Fluorescence Decay Kinetics of Solubilized Pigment Protein

Feb 11, 2004 - P., Ed.; Kluwer Academic Publishers: Dordrecht, 1995; Vol II, p 275. (50) Bergmann, A.; Eichler, H.-J.; Eckert, H.-J.; Renger, G. Photo...
0 downloads 0 Views 422KB Size
3326

J. Phys. Chem. B 2004, 108, 3326-3334

Fluorescence Decay Kinetics of Solubilized Pigment Protein Complexes from the Distal, Proximal, and Core Antenna of Photosystem II in the Range of 10-277 K and Absence or Presence of Sucrose J. Huyer,† H.-J. Eckert,‡ K.-D. Irrgang,‡ J. Miao,‡ H.-J. Eichler,† and G. Renger*,‡ Optical Institute and Max-Volmer-Laboratories for Biophysical Chemistry, Technical UniVersity Berlin, Strasse des 17. Juni 135, 10623 Berlin ReceiVed: July 31, 2003; In Final Form: October 27, 2003

The decay kinetics of chlorophyll (Chl) fluorescence of solubilized pigment protein complexes of the distal, proximal, and core antenna of photosystem II from higher plants have been analyzed in the temperature range of 10-277 K using buffer solutions containing or lacking sucrose as cryoprotectant. It was found that (i) at 277 K the 1Chl* decay of the complexes LHCIIb (distal), CP29 (proximal), and CP47 (core) is characterized by a biphasic kinetics with characteristic lifetimes in the range of 1.5-2.5 ns (fast phase) and 4-4.8 ns (slow phase), (ii) the slow phase dominates in all three complexes with normalized amplitudes of g0.65, (iii) in solutions containing sucrose the lifetime of the slow phase increases with decreasing temperature and reaches values in the range of 5.2-5.8 ns at 10 K, whereas those of the fast phase exhibit a more complex temperature dependence with a pronounced minimum value in the range of 150-200 K, (iv) markedly different temperature dependencies with pronounced minima in the range of 150-200 K are observed for both lifetime and normalized amplitude of the slow phase when the pigment protein complexes are dissolved in buffers without sucrose. The results are interpreted as evidence for two spectroscopically and kinetically distinguishable subpopulations in solubilized LHCIIb, CP29, and CP47 that are characterized by different rate constants of radiationless decay into the ground state of Chl. Possible mechanisms are discussed.

Introduction The exploitation of solar radiation as a free energy source of living matter takes place in photosynthetic organisms. In general, the overall process comprises three types of reaction sequences (for reviews see refs 1-3): (i) formation of electronically excited states by light absorption of pigment ensembles and efficient transfer to the photochemically active pigment PRC that is located within a special functional unit, the reaction center, (ii) dissociation of the excited singlet state at 1PRC* into a radical ion pair PRC+• Acc1-• followed by stabilization of the primary charge separation via rapid electron transfer from acceptor Acc1-• to another component Acc2-•, and (iii) dark reaction sequences energetically driven by PRC+• and Acc2-• leading to formation of stable products with high energy content. Photosynthesizing organisms are illuminated with light of different intensities and spectral distribution. Therefore, suitable adaptation mechanisms are required in order to achieve both, efficient excited-state transfer (EET) to PRC at limiting photon flux densities1,2 and protection to photoinhibition (PI) emerging under exposure to strong light with preferential sensitivity of PS II in all oxygen evolving organisms.5-7 In particular, land plants exposed to solar radiation of drastic diurnal and seasonal variations are unable to respond by large scale phototrophic movements such as some microorganisms (for a review see ref 4). The adaptation to varying illumination predominantly occurs at the level of electronically excited states formed by light absorption within pigment protein complexes. The pigment * To whom correspondence should be addressed. Fax: +49-30-31421122. E-mail: [email protected]. † Optical Institute. ‡ Max-Volmer-Laboratories for Biophysical Chemistry.

proteins constitute antenna systems that are functionally connected with PRC via excited energy transfer.1-3 The antenna systems are built up by two essentially different types of pigment proteins: (i) the core antenna that is an integral part of the reaction centers/ photosystems which contain PRC, Acc1, and Acc2 and (ii) the proximal/distal antenna that does not contain these cofactors. During the evolutionary development of oxygen evolving organisms from cyanobacteria up to the level of higher plants, the primary structures of the core antenna proteins designated as CP43 (PsbC) and CP47 (PsbB) and the number of pigments bound to them were very likely not changed, whereas the proximal/distal antenna complexes reveal rather large structural divergencies. In the phylogenetically older cyanobacteria,8 discoidal or hemispherical phycobilisomes containing covalently bound open chain tetrapyrroles form the distal antenna, which are extrinsically bound to the cytoplasmic site of the PS II complexes (for reviews see ref 9 and 10). A key step in evolution of the distal/proximal antenna was the development of integral pigment proteins referred to as light harvesting complexes (LHCs). This event occurred at the level of red algae containing both phycobilisomes and LHCs.11,12 The common structural motif of all LHCs are three transmembrane R helices and noncovalent binding of chlorophylls (Chls) and carotenoids (Cars). All members of the LHC family so far known contain Chl a. In addition, further pigments are bound that are different in various taxonomic groups: zeaxanthin in most rhodophytes, Chl c and peridinin in dinophytes, Chl c and fucoxanthin in chromophytes, and Chl b and lutein in chlorophytes.13 These findings are indicative of some flexibility of pigment binding by the polypeptide matrix of the LHCs. Based on recent studies, the existence of eight Chl binding sites per

10.1021/jp030944l CCC: $27.50 © 2004 American Chemical Society Published on Web 02/11/2004

