© Copyright 2000 by the American Chemical Society
VOLUME 104, NUMBER 34, AUGUST 31, 2000
LETTERS Single-Molecule Spectroscopy on Photosystem I Pigment-Protein Complexes F. Jelezko,*,† C. Tietz,† U. Gerken,† J. Wrachtrup,† and R. Bittl‡ 3. Physikalisches Institut, UniVersita¨ t Stuttgart, Pfaffenwaldring 57, 70569 Stuttgart, Germany, and Technische UniVersita¨ t Berlin, Max-Volmer-Institut fu¨ r Biophysikalische Chemie und Biochemie, Strasse des 17. Juni 135, 10623 Berlin, Germany ReceiVed: April 6, 2000; In Final Form: May 30, 2000
Photophysical properties of low-energy antenna chlorophylls were probed in individual Photosystem I complexes from the cyanobacterium Synechococcus elongatus. Temperature activated fluorescence quenching unravels energy transfer pathways within the red-most pigment pool and to the reaction center. Low-temperature spectroscopy indicates that two subgroups of pigments which are present in the red antenna pool differ not only by their spectral position but also by the strength of electron-phonon coupling. Possible dimeric nature of the strongly phonon coupled red-most state is discussed in context of hole-burning data (Ra¨tsep, M.; Johnson, T. W.; Chitnis, P. R.; Small, G. J. J. Phys. Chem. B 2000, 104, 836).
Introduction Photosystem I (PSI) of cyanobacteria and green plants is a pigment-protein complex that consists of reaction center (RC) and antenna parts. The first step of the photosynthetic reaction is the absorption of a photon in the antenna pigments of the PSI with subsequent energy transfer to the reaction center. The excitation energy is used to induce transmembrane charge separation. The PSI uses plastocyanine of chytochrome as electron donors and ferrodoxin as electron acceptors finally reducing the energy carrier NADP+. The structure of PSI from the cyanobacteria Synechococcus elongatus was recently determined by X-ray diffraction.1,2 Each PSI monomer contains 11 protein subunits binding approximately 100 chlorophyll a (Chl) and 10-12 carotenoid molecules. A pair of chlorophylls located in the central area of the photosynthetic unit was assigned to the primary electron donor (called P700). The electronic transitions of the majority of Chl antenna molecules are at higher transition energies than those * Corresponding author. E-mail:
[email protected]. † 3. Physikalisches Institut. ‡ Technische Universita ¨ t Berlin.
of P700 and form an absorption band around 680 nm (C680). However, there is also a considerable number of chlorophyll molecules absorbing at wavelength longer than 700 nm. These chlorophylls are often referred to as the red pool. Different spectroscopic methods were successfully applied to unravel the energy pathway in PSI aggregates. Time-resolved absorption measurements indicate that the energy equilibration between the main C680 and the red pool of pigments takes place on a picosecond time scale. Several models have been applied to describe energy trapping dynamics in PSI. A trap-limited model predicts that the overall kinetics in PSI is determined by the rate of charge separation in the reaction center part.3 A transferto-trap limited mechanism assumes that energy transfer from the antenna pigments to P700 is rate limiting.4 Under ambient conditions energy captured by low-energy Chls can be efficiently transferred to P700 via thermally populated vibrational levels. This process is highly efficient at physiological temperatures as shown by various techniques (see e.g. ref 5 and references therein). Photophysical properties of red pool Chls have been subject to intense spectroscopic studies during the past few years (for a recent review see ref 6). The amount of PSI low-energy pigments and their spectral forms
10.1021/jp001332t CCC: $19.00 © 2000 American Chemical Society Published on Web 08/05/2000
8094 J. Phys. Chem. B, Vol. 104, No. 34, 2000
