitation at as little as 5 nm shorter wavelength results in a completely different, very narrow and excitation-wavelength-centered DAS (Figure 2G), suggesting that the molecules responsible for changes seen at 700 nm excitation are spectrally well covered by a pool of Chls absorbing at ∼695 nm, seen in the absorption spectrum in Figure 1. In the RT paper, the broad initial transient spectrum was ascribed to a group of excitonically coupled molecules most likely ETC Chls. However, the resolution of the data at RT did not allow us to distinguish clearly any subbands. At 10 K, the structure of this spectrum is clear, with four bands peaking between 630 and 665 nm in the positive part and four bands between 665 and 710 nm in the negative part of the spectrum. We propose to ascribe these latter four bands to photobleaching of four one-exciton states resulting from interaction between six ETC Chls (Figure 5). As excitation is at 700 nm, one can expect that only the very lowest one-exciton state, at 695 nm, may be populated under the experimental conditions. Consequently, stimulated emission may contribute only to this ∆A

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Figure 5. Hypothetical energetic scheme explaining the origin of the multi-band, structured transient spectrum presented in Figure 3B following excitation at 700 nm. The four one-exciton and four twoexciton states are presented as rectangles with their thickness corresponding to the experimental error assumed to be (1 nm (∼25 cm-1). The arrows from the ground state to the four one-exciton states represent four transitions being bleached when 700 nm excitation is used (left side). The arrows from the very lowest one-exciton state to the four two-exciton states correspond to the four excited state absorption bands (right side). For comparison, on the very left side, there are six theoretically calculated one-exciton levels resulting from interaction between six ETC Chls in PS I, shifted 100 cm-1 to lower energy (compare Figure 3B in Beddard 1998 - the estimated from this figure, energy levels are as follows: 14 490 (the lowest one), 14 740, 14 940, 15 060, 15 250, and 15 320 cm-1 (the highest one)). The distances between energy levels are proportional to energy gaps between them except for two spots where E axis is broken.

band and not to the 667, 675, and 683 nm bands. On the other hand, the bands in the positive parts of the spectrum may originate from excited-state absorption from the very lowest oneexciton state to four two-exciton states. Figure 5 shows four transitions bleached by the excitation and four transitions responsible for excited-state absorption. For comparison, there are also shown six one-exciton states calculated by Beddard37 for six ETC Chls in PS I based on the crystallographic structure.1,2 Four of the one-exciton states calculated by Beddard correspond very well to those directly detected in this work except for being shifted about 100 cm-1 to higher energy. The relative energy gaps between particular exciton states calculated and measured experimentally are the same within the experimental error. This supports the proposition that the observed spectrum originates from excitonically coupled ETC Chls. However, theoretically two more one-exciton states were calculated. Their lack of appearance in the experiment may be explained in two ways. The first possibility is that the oscillator strength of the two remaining transitions is too small to result in observable bands. Second, the two bands may be spectrally overlapped with bands resulting from excited-state absorption. Indeed, the energy of the two highest one-exciton levels calculated by Beddard (15 250 and 15 320 cm-1) drop into the range of the four two-exciton states observed in the experiment (15 130-15 750 cm-1), even if one takes into account the 100 cm-1 shift between experimental and theoretical results. In principle, it is also possible that the spectral position of two missing transitions may be very close to some of the four, which are observed, and thus are not resolved. It remains to be determined if calculation based on the newly available 2.5 Å PS I structure will result in an exciton structure similar to that