Kinetics of Solubilized Pigment Protein Complexes polypeptide and its capability to bind different pigments appear to be common features of LHCs.14 In green plants, the promiscuity of pigment binding is nicely illustrated by in vitro reconstitution experiments with different stoichiometric ratios of Chl a, Chl b, and various Cars.15-17 It has been suggested that these characteristics of LHCs enabled a widespread distribution of eucaryotic photosynthetic organisms in different aquatic regions and, most importantly, the occupation of the land by green plants.14 Among the LHCs of chromophytes, rhodophytes, dinophytes, and “minor” Chl a/b LHCbs of chlorophytes (CP24, CP26, and CP29), the LHC II complex of plants is a marked exception. This complex which binds about 50% of the total Chl of the thylakoid membrane is normally isolated as trimers that are assumed to be also the predominant form of LHC IIb in vivo. The structure of the monomeric subunits each containing 12 Chls (7 Chl a and 5 Chl b) and at least 2 carotenoids has been gathered from analyses of electron diffraction patterns at a resolution of 3.4 Å.18 The proteins of LHC IIb are encoded by three different nuclear genes (lhcb1-lhcb3) leading to primary structures of 223-232 amino acids and apparent molecular masses of 24-28 kDa.19,20 LHC IIb was found to oligomerize into different homo- and heterotrimers.21 Recent proteomics analyses using reversed phase high performance liquid chromatography, in combination with electrospray ionization mass spectrometry (ESI-MS), reveals a high copy number of genes and a great variability of the hydrophobic properties of the Lhcb1-Lhcb6 proteins. This striking feature may be a strategy of plants for tuning the antenna system to achieve optimal adaptability22 to different illumination conditions. Another recently discovered feature is the existence of mixed pigment binding sites occupied by either a Chla-Chlb or a Chlb-Chlb pair23 that could be a complementary way of fine-tuning of the functional role of LHCIIb. In excess light, the harmless decay of the superfluous population of electronically excited states is of central relevance for protection to photoinhibition. This goal is achieved by enhanced deexcitation of 1Chl* via dissipative channels thus giving rise to nonphotochemical quenching (NPQ) of chlorophyll fluorescence.24 Among different contributions to NPQ, the “energy quenching” qE is of special relevance because it permits a short-term adaptation (see ref 25 and references therein). Three factors are essential for the extent of qE:24-30 acidification of the thylakoid lumen, low pH induced deepoxidation of violaxanthin to zeaxanthin with antheraxanthin as intermediate, and the presence of the PsbS protein that becomes protonated. In principle, two basically different types of mechanisms can be used to decrease high populations of electronically excited pigments: (i) rapid transfer from 1Chl* to pigments that act as efficient quenchers or (ii) opening of channels of radiationless decay of excited singlet states of Chls. At present, the mechanism of qE is not yet fully understood and especially for the role of the violaxanthin (V) S zeaxanthin (Z) cycle both of the above-mentioned mechanisms are discussed.26,30-33 Different lines of evidence favor the idea that conformationally induced changes in the protein matrix open channels for radiationless decay of 1Chl* into the ground state. These structural changes exhibit a striking cooperativity to H+ binding from the lumen and are controlled by the population with the different species of the V/Z cycle.33,34 In an attempt to address questions on radiationless decay pathways in the PS II antenna, the decay kinetics of 1Chl* were analyzed in three different purified detergent-solubilized pigment

J. Phys. Chem. B, Vol. 108, No. 10, 2004 3327 protein complexes being representative subunits of the peripheral, proximal, and core antenna. Experimental Section Preparation of Samples. The starting materials for the preparation of LHC II, CP29, and CP47 were PS II membrane fragments isolated from spinach chloroplasts according to the procedure described by Berthold et al.35 with some modifications outlined in Vo¨lker et al.36 LHC II samples were isolated by solubilization of salt-washed PS II membrane fragments from spinach in the presence of n-βdodecyl maltoside (β-DM) and separation by sucrose density gradient centrifugation as described in detail in Irrgang et al.37 The Chl a/Chl b (w/w) ratio was determined to be 1.29 ( 0.05 using the method of Porra et al.38 Densitometric scanning of silver stained SDS/urea/polyacrylamide gels revealed that LHC II samples consisted to about 90% of Lhcb1, Lhcb2, and Lhcb3. About 10% were due to minor Chl a/Chl b proteins (Lhcb5, Lhcb6) as immunologically identified and outlined previously in Irrgang et al.37 and Vasil’ev et al.39 PS II core complex proteins were not detectable. CP29 was isolated from PS II membrane fragments and purified following a modified protocol of Henrysson et al.40 in the presence of 2 mM benzamidine (Sigma), 5 mM -aminocaproic acid (Sigma) and 1 mM Pefabloc (Merck, Whitehouse Station, NJ) as protease inhibitors. Sulfobetaine 12 (SB 12) was used in place of sulfobetaine 14 (SB 14) and the column equilibration and gradient buffers contained 0.1% (wt/vol) SB 12 and 0.05% (wt/vol) n-β-dodecyl maltoside (β-DM). Chromatography was run using a CM-Sepharose Fast Flow column under dim green light at 4 °C. One to two rechromatographies were necessary to obtain purified CP29 as described in Pieper et al.41 The purified pigment-protein complex was concentrated by centrifugation in Centriprep 10 tubes to the desired Chl concentration. The purity was analyzed by sodium dodecyl sulfate (SDS)/ urea/PAGE using a 6% polyacrylamide stacking and a 15% polyacrylamide separating gel. Both gels contained 0.1% (wt/ vol) SDS and 5 M urea, and the buffer system of Laemmli was applied.42 Western blotting was carried out according to Towbin et al.43 using monoclonal antibodies as in Pascal et al.44 Gels were stained with silver as in Heukeshoven and Dernick.45 Chl a and b were determined according to Porra et al.,38 and carotenoids were spectroscopically analyzed by measuring the absorbance at 470 nm using the extinction coefficients of Wellburn and Lichtenthaler46 and Davis.47 Furthermore, the pigment composition was analyzed by reverse-phase highperformance liquid chromatography as described in Scho¨del et al.48 Purified homogeneous CP29 contained 6 Chla, 2 Chlb, and 2-3 carotenoids. Crude CP47 obtained after the first CM-Sepharose column chromatography was further purified by anion exchange chromatography using a Mono Q HR (5/5) in combination with a FPLC system (AmershamPharmacia Biotech). The purity of the pigment-protein complex has been tested by SDS/urea/PAGE and immuno analysis using epitope-directed antibodies as outlined in Irrgang.49 The chromophore analysis was carried out as described above. Usually 14 ( 1 Chl a and 3-4 β-carotenes were identified per CP47, no Chlb or xanthophylls were detectable (unpublished results). Protein integrity of all samples were furthermore routinely analyzed by matrix-assistedlaser desorption ionization time-off-flight mass spectrometry (MALDI-TOF-MS) using a RETOF-MS (Bruker-Franzen). Measurements were performed in a linear mode using a pulsed