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depend on the type of photosynthetic unit. Deconvolution of Synechocystis mutant absorption spectra shows that the red pigments pool cannot be associated with peripheral protein subunits.7 Recently reported absorption spectra of Synechococcus elongatus show that low-energy Chls may belong to the connecting domain of the monomeric PSI within the trimer.5 It was estimated that five Chls C708 and six chlorophylls C719 contribute to the absorption in this spectral region.8 The spectral assignment of the red-most pigments is tentative because of the strong overlap of spectral bands.8 Absorption and fluorescence emission bands of long-wavelength pigments are strongly inhomogeneously broadened. Holeburning spectroscopy has proven to be a powerful method for probing the electronic structure and photophysical properties of many photosynthetic complexes.9 Hole-burning data provide evidence for strong electron-phonon coupling in reaction center containing photosynthetic units if compared to light-harvesting antenna (LH2, LHCII).10 Single-molecule spectroscopy dealing with single photosynthetic aggregates provides a possibility to resolve the optical transition of individual pigments, even under nonselective excitation of antenna Chls (for recent review of the method see ref 11). This powerful approach was successfully applied to the purple bacterial light harvesting complex LH212-17 and plant antenna LHCII.18 We present here the first results of single-molecule spectroscopy on the reaction-center containing photosynthetic complex PSI from the cyanobacteria Synechococcus elongatus. The aim of this work is to probe the excitation dynamics in individual PSI complexes. Experimental Section PSI from the cyanobacterium Synechococcus elongatus has been isolated as described in ref 19. Purified PSI trimers were diluted in buffer containing 20 mM Tricine, pH 7.5, 25 mM MgCl2, and 0.02% (w/w) of detergent (β-DM, Sigma) to reach a final concentration of approximately 10-6 M Chl. Detergent concentration was slightly above micelle formation to avoid PSI aggregation. For measurements, PSI containing buffer was diluted again (1/100) in a solution containing 1% (w/w) PVA, 25 mM MgCl2, and 5 mM Na-ascorbate and spin coated on a cover glass to obtain thin PSI doped films. The low final PSI concentration allows the spatial selection of individual PSI trimers. Sample preparation and mounting were accomplished in the dark. Experiments were carried out using a home-built beam scanning confocal microscope able to operate from 1.8 to 300 K. The microscope objective (63X, N. A. 0.85, Melles Griot) and the sample were immersed in liquid helium when operating at temperatures below 4.2 K. The excitation source was a Coherent CR 699-21 dye laser operating with DCM and pyridine dyes (broadband mode, line width 1 cm-1). Fluorescence emission of single PSI particles was filtered using a holographic notch filter (Kaiser) and detected by an avalanche photodiode (EG&G). Fluorescence spectra were measured with an Acton Research 1/4 m spectrograph equipped with a back illuminated CCD camera (PI, model 100EB), typical acquisition time was a few tens of seconds. Fluorescence excitation spectra were recorded using multiple fast (approximately 1 nm/s) laser sweeps across the red pool absorption band in order to minimize the effect of photoinduced spectral diffusion. Results and Discussion Figure 1a shows fluorescence images of single PSI complexes recorded at 17 and 236 K. Every bright spot corresponds to the fluorescence emission of a single PSI trimer. Note the drastic decrease of the fluorescence intensity upon temperature increase.
Figure 1. Temperature dependence of single PSI fluorescence intensity. (A)Confocal fluorescence images of single PSI isolated in PVA film. Images have been recorded at temperatures as indicated. Fluorescence intensity is decoded in gray scale. The excitation wavelength was 680 nm. (B) Fluorescence intensity of two single PSI trimers as a function of temperature. Experimental data have been fitted with Arrhenius equation: (A + B exp(-∆E/kBT))-1 (according to ref 16). The activation energy (∆E) for the two complexes is ∆E1 ) 550 cm-1 and ∆E2 ) 490 cm-1.