Gibasiewicz et al. calculated from the low resolution structure37 and measured here. It would also be interesting to consider energies of possible twoexciton states and determine if these support the interpretation of the absorbance changes data equally well. To study further the nature of this excitonic coupling experimentally, we plan to perform similar transient absorption measurements on C. reinhardtii mutants in which the interaction of the primary donor Chls is changed.39,41 The mutations are expected to disturb the normal excitonic interaction between the ETC Chls. The wide and structured spectrum of the ETC Chls may be of great significance for the effectiveness of energy transfer from the antenna to the RC. It is expected to increase the range of spectral overlap between these two pools of Chls, thus possibly promoting the effective energy transfer between different spectral forms of antenna Chls and ETC Chls. However, this concept is in contrast to the idea of connecting Chls being the bottleneck linkers in energy transfer between the antenna and RC.2,3 Which of these two models of energy funneling better explains excitation energy transfer for PS I is currently being explored. It is also of interest to note that the accessory Chls may play an indispensable role in organizing the RC chlorophylls into one excitonically coupled system and this may be an equally important role as their possible mediating in electron transfer between the primary donor and primary acceptor. Trapping. The observation that the spectra of the trapping component are essentially independent of the excitation wavelength when exciting at 670 and 680 nm, yet quite exciatation wavelength dependent when exciting at longer wavelengths (Figure 4A), is consistent with the finding that in the former case, downhill energy transfer to the Chl pool absorbing at ∼685 nm takes place and in the latter case uphill energy transfer to the same pool is practically blocked by a very unfavorable Boltzmann distribution at 10 K. At RT, independent of excitation wavelength (between 670 and 710 nm), energy was always equilibrated over a pool of Chl molecules absorbing at 681683 nm,27 which may correspond to the pool centered at ∼685 nm seen at 10 K. The small red shift of the maximum, compared to that at RT, may indicate that in the 681-685 nm pool, lower energy Chls are preferentially excited at 10 K after equilibration is complete. In addition, in the trapping component there is some contribution from the charge separated state (with a maximum at ∼691 nm; see below), which compensates a red part of photobleaching caused by equilibrated excited antenna states. Thus, the maximum of the trapping DAS is blue shifted compared to the absorption maximum of the Chl pool over which the excitation energy is finally equilibrated. This blue shift is expected to be larger at RT, where the contribution from charge separated state is higher than at 10 K (see below). The trapping time from this pool, 29-30 ps, tends to be only slightly longer compared to that at RT (20-23 ps). As mentioned above, the pool of the 685 nm Chls clearly includes only a small subpopulation of antenna Chls. Moreover, this pool is very well coupled to the RC, as at ∼100 ps after the excitation, the amplitude of the transient spectra at ∼685 nm is very low (Figure 2A-D). This may indicate that this pool is located close to the RC. Other spectral pools of antenna Chls are apparently not so well coupled to the RC at 10 K (see below). Trapping components found at 695- and 700-nm-excitation, when the energy cannot flow to the 685-nm-pool, peak at about the excitation wavelengths: 693 and 697 nm, respectively (Figure 2G and 2H). It is interesting to note that even from those pools (perhaps very small ones) trapping occurs in a significant percentage of PS I complexes and that the trapping time is again

Excitation Dynamics in Eukaryotic PS I not very different, 35-44 ps, from that found at shorter wavelengths excitations. If the origin of excitation decay is its quenching by P700 or P700+, then it means that there are different, roughly equally effective pathways for excitation to reach the RC: via 685-nm pool, via 693-nm pool, and via 697nm pool. It is not excluded that there are quite different mechanisms of excitation energy quenching for the 685 nm pool compared to the longer wavelength pools. For example, the pool at 685 nm may be composed of many Chls and the energy may flow easily and many times between this pool and ETC Chls. On the other hand, there may be only a few molecules absorbing at 693 and 697 nm, in line with the low absorption in this region, more distant from the RC, which need to wait quite a long time before transferring the energy to the RC. The efficient energy transfer from different spectral forms of antenna Chls to the RC would be consistent with the idea put forward above that the broad spectrum of the RC improves the spectral overlap between the ETC and a variety of antenna Chls. In principle, excited states of different Chls as these absorbing at 667 and 695 nm may still be isoenergetic with different exciton levels of the ETC Chls. Nondecaying Spectra. The observation that the nondecaying spectra are strongly excitation wavelength dependent and that their maxima are close to the excitation wavelengths (Figure 4B) indicates that, in a fraction of PS I particles, energy is neither transferred to the RC nor even equilibrated over Chls that are spectrally different from the initially excited groups of Chls. This is quite different from what was observed at RT. There, the excitation at 695 and 700 nm results in complete energy transfer to the RC, and excitation at 670 and 680 nm leads to some fraction of uncoupled Chls which, however, has the ∆A band peaking at ∼677 nm that clearly results from earlier energy transfer from initially excited pools of 670 and 680 nmabsorbing Chls. A very rough estimation of the amount of energy which cannot be further transferred, may be done by comparison of the amplitudes of trapping components, which are believed to originate from decay of the excited Chls well coupled to the RC, and of main bands of nondecaying components - due to energetically uncoupled Chls (Figure 2E-H). At the 670 and 680 nm excitation, the maximum amplitudes of these two components are roughly the same, indicating that up to 50% of antenna Chls do not exchange the excitation energy with neighbors (at least on the time scale of 100 ps). Compared to excitation at the shorter wavelengths, the contribution of uncoupled Chls seems to be clearly lower at λex ) 695 nm (Figure 2G), and higher at λex ) 700 nm (Figure 2H). This may be due to selective excitation of relatively lower and higher amounts of Chls uncoupled at low temperature. Some contribution to the ND spectra in Figure 2E-H in the region above 690 nm may come from absorption changes caused by the oxidation of the primary donor (P700+-P700). However, it is not well established what fraction of PS I recovers to the state with P700 oxidized and what fraction is permanently in the state with P700 reduced at 10 K under repetitive flashes, and in consequence what fraction can contribute to the ∆A(P700+P700) signal. At 77 K, it was reported that only in ∼45% of PS I particles from S. elongatus, reoxidation of P700+ (due to charge recombination between P700+ and A1-) is fast enough (t1/2 ) 170 µs;42) to ensure that each flash in our experiment excites PS I with P700 neutral. In the remaining PS I particles, rereduction of P700+ does not occur (35%) or is too slow compared to the repetition rate (20%). If one assumes similar numbers for 10 K in PS I from C. reinhardtii, it could be expected that the maximal amplitude of the P700+-P700 ∆A