3328 J. Phys. Chem. B, Vol. 108, No. 10, 2004 UV laser (N2-laser, λ ) 337 nm, 3 ns pulse width). Calibration was carried out using bovine serum albumin (66430.0 [M+H+]+), myoglobin (17568.0 [M+H+]+)/apomyoglobin (16951.0 [M+H+]+), and insulin (5734.0 [M+H+]+). Mass deviations from the theoretically predicted values were less than 0.3% (unpublished results, data not shown). Gel filtration experiments were performed on a Superose 6 HR 10/30 column in combination with a FPLC system (Amersham Pharmacia Biotech) verifying that no oligomeric forms or free pigments were in the samples used for spectroscopic analyses. The following buffers were used for fluorescence lifetime measurements: (1) LHCII and CP29 samples were dissolved in buffer solutions containing 10 mM MES-NaOH pH ) 6.5, 10 mM NaCl, 0.05% (wt/vol) n-β-DM, 0.05% (wt/vol) SB12, 2 mM benzamidine, 5 mM -aminocaproic acid and (30% (wt/vol) sucrose (buffer A); (2) CP47 was investigated in 50 mM MES-NaOH pH ) 6.5, 30 mM NaCl, 0.025% (wt/vol) n-β-DM, 2 mM benzamidine, 5 mM -aminocaproic acid and (30% (wt/vol) sucrose (buffer B). The buffers applied in these measurements were adjusted to those which have been used during the isolation and purification of these pigment protein complexes except for LHCII. In the latter case, the same buffers were used as for CP29. In all cases, the pigment-protein complexes were stable and did not show any denaturation or pigment release. Lifetime Measurements. The fluorescence lifetime was measured with a home-built compact fluorescence spectrometer that is described in detail in Bergmann et al.50 In this system, fluorescence is excited at 45° with 80 ps (fwhm) light pulses from a diode laser with an energy of a few pJ and a wavelength of 654 nm. The cuvette (path length 3 mm) was positioned in a variable-temperature cryostat (10-300K, CTI-Cryogenics 8001/8300). Time and space correlated single photon counting (TSCSPC) was achieved by using a microchannel plate photomultiplier (MCP-PMT) with delay line anode (Europhoton GmbH, Berlin). The delay-line splits the electric pulse emanating from the second MCP and the difference in propagation time to both outputs of the delay line yields the space-coordinate of the photon. In combination with a 120 mm crossed CzernyTurner polychromator (MultiSpec, LOT) as dispersive element, this detector allows simultaneous monitoring of the time and wavelength dependence of the chlorophyll fluorescence with ps time resolution. The electronic equipment consists of two 1GHz preamplifiers (Ortec 9306) which connect the two outputs of the delay line anode with constant fraction discriminators (Tennelec TC 454). One output provides the start signal for two time-to-amplitude converters (TACs) (Ortec 457, space domain, and Tennelec TC 464, time domain). The stop signal for the space domain-TAC is provided by the second output of the delay line. The stop signal for the time domain-TAC is provided by a trigger signal from the laser diode. A personal computer is used for data processing in order to obtain the corrected signals and to achieve suitable transfer into a twodimensional matrix of 256 channels for the space domain (spectral resolution) and 1024 channels for the time domain. Special attention was paid to calibrate the time and wavelength axes by implementation of two calibration modes. For calibration of the time axis, the stop signal can be delayed by introducing a fixed time delay (e.g., ∆t ) 8 ns, Canberra type 2058 ns delay) in the electric circuit between the laser diode and the time domain-TAC. This time delay is switched on in the middle of one measurement of a fluorescence decay, thereby shifting the fluorescence signal by 8 ns. Using this procedure, one obtains a fluorescence decay curve with two peaks which are separated by the number of time channels that correspond

Huyer et al. to 8 ns. From the time difference and the number of channels between the two maxima, the time per channel for the time axis is calculated. The wavelength axis was calibrated by using the spectral lines of a neon gas lamp to link the 256 channels of the space axis of the two-dimensional data matrix to wavelengths. For reproducibility, the measurements were repeated up to 4 times using a new sample. With each sample, the measurement cycle started at 277 K and was then repeated at different temperatures down to 10 K. Each cycle was ended with a final measurement at 277 K. For data analysis, the interesting part of the 256 space () wavelength) channels was selected and binned into 12 groups representing the fluorescence decay curves at different wavelengths (width about 3.5 nm). To obtain decay-associated spectra (DAS), these 12 decay curves were globally fitted to a threeor two-exponential decay (lifetimes linked) by using the GLOBALS UNLIMITED software program (University of Illinois, Urbana), and the initial amplitudes of the decay components were plotted as a function of the wavelength. Results The top panel of Figure 1 shows typical contour plots of twodimensional (wavelength and time) fluorescence decay curves monitored at 277 K on samples of solubilized LHC II, CP29, and CP47 dissolved in buffer A (LHC II, CP29) or B (CP47). The time course of the fluorescence decay at 680 nm is depicted in the middle panel of Figure 1. A deconvolution of these curves reveals that for all three sample types the decay can be satisfactorily described by a biphasic kinetics of the form

F(t) ) afaste-t/τfast + aslowe-t/τslow

(1)