This strong temperature dependence of the fluorescence intensity was observed in ensemble experiments5,20 and is a signature of thermally activated uphill energy transfer from the low-energy Chl pool to the reaction center. At physiological temperature, energy trapped by the red pool of Chl can be transferred to the reaction center part via thermally populated vibrational levels. At low temperature this channel is blocked leading to a more intense fluorescence emission. The quenching behavior therefore contains information concerning the energy barrier between red pool pigments and P700. Analysis of temperature activated
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Figure 2. Low-temperature fluorescence excitation spectra of red-most antenna pigments. (A) A fluorescence excitation spectrum of a single PSI aggregate at 2 K. The spectrum was obtained summing 30 consecutive scans and detecting the fluorescence at λ > 725 nm. The background is limited by the dark count rate of the detector. (B) Spectral distribution of single Chl zero-phonon lines (spectral region between 705 and 717 nm has been studied).
fluorescence quenching was performed for 15 aggregates. In Figure 1b we show results for two typical complexes. The fluorescence intensity decreases by an order of magnitude as the temperature is increased from 17 to 280 K. A simple Arrhenius law has been used to fit experimental data (for details see ref 20). The average activation energy derived from all data on single PSI complexes is 500 cm-1. This value is higher than that obtained using ensemble measurements (363 cm-1). The difference may be due to the method chosen. When dealing with single molecules we select those having high enough fluorescence quantum yield in order to be able to detect them. These are aggregates, which may be characterized by a low rate of uphill energy transfer to the RC and hence a high energy gap between the red Chl pool and RC. Classical measurements are the results of averaging over the whole molecular ensemble, also containing low-energy gap aggregates. The slight difference in activation energy for the two aggregates presented in Figure 1b can be explained in terms of differences in P700 and/or pigment energy levels among the complexes. From hole-burning data21 it is known that the P700 absorption band is distributed over roughly 100 cm-1. This is on the order of the observed differences in ∆E in our single molecules. It is noticeable that single PSI fluorescence emission can be detected at room temperature regardless of the low fluorescence quantum yield. The high photostability of PSI at low temperature make them a good system for detailed spectroscopic study. To probe the spectroscopic properties of the low-energy antenna we recorded fluorescence excitation spectra of single PSI complexes. Therefore, the excitation laser was scanned in the low-energy antenna region (705-720 nm). Fluorescence was detected at wavelength longer than 725 nm. The spectra show narrow excitation lines (Figure 2a). The line width of spectra (∼1 cm-1) recorded using a single fast laser sweep across the resonance was limited by our experimental resolution. Those lines may correspond to the zero-phonon transitions of individual Chl molecules. Significant line broadening was observed during multiple scans due to spectral diffusion. The number of the narrow Chl lines observed in the excitation spectra varies from aggregate to aggregate (typically we observed 1-2 lines in the region 705-715 nm, however for some aggregates up to 4 lines were visible). The quantitative analysis of the spectral pool composition is difficult because of the intense spectral diffusion. Spectral jumps of single chlorophylls may be caused by the rearrangement of the local
Figure 3. A fluorescence emission spectrum of single PSI complex at 2 K. The fine structure in the 712 nm region corresponds to zerophonon transitions of individual Chl molecules.