J. Phys. Chem. B, Vol. 106, No. 24, 2002 6329 signal should decrease approximately twice relative to the case at RT, where ∼100% of RCs are believed to be in the state with reduced P700 before each flash.27 A straightforward comparison of these amplitudes is impossible. However, if one compares the maximum amplitudes of the ND bands peaking in the 690-693 nm, ascribed to P700+-P700, relative to the respective trapping component at 10 K (Figure 2E) and at RT (Figure 4A in ref 27), indeed a drop of a factor of ∼2 is observed at 10 K relative to RT. Comparison of the P700+-P700 ∆A maximum relative to the respective maximum of the trapping components is justified because quenching of excited states by P700+ was reported to be equally fast and efficient as by P700,20,43 although the reason for that is not well understood. Thus, our results at λex ) 670 nm (and also at λex ) 680 nm) indirectly demonstrate that roughly in 50% of PS I particles with good connection between antenna and RC, charge separation still occurs at 10 K, and that the fast ∼30 ps quenching occurs even in the remaining ∼50% of PS I with the primary donor permanently oxidized. Considering the areas under the respective ∆A bands instead of maximal amplitudes leads to similar conclusions. Measurements with λex ) 695 nm and λex ) 700 nm are not conclusive concerning this issue because the P700+-P700 ∆A signal is spectrally overlapped by the ∆A signal from uncoupled Chls. It is interesting to point out that according to Pålsson et al.34 and Hayes et al.,44 in cyanobacterial PS I about 50% of excitons are irreversibly trapped by red Chls at low temperatures resulting in ∼50% reduction of charge separation. This is clearly not the case in our preparation from C. reinhardtii where no long-lived ∆A signal ascribed to red Chls was detected after excitation of bulk Chls. Instead, some reduction in charge separation is caused by trapping of excitation by Chls that do not exchange energy with their neighbors (see above). Acknowledgment. We thank Prof. A. Freiberg for a valuable discussion. This work was supported by DOE Grant No. DEFG03-99ER20349 to A.W. and N.W. References and Notes (1) Fromme, P.; Witt, H. T.; Schubert, W. D.; Klukas, O.; Saenger, W.; Krauss, N. Biochim. Biophys. Acta 1996, 1275 (1-2), 76. (2) Schubert, W.-D.; Klukas, O.; Krauss, N.; Saenger, W.; Fromme, P.; Witt, H. T. J. Mol. Biol. 1997, 272, 741. (3) Jordan, P.; Fromme, P.; Witt, H. T.; Klukas, O.; Saenger, W.; Krauss, N. Nature 2001, 411, 909. (4) Joliot, P.; Joliot, A. Biochemistry 1999, 38, 11 130. (5) Guergova-Kuras, M.; Boudreaux, B.; Joliot, A.; Joliot, P.; Redding, K. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 4437. (6) Brettel, K. Biochim. Biophys. Acta 1997, 1318, 322. (7) Klukas, O.; Schubert, W.-D.; Jordan, P.; Krauss, N.; Fromme, P.; Witt, H. T.; Saenger, W. J. Biol. Chem. 1999, 274, 7361. (8) Gobets, B.; van Grondelle, R. Biochim. Biophys. Acta 2001, 1507, 80. (9) White, N. T. H.; Beddard, G. S.; Thorne, J. R. G.; Feehan, T. M.; Keyes, T. E.; Heathcote, P. J. Phys. Chem. 1996, 100, 12 086. (10) Kumazaki, S.; Ikegami, I.; Furusawa, H.; Yasuda, S.; Yoshihara, K. J. Phys. Chem. B 2001, 105, 1093. (11) Holzwarth, A. R.; Schatz, G.; Brock, H.; Bittersman, E. Biophys. J. 1993, 64, 1813. (12) Hastings, G.; Hoshina, S.; Webber, A. N.; Blankenship, R. E. Biochemistry 1995, 34, 15 512. (13) Hastings, G.; Reed, L. J.; Lin, S.; Blankenship, R. E. Biophys. J. 1995, 69, 2044. (14) Melkozernov, A. N.; Lin, S.; Blankenship, R. E. Biochemistry 2000, 39, 1489. (15) Hastings, G.; Kleinherenbrink, F. A. M.; Lin, S.; Blankenship, R. E Biochemistry 1994, 33, 3185. (16) Turconi, S.; Schweitzer, G.; Holzwarth, A. R. Photochem. Photobiol. 1993, 57, 113. (17) Turconi, S.; Kruip J.; Schweitzer, G.; Ro¨gner, M.; Holzwarth, A. R. Photosynth. Res. 1996, 49, 263.

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