Furthermore, the lifetimes τfast and τslow are virtually independent of the emission wavelength in the range of 665-705 nm. The bottom panel of Figure 1 presents the spectra of the decay components gathered from a numerical fit of the experimental data shown in the top panel of Figure 1. An inspection of these data reveals three features: (i) the lifetime of the dominating slow component is very similar in the three different pigmentprotein complexes covering a range from 4.2-4.8 ns, whereas the fast component exhibits a greater variation from 1.5 ns for LHCII to 2.4 ns for CP29 with an intermediate value of 1.9 ns for CP47, (ii) the amplitude ratio of the slow to fast component aslow/afast varies between g8 for LHCII and values of about 2 and 3 for CP29 and CP47, respectively, and (iii) the spectra exhibit marginally small differences between the two components of each complex but the emission maximum of CP47 is red shifted by about 3 nm compared with the other two complexes. The striking feature of a biphasic relaxation kinetics in all three complexes raises questions on the origin of this phenomenon. At a first glance, it seems attractive to assume that the similar features of a biphasic fluorescence decay kinetics originate from the structure of the detergent micelles encapsulating the solubilized pigment protein complexes rather than reflecting the intrinsic properties of the biological material. However, for several reasons, this idea appears to be highly unlikely: (i) the general feature of a biphasic decay kinetics with lifetimes of 1.5-2.4 ns and 4.2-4.8 ns with the dominance of the latter component exhibit a striking similarity with findings recently reported from another research group (61), (ii) it is not easily understandable why only two distinct micelle structures with very similar distribution probabilities should exist under

Kinetics of Solubilized Pigment Protein Complexes

J. Phys. Chem. B, Vol. 108, No. 10, 2004 3329

Figure 1. Contour plot of two-dimensional fluorescence decay (top panel) at 277 K, decay kinetics at emission maximum (middle panel), and decay associated (DAS) spectra of fast and slow decay components (bottom panel) of solubilized LHCII (left side), CP29 (middle), and CP47 (right side) dissolved in buffer A (LHCII, CP29) or B (CP47) containing 30% w/v sucrose (see the Materials and Methods section). The panel with the decay kinetics of LHCII complexes also contains the instrument response function of the fluorescence spectrometer.

different conditions, (iii) incorporation into liposomes of the solubilized Lhcb-type pigment protein complexes gives rise to a multiexponential kinetics comprising much faster decay components.61 Based on these considerations, it is more reasonable to ascribe the observed phenomena to characteristics of the pigment protein complexes. In this case, in principle, two basically different mechanisms can be considered: (a) heterogeneous pigment binding within each monomeric subunit thus giving rise to two pools of Chls with distinctly different lifetimes or (b) existence of two populations of each type of pigment protein complexes (LHCII, CP29, and CP47) with different binding modes of at least one Chl that determines the fluorescence decay kinetics of the whole pigment ensemble within the monomeric unit (CP29 and CP47) or in LHCII trimers. The first alternative can be readily excluded because in all three types of antenna complexes analyzed in this study (LHCII, CP29, and CP47) the Chls within each monomer were shown to be connected via excited energy transfer (EET) in the picosecond (subpicosecond) time domain51-55 so that at room temperature during an overall lifetime of at least 1 ns a perfect equilibration is achieved and single exponential decay kinetics are expected. Therefore, only the second alternative has to be analyzed. The mechanism (b) tacitly implies that the noncovalent binding of Chls to a protein opens the road for installing nonradiative dissipative pathways to the ground state with rate

constants that are determined by its conformation. Accordingly, two subpopulations symbolized by “fast” and “slow” have to exist that are distinctly different with respect to the 1Chl* lifetimes. Furthermore, possible interconversions between these two populations have to be slow compared with the decay of 1Chl*. In an attempt to obtain further information on this striking “two state” phenomenon, experiments were performed where the nature of pigment protein interactions and their dynamics are modulated. This manipulation is expected to give rise to changes of the kinetics predominantly by variation of the contribution owing to nonradiative decay processes. One widely applied method of noninvasive changes is the freezing of protein modes. This gives rise to well-known changes of fluorescence emission originating from decreasing effects of electron-phonon coupling at lower temperatures [see ref 56 and references therein]. Likewise, radiationless decay channels are expected to be affected. As a result of these considerations, fluorescence decay kinetics were monitored as a function of temperature. Figure 2 compiles typical results for the emission spectra gathered from measurements performed at 277, 200, 77, and 10 K on samples of LHCII, CP29, and CP47. Apart from the expected sharpening of the spectra at lower temperatures, the data indicate that in all three complexes the

3330 J. Phys. Chem. B, Vol. 108, No. 10, 2004

Huyer et al.

Figure 2. Spectra of fast and slow decay components at 10, 77, 200, and 277 K of solubilized LHCII (top panel), CP29 (middle panel), and CP47 (bottom panel) in buffers A (LHCII, CP29) or B (CP47) containing 30% w/v sucrose. For experimental details, see the Materials and Methods section.

lifetime of the dominating slow component increases and reaches very similar levels within a range of 5.2-5.8 ns at 10 K. This value corresponds with that of monomeric Chl a in different solvents at room temperature.57 In marked contrast to the “slow” state conformation, the lifetime of the “fast” state subpopulation is not characterized by a monotonic increase toward longer values at decreasing temperatures but rather attains minimum values in the range of 150-200 K. The amplitude ratio aslow/ afast is only slightly dependent on temperature (CP29 and CP47) or virtually constant (LHCII); that is, the slow kinetics always dominates the overall fluorescence decay. A more detailed description is shown in Figure 3 where the temperature dependencies of τfast, τslow, the relative contributions Afast, Aslow, and the peak wavelengths λmax(fast) and λmax(slow) are summarized for the range of 10-277 K. To eliminate effects due to changes of band shape and peak position, the amplitudes afast and aslow are replaced by the relative contributions corresponding to the areas Afast and Aslow of the bands. An inspection of these data not only confirms the general trends gathered from Figure 2 but provides in addition further interesting information. The emission maxima of the fast and slow components are nearly the same for LHCII and CP29 at all temperatures, whereas in CP47, the difference between λmax(slow) and λmax(fast) increases when the temperature decreases and this shift reaches values of about 5 nm at 10 K. A similar red shift of the stationary fluorescence spectrum of CP47 has been described in an earlier report.58 The results also show that the fast component attains its minimum lifetime in the temperature range