protein environment. Such processes may be a result of the optical excitation because the emission of Stokes-shifted photons by Chl molecule is accompanied by heat dissipation in their local environment. The absence of a visible phonon structure in the excitation spectra is remarkable. This effect is related to the difference in absorption cross section in the zero-phonon line and phonon wing. Even in the case, when the major part of the total spectral intensity is concentrated in a phonon wing, the absorption cross section of a pure electronic transition can be higher because its oscillator strength is concentrated in a narrow spectral interval. The spectral distribution of zero-phonon transitions (Figure 2b) shows a maximum at 712 nm with a considerable spread. Upon tuning the laser to the maximum of the bulk Chl absorption band (680 nm), only emission from red-most Chl is detected in low-temperature fluorescence emission spectra (see Figure 3). Individual Chl lines resolved in fluorescence excitation spectra are also visible in fluorescence emission spectra (fine structure in the spectral region between 710 and 715 nm). The spectral positions of the observed lines vary from one aggregate to another (this effect is related to differences in the local environment of pigments leading to an inhomogeneous broadening in ensemble experiments). Note that the singlemolecule approach provides the possibility to unravel spectral fine structure using nonselective excitation. Close to the narrow emission lines of individual Chl molecules we observe a broad spectral band peaking at 730 nm (see Figure 3). Since there are no intense Chl vibrations in the low-frequency domain we believe that the broad structure is related to a pigment-protein system with strong electron-phonon coupling. Two possible origins of the 730 nm band can be considered. First, the broad spectral band may be a phonon wing of the zero-phonon line found in the 710-715 nm region of Figure 3. Because of strong electron-phonon coupling, a large Stokes shift may be associated with the low-frequency phonon modes of the protein environment. Second, the 730 nm band corresponds to a different pigment site. On the basis of hole-burning studies of pigment-protein complexes10 we assume that the main phonon frequency responsible for the formation of a phonon wing is 16 cm-1. A large Stokes shift (300 cm-1) would require a
8096 J. Phys. Chem. B, Vol. 104, No. 34, 2000 Huang-Rhys factor of the 712 nm state of about 20. Under these conditions the zero-phonon lines should not be observable because of an extremely low Franck-Condon factor (exp (-20)). On the basis of the ensemble data8 we can tentatively assign the two observed bands to the earlier reported C708 and C719 groups of pigments. We expect a ratio of the oscillator strength of about 5:6 for C708 and C719 Chl sites, respectively. The high total intensity of the 730 nm band may be related to the difference in the triplet quenching efficiency between two pigment pools. (The efficiency of the triplet state quenching is particularly important for the single molecule fluorescence intensity because experiments are usually carried out under conditions close to the saturation). The stronger quenching of the red-most state seems to be physiologically motivated because of the efficient energy transfer to the red-most state at room temperature. Hence two different Chls pools probably play a role in the formation of the long-wavelength antenna part of PSI. The absence of a narrow zero-phonon lines in the red-most pool may be explained in terms of an intense spectral diffusion or/ and strong coupling leading to low zero-phonon lines intensity. The efficiency of electron-phonon coupling in pigment-protein complexes depends on the change in permanent dipole moment (∆m).22 Recently available hole-burning data on red pool Chls of PSI from Synechocystis show that ∆m is increasing when the burn wavelength increase from 700 to 715 nm leading to significant broadening of hole spectra and disappearance of zerophonon structure on the red edge of the PSI absorption band.10 A charge-transfer character