between 150 and 200 K and markedly increases upon further lowering of the temperature. This property strikingly differs from the continuous increase of the lifetime of the slow component with decreasing temperature. With respect to the ratio of the relative contributions Afast/ Aslow, LHCII appears to differ from CP29 and CP47. In the former sample, type Afast/Aslow is virtually independent of the temperature, whereas the other two preparations exhibit a slight increase of this ratio due to freezing down to 150-200 K. Freezing of pigment protein complexes is expected to lead predominantly to a restriction of the protein flexibility without major effects on the time averaged “static” structure of the protein. Significant conformational changes without disturbing the native protein folding can be induced by the addition/ omission of substances in the buffer solution that are known to affect hydrogen bond networks within proteins. Many of these compounds simultaneously enhance the structural stability of proteins toward temperature variation. Among a great variety of substances naturally produced, several polyalcohols, especially sugars, are of central relevance because they are able to form hydrogen bonds and to give rise to cryoprotection. Polyols have been shown to stabilize special protein structures such as the molten globule state of water soluble cytochrome c at low pH.59,60 Therefore, it appears worth analyzing effects on the fluorescence decay kinetics in samples dissolved in buffer solutions that contain or lack these type of substances. All experiments so far described were performed on samples containing 30% w/v sucrose. Accordingly, different results are

Kinetics of Solubilized Pigment Protein Complexes

J. Phys. Chem. B, Vol. 108, No. 10, 2004 3331

Figure 3. Temperature dependence of lifetime, relative contribution, and peak wavelength of fast and slow phase of fluorescence emission from LHCII, CP29, and CP47 dissolved in buffers containing 30% w/v sucrose.

expected in the absence of sucrose provided that the dynamics of pigment-protein interactions and their temperature dependence are changed by the absence of this cryoprotectant. Figure 4 shows representative results obtained when using probes of the same composition as in the former experiments, except that sucrose was omitted. At room temperature, significant qualitative differences are not observed between samples with or without sucrose but quantitative shifts toward the contribution of the fast phase are discernible. In marked contrast, however, striking effects emerge at 200 K. In this case, samples without sucrose exhibit a drastic increase of the relative extent of the fast decay component at the expense of the slow phase. Furthermore, the lifetime of the slow component is significantly shorter than the corresponding values at 277 K in all three complexes. Accordingly, these effects are likely to originate from changes inside the monomeric units rather than from a less specific aggregation that would be expected to be different in the various sample types. The fast component exhibits the same trend of lifetime shortening only in CP29 and CP47 but not in LHCII. Apart from these general features, a specific effect emerges for CP29. At low temperatures (e77 K) biphasic kinetics are not sufficient to describe the fluorescence decay, i.e., an additional very fast component of about 700 ps is observed. The origin of this fast component in CP 29 remains to be clarified. To analyze these temperature dependencies in more detail, experiments were performed in the range from 10 to 277 K. The results obtained are summarized in Figure 5. A comparison of these data with the corresponding values depicted in Figure 3 reveals several significant differences between the complexes dissolved in buffer solutions either containing or lacking sucrose: (i) in samples without sucrose the lifetime of the slow component decreases upon freezing down to 200 K and subsequently increases when the temperature is further lowered to 10 K but does not reach the value of 5.5 ( 0.3 ns (in LHCII and CP29) that is observed in probes containing sucrose and

(ii) the relative contribution of the slow decay kinetics exhibit qualitatively an analogous temperature dependence, i.e., a decrease at 150-200 K followed by an increase upon further lowering of the temperature and are generally significantly smaller than the corresponding values obtained in sucrose containing buffer. Discussion The results of the present study unambiguously show that the lowest excited singlet states of Chla in solubilized complexes of the distal (LHCII), proximal (CP29), and core (CP47) antenna of PS II exhibit a striking biphasic decay kinetics. This finding is fully consistent with a recent report of Moya et al.61 on fluorescence lifetime measurements performed at 77 and 293 K on pigment-protein complexes of the distal and proximal antenna (LHCII, CP29, CP26, and CP24) in detergent micelles and liposomes. Here we show that the biphasic decay is a general feature observed over the wide temperature range from 10 to 277 K and not restricted to pigment-protein complexes of the Lhcb family but also includes the core antenna complex CP47 (PsbB protein). In agreement with recent suggestions,61 the biphasic kinetics is assumed to originate from the existence of two different conformational states referred to as “fast” and “slow” according to their marked difference in the lifetime τ of 1Chla*. Because the EET processes between the chlorophyll molecules in each complex are much faster than τfast and τslow, the latter parameters can be described by a simple relation:

τi ) (kF + kISC + kdiss + kQ)i-1

(2)

where kF, kISC, and kdiss are the rate constants for radiative emission, intersystem crossing, and radiationless decay, respectively, kQ symbolizes the decay owing to interaction of 1Chl* with quenchers, and i symbolizes the conformational state (i )

3332 J. Phys. Chem. B, Vol. 108, No. 10, 2004

Huyer et al.

Figure 4. DAS spectra of fast and slow decay components at 10, 77, 200, and 277 K of solubilized LHCII (top panel), CP29 (middle panel), and CP47 (bottom panel) dissolved in buffers A (LHCII, CP29) or B (CP47) without sucrose. For experimental details, see the Materials and Methods section.

“fast” or “slow”). The formation of stable quenchers (e.g., carotenoid triplets) appears to be unlikely under our experimental conditions (very weak laser pulses, see the Materials and Methods section), and therefore, kQ is assumed to be negligible. As a consequence, the differences of the 1Chl* lifetimes in complexes attaining either conformation “fast” or “slow” are ascribed predominantly to effects of the protein matrix on kdiss of Chls. Furthermore, all Chls in LHCII, CP29, and CP47 are connected via efficient EET, and therefore, the modulation of kdiss of a single Chl molecule in each subunit is sufficient to affect the fluorescence lifetime of the whole Chl ensemble in this complex. The lifetime of all complexes in the “slow” conformation suspended in buffer solutions containing the cryoprotectant sucrose reaches very similar values within a range of τslow ) 5.5 ( 0.3 ns at 10 K. Interestingly, this value corresponds with the lifetime of 1Chla* in dilute solutions at room temperature57 where radiationless decay is negligibly small.62 Three conclusions can be drawn from this finding: (a) the fluorescence lifetime in all three complexes in the “slow” conformation state is limited by the decay reactions in Chl(s) that are virtually not coupled with another pigment to form a quenching species, (b) in sucrose containing buffer radiationless decay is dominated by the coupling with protein vibrations in complexes kept in the “slow”-conformation state, and (c) in the “fast”-conformation state an additional pathway is opened that enhances kdiss in at least one Chl molecule acting as dissipative trap of excited singlet states.

When neglecting kQ in eq 2, the rate constant kdiss,i is obtained by the relation

kdiss,i )

1 - (kF + kISC)i τi

(3)

If one assumes that kF is neither significantly affected by temperature nor by binding to the protein and also kISC is virtually invariant because the protein does not contain heavy atoms that could affect spin-orbit coupling (see textbooks of Physical Chemistry), a rough value of kdiss can be gathered from eq 3 by using (kF + kISC)-1 ) (5.5 ( 0.3) ns. Thus, values in the range of 107s-1 are obtained for kdiss,slow of solubilized LHCII, CP29, and CP47 at 277 K when attaining the “slow” conformation in buffer solutions containing sucrose. This finding indicates that the protein dynamics opens a radiationless channel for excited singlet state decay via heat dissipation with a rate constant of the same order of magnitude as the radiative pathway. Freezing of the protein vibrations eliminates this dissipative channel. The underlying mechanism of a protein dynamics induced kdiss is unknown. Recently, a first molecular dynamics simulation has been reported that analyzes effects of protein conformational flexibility on the electronic properties of chlorophylls in the light harvesting peridinin-chlorophyll-protein complex of dinoflagellate Amphidinium carterae.63 Vertical excitation energies were calculated as a function of dynamical perturbation

Kinetics of Solubilized Pigment Protein Complexes

J. Phys. Chem. B, Vol. 108, No. 10, 2004 3333

Figure 5. Temperature dependence of lifetime, relative contribution, and peak wavelength of fluorescence emission from LHCII, CP29, and CP47 dissolved in buffers without sucrose.

effects of the protein by using a first principle method (perturbed matrix method) to describe a rigidlike chromophore interacting with a stable classical environment. An extension of this method to higher order effects (e.g., including chromophore flexibility and environment polarizability) might provide a tool to understand and calculate kdiss as a function of protein dynamics. When applying the same considerations and eq 3 to describe the properties of the minor “fast”-state subpopulation, two interesting features emerge: (i) the estimated kdiss,fast values are 1 order of magnitude larger than kdiss,slow of the major “slow” subpopulation and also exhibit a greater variation between the complexes with kdiss,fast ≈ 5 × 108, 2 × 108, and 3.5 × 108 s-1 at 277 K for LHCII, CP29, and CP47, respectively, and (ii) the temperature dependence markedly differs from that of kdiss,slow with maximum values of kdiss,fast of about 5 × 108 s-1 at 150200 K for all three complexes and minimum values of (2-3) × 108 s-1 at 10 K. The 10 K values clearly show that in the “fast”-state subpopulation a significant dissipative pathway still exists with kdiss,fast (10 K) markedly larger than kdiss,slow (277 K). This finding indicates that the mechanism of generating kdiss,fast is dominated by factors other than the protein dynamics. Furthermore, the striking temperature dependence suggests that probably at least two effects contribute to kdiss,fast. If one assumes that the protein dynamics affect kdiss,i in a similar manner in both “slow”- and “fast”-state populations, the remaining and dominating contribution to kdiss,fast is characterized by an increase above a critical temperature of 150-200 K and a pronounced decrease below this threshold when the temperature decreases. A temperature of 150-200 K is reminiscent of characteristic changes of fluorescence spectra in LHCII64 and of the bands of B850 and B875 in LH2 and LH1, respectively, of anoxygenic purple bacteria.65,66 These changes of spectroscopic properties are ascribed to conformational changes of the protein.64 Therefore, it appears reasonable to assume that this transition is also responsible for a marked change of the temperature dependence of kdiss,fast.

The idea of structural effects on the extent of radiationless decay into the ground state is highly supported by the marked differences between the results gathered from the samples dissolved in buffers either containing or lacking sucrose (compare Figures 3 and 5). The most prominent features are the striking differences in the extent and temperature dependence of the relative contributions of the fast and slow phase that are ascribed to the population of the complexes in conformational states “fast” and “slow”. The molecular mechanism of the sucrose effect is not clarified. Apart from the particular situation of LHCII that is known to form aggregates with short fluorescence lifetimes,39,64,71 the molecular configurations mediated by xanthophyll species bound to the L2 site of Lhc proteins have been suggested to be responsible for the existence of the “fast”- and “slow”-state population in Lhc-type pigment protein complexes.61,67 This interesting hypothesis, however, does not explain the analogous “fast”/”slow”-state feature in CP47 because the protein matrix with six transmembrane helices markedly differs from that of Lhcb proteins such as CP 29 and LHC IIb monomers characterized by three transmembrane helices and most likely also different types and arrangements of the carotenoids. Furthermore, there exists a pronounced temperature dependence of the relative contributions in a buffer without sucrose. Therefore, a more extended pattern has to be considered. If one accepts that excited singlet state equilibration within each of the subunits is always 1-2 orders of magnitude faster than its decay, at least one Chl molecule in each pigment-protein complex has to attain two states with distinctly different rate constants kdiss for the nonradiative decay into the ground state. In agreement with former suggestions,61 it appears most likely that the magnitude of kdiss of the chromophore is regulated by the conformation of the protein microenvironment. In general, two different factors have to be taken into account: (i) conformationally distinct Chls and (ii) formation of radiationless decay channels by pigmentpitment and/or pigment-protein interactions. The former mech-