of excited antenna state can be associated with a strong electron-exchange coupling between closely spaced Chl.23 Unfortunately Stark-spectroscopy data are not available for Synechococcus elongatus and we can only speculate about the nature of the strong coupling in this system. Nevertheless our data suggest that the discussion concerning the excitonic nature of the low-energy C714 state of Synechocystis (see refs 10 and 24) can be expanded to the redmost state of Synechococcus elongatus. Summarizing, the spectral analysis suggests that two antenna Chl pools are responsible for the red-most absorption of PSI from Synechococcus elongatus. The electronic transition of 712 nm state is not strongly coupled to the phonon bath of the protein environment whereas electron-phonon coupling of the red-most pool leads to a complete disappearance of the zero-phonon structure. Strong coupling may be associated with excitonic interaction between chlorophylls in the red-most pool. With a variable temperature confocal microscope we are able to follow the fluorescence intensity of a single complex from 1.5 to 280 K, demonstrating differences in the activation energy for the transitions from the red chlorophyll pool to the reaction
Letters center among different complexes. An unexpected result was that the fluorescence of single PSI complexes can be detected at room temperature, opening the door toward the probing of a single PSI complex at work. Acknowledgment. We thank Dr. E. Schlodder for stimulating discussions, Prof. H. T. Witt and Dr. P. Fromme (all TU Berlin) for the PS I preparations, and Prof. H.-W. Trissl (University Osnabru¨ck) for helpful comments. This work was supported by Volkswagen Stiftung and Fonds der Chemischen Industrie (to R.B.). References and Notes (1) Krauss, N.; Schubert, W.-D.; Klukas, O.; Fromme, P.; Witt, H. T.; Saenger, N. Nat. Struct. Biol. 1996, 3, 965. (2) Schubert, W.-D.; Klukas, O.; Krauss, N.; Saenger, W.; Fromme, P.; Witt, H. T. J. Mol. Biol. 1997, 272, 741. (3) Holzwarth, A. R.; Schatz, G.; Brock, H.; Bittersman, E. Biophys. J. 1993, 64, 1813. (4) van Grondelle, R.; Dekker: J. P.; Gillbro, T.; Sundstro¨m, V. Biochim. Biophys. Acta 1994, 1187, 1. (5) Palsson, L. O.; Flemming, C.; Gobets, B.; van Grondelle, R.; Dekker: J. P.; Schlodder, E. Biophys. J. 1998, 74, 2611. (6) Karapetyan, N. V.; Holztwarth, A. R.; Ro¨nger, M. FEBS Lett. 1999, 460, 395. (7) Soukalis, V.; Savikhin, S.; Xu, W.; Chitnis, P. R.; Struve, W. S. Biophys. J. 1999, 76, 2711. (8) Palsson, L.-O.; Dekker: J. P.; Schlodder, E.; Monshouwer, R.; van Grondelle, R. Photosynth. Res. 1996, 48, 239. (9) Moerner, W. E., Ed. Persistent Spectral Hole Burning: Science and Applications; Topics in Current Physics 44; Springer-Verlag: New York, 1987. (10) Ra¨tsep, M.; Johnson T. W.; Chitnis P. R.; Small G. J. J. Phys. Chem. B 2000, 104, 836. (11) Tamarat Ph.; Maali, A.; Lounis, B.; Orrit, M. J. Phys. Chem. A 2000, 104, 1. (12) Orrit, M. Science 1999, 285, 349. (13) Tietz, C.; Chekhlov, O.; Dra¨benstedt, A.; Schuster, J.; Wrachtrup, J. J. Phys. Chem. B 1999, 103, 6328. (14) Tietz, C.; Dra¨benstedt, A.; Schuster, J.; Wrachtrup, J. Proc. SPIE 1999, 3607, 68. (15) van Oijen, A. M.; Ketelaars, M.; Ko¨hler, J.; Aartsma, T. J.; Schmidt, J. Science 285, 400. (16) van Oijen, A. M.; Ketelaars, M.; Ko¨hler, J.; Aartsma, T. J.; Schmidt, J. J. Phys. Chem. B 1998, 102, 9363. (17) van Oijen, A. M.; Ketelaars, M.; Ko¨hler, J.; Aartsma, T. J.; Schmidt, J. Biophys. J. 2000, 78, 1570. (18) Tietz, C.; Gerken, U.; Jelezko, F.; Wrachtrup, J. Single Mol. 2000, 1, 15. (19) Fromme P. Witt H. T. Biochem. Biophys. Acta 1998, 1365, 175. (20) Tusov, V. B.; Korvatovskii, B. N.; Panchenko, V. Z.; Rubin L. B. Doklady Biophysics 1980, 252, 112. (21) Gillie, J. K.; Lyle, P. A.; Small, G. J.; Golbeck, J. H. Photosyn. Res. 1989, 22, 233. (22) Small, G. J. Chem. Phys. 1995, 197, 239. (23) Beekman, L. M. P.; Frese, R. N.; Fowler, G. J. S.; Picorel, R.; Cogdell, R. J.; van Stokkum, I. H. M.; Hunter, C. N.; van Grondelle, R. J. Phys. Chem. B 1997, 101, 7293. (24) Melkozernov, A. N.; Lin, S.; Blankenship, R. E. J. Phys. Chem. B 2000, 104, 1651.