3334 J. Phys. Chem. B, Vol. 108, No. 10, 2004 anism could originate from a distortion of the ring planarity because saddled and ruffled porphyrines are characterized by significantly shortened lifetimes.68-70 It is easily conceivable that the protein backbone can effect the structure of the ring plane. Regardless of the detailed mechanism, the major question to be answered is why do only two different conformations “fast” and “slow” occur in these three types of solubilized antenna subunits in sucrose containing buffers? Further studies are underway to analyze this feature and its origin in detail. Acknowledgment. The authors thank K. Scharf and S. Kussin for their expert technical assistance. Financial support by the DFG (SFB 429 TP A1 and A3) and European Community Program Biotechnology (BIO4-CT97-2177) is gratefully acknowledged. References and Notes (1) Renger, G. In Topics in Photosynthesis, The Photosystems: Structure, Function and Molecular Biology; Barber, J., Ed.; Elsevier: Amsterdam 1992; p 45. (2) van Grondelle, R.; Dekker, J. P.; Gilbro, T.; Sundstro¨m, V. Biochim. Biophys. Acta 1994, 1187, 1. (3) Renger, G. In Concepts in Photobiology: Photosynthesis and Photomorphogenesis; Singhal, G. S., Renger, G., Govindjee., Irrgang, K.D., Sopory, S. K., Eds.; Kluwer Academic Publishers: Dordrecht; Narosa Publishing Co.: Delhi, 1999; p 52. (4) Ha¨der, D. P.; Tevini, M. Photobiology, Pergamon: Oxford 1987. (5) Prasil, O.; Adir, N.; Ohad, I. In The Photosystems: Structure, Function and Molecular Biology; Barber, J., Ed.; Elsevier: Amsterdam 1992; p 295. (6) Aro, E.-M.; Virgin, I.; Andersson, B. Biochim. Biophys. Acta 1993, 1143, 113. (7) Chow, W. S. In Molecular Processes of Photosynthesis, Vol. 9 of Bittar E; Barber, J., Ed.; AdVances in Molecular and Cell Biology series Ed.; JAI Press Inc.: Connecticut 1994. (8) Morcira, D.; Le Guyader, H.; Phillipe, H. Nature (London) 2000, 405, 69. (9) Gantt, E. In Encyclopedia of Plant Physiology, New Series; Staehelin, L. A., Arntzen, C. J., Eds.; Springer: Berlin, 1986; Vol. 19, p 260. (10) Mimuro, M. In Concepts in Photobiology: Photosynthesis and Photomorphogenesis; Singhal, G. S., Renger, G., Govindjee., Irrgang, K.D., Sopory, S. K., Eds.; Kluwer Academic Publishers: Dordrecht; Narosa Publishing Co.: Delhi 1999; p 104. (11) Green, B. R.; Dunford, D. G. Annu. ReV. Plant Physiol Plant Mol. Biol. 1996 47, 685. (12) Larkum, T.; Howe, C. J. AdV. Bot. Res. 1997, 28, 257. (13) Dunford, D. G.; Deane, J. A.; Tan, S.; McPadden, G. L.; Gantt, E.; Green, B. R. J. Mol. EVol. 1999, 48, 59. (14) Grabowski, B.; Cunningham, F. X.; Gantt, E. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 2911. (15) Bassi, R.; Croce, R.; Cugini, D.; Sandora, D. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 10056. (16) Rogl, H.; Ku¨hlbrandt, W. Biochemistry 1999, 38, 16214. (17) Hobe, S.; Niemeier, H.; Bender, A.; Paulsen, H. Eur. J. Biochem. 2000, 267, 616. (18) Ku¨hlbrandt, W. Curr. Opin. Struct. Biol. 1994, 4, 519. (19) Jansson, S. Biochim. Biophys. Acta 1994, 1184, 1. (20) Jansson, S. Trends Plant Sci. 1999, 4, 236. (21) Jackowski, G.; Pielucha, K. J. Photochem. Photobiol. B: Biol. 2001, 64, 45. (22) Zolla, L.; Timperio, A.-M.; Walcher, W.; Huber, C. G. Plant Physiol. 2003, 131, 1. (23) Salverda, J. M.; Vengris, M., Krueger, B. P.; Scholes, G. D.; Czarnoleski, A. R.; Novoderezhkin, V.; van Amerongen, H., van Grondelle, R. Biophys. J. 2003, 84, 450. (24) Krause, G. H.; Weiss, E. Annu. ReV. Plant. Physiol. Plant Mol. Biol. 1991, 42, 313. (25) Horton, P.; Ruban, A. V.; Walters, R. G. Annu. ReV. Plant Physiol. Plant Mol. Biol. 1996, 47, 655. (26) Demming-Adams, B.; Gilmore, A. M.; Adams, W. FASEB J. 1996, 10, 403. (27) Yamamoto, H. Y.; Bugos, R. C.; Hieber, A. D. In The Photochemistry of Carotenoids; Frank, H. A., Young, A. J., Britton, G., Cogdell, R. J., Eds.; Kluwer: Dordrecht, 1999: p 293. (28) Gilmore, A. M.; Hazlett, T.; Govindjee Proc. Natl. Acad. Sci. U.S.A. 1996, 92, 2273. (29) Li, X.; Phippard, A.; Pasari, J.; Niyogi, K. K. Funct. Plant Biol. 2002, 29, 1131.

Huyer et al. (30) Li, X.; Mu¨ller-Moule, P.; Gilmore, A. M.; Niyogi, K. K. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 15222. (31) Frank, H. A.; Cua, A.; Chynwat, V.; Young, A.; Gosztola, D.; Wasielewski, M. R. Photosynth. Res. 1994, 29, 1131. (32) Gilmore, A. M.; Shinkarev, V. P.; Hazlett, T. L.; Govindjee. Biochemistry 1998, 37, 13582. (33) Ruban, A. V.; Wentworth, M.; Horton, P. Biochemistry 2001, 40, 9896. (34) Wentworth, M.; Ruban, A. V.; Horton, P. Biochemistry 2001, 40, 9902. (35) Berthold, D. A.; Babcock, G. T.; Yocum, C. F. FEBS Lett. 1981, 134, 231. (36) Vo¨lker, M.; Ono, T.; Inoue, Y.; Renger, G. Biochim. Biophys. Acta 1985, 806, 25. (37) Irrgang, K.-.D.; Boekema, E. J.; Vater, J.; Renger, G. Eur. J. Biochem. 1988, 178, 209. (38) Porra, R. J. W.; Thompson, W. A.; Kriedemann, P. E. Biochim. Biophys. Acta 1989, 975, 384. (39) Vasil’ev, S.; Irrgang, K.-D.; Schro¨tter, T.; Bergmann, A.; Eichler, H.-J.; Renger, G. Biochemistry 1997, 36, 7503. (40) Henrysson, T.; Schro¨der, W. P.; Sprangfort, M.; A° kerlund, H.-E. Biochim. Biophys. Acta 1989, 977, 301. (41) Pieper, J.; Irrgang, K.-D.; Ra¨tsep, M.; Voigt, J.; Renger, G.; Small, G. J. Photochem. Photobiol. 2000, 71, 574. (42) Laemmli, U. K. Nature 1970, 227, 680. (43) Towbin, H.; Staehelin, T.; Gordon, J. Proc. Natl. Acad. Sci. U.S.A. 1979, 76, 4350. (44) Pascal, A.; Gradinaru, C.; Wacker, U.; Peterman, E.; Calkoen, F.; Irrgang, K.-D.; Horton, P.; Renger, G.; van Grondelle, R.; Robert, B.; van Amerongen, H. Eur. J. Biochem. 1999, 262, 817. (45) Heukeshoven, J.; Dernick, R. Electrophoresis 1985, 6, 103. (46) Wellburn, A. R.; Lichtenthaler, H. In AdVances in Photosynthesis Research; Sybesma, C., Ed.; Martinus Nijhoff Publ.: The Hague, 1984; Vol. II, p 9. (47) Davis, B. H. In Chemistry and Biochemistry of Plant Pigments, 2nd ed.; Goodwin, T. N., Ed.; Academic Press: London, 1976; Vol. II, p 38. (48) Scho¨del, R.; Irrgang, K.-D.; Voigt, J.; Renger, G. Biophys. J. 1999, 75, 3143. (49) Irrgang, K.-D. In Photosynthesis: from Light to Biosphere; Mathis, P., Ed.; Kluwer Academic Publishers: Dordrecht, 1995; Vol II, p 275. (50) Bergmann, A.; Eichler, H.-J.; Eckert, H.-J.; Renger, G. Photosynth. Res. 1998, 58, 305. (51) Bittner, T.; Irrgang, K.-D.; Renger, G.; Wasielewski, M. R. J. Phys. Chem. 1994, 98, 11821. (52) van Amerongen, H.; van Grondelle, R. J. Phys. Chem. B 2001, 105, 604. (53) Cinque, G.; Croce, R.; Holzwarth, A.; Bassi, R. Biophys. J. 2000, 79, 1706. (54) Chang, H.-C.; Jankowiak, R.; Yocum, C. F.; Picorel, R.; Alfonso, M.; Seibert, M.; Small, G. J. J. Phys. Chem. 1994, 98, 7717. (55) de Weerd, F. L.; van Stokkum, I. H. M.; van Amerongen, H.; Dekker: J. P.; van Grondelle, R. Biophys J. 2002, 83, 1586. (56) Pieper, J.; Voigt, J.; Renger G.; Small, G. J. Chem. Phys. Lett. 1999, 310, 296. (57) Vasil’ev, S.; Schro¨tter, T.; Bergmann, A.; Irrgang, K.-D.; Eichler, H.-J.; Renger, G. Photosynthetica 1997, 33, 553. (58) Groot, M. L.; Peterman, E. J.; van Stokkum, I. H.; Dekker, J. P.; van Grondelle, R. Biophys J. 1995, 68, 281. (59) Saunders, A. J.; Davis-Searles, P. R.; Allen, D. L.; Pielak, G. J.; Erie, D. A. Biopolymers 1999, 53, 293. (60) Konni, T.; Iwashita, J.; Nagayama, K. Protein Sci. 2000, 9, 564. (61) Moya, I.; Silvestri, M.; Vallon, O.; Cinque, G.; Bassi, R. Biochemistry 2001, 40, 12552. (62) Parker, C. A.; Joyce, T. A. Photochem. Photobiol. 1967, 6, 395. (63) Spezia, R.; Aschi, M.; Di Nola, A.; Di Valentin, M.; Carbonera, D.; Amadei, A. Biophys. J. 2003, 84, 2805. (64) Pieper, J.; Scho¨del, R.; Irrgang, K.-D.; Voigt, J.; Renger, G. J. Phys. Chem. 2001, 105B, 7115. (65) Wu, H.-M.; Ra¨tsep, M.; Lee, I.-J.; Cogdell, R. J.; Small, G. J. J. Phys. Chem. B 1997, 101, 7654. (66) Wu, H.-M.; Ra¨tsep, M.; Jankowiak, R.; Cogdell, R. J.; Small, G. J. J. Phys. Chem. B 1998, 102, 4023. (67) Frank, H. A.; Das, S. K.; Bautista, J. A.; Gosztola, D.; Wasielewski, M. R.; Crimi, M.; Croce, R.; Bassi, R. Biochemistry 2001, 40, 1220. (68) Rivikanth, M.; Reddy, D.; Chandrashekar, T. K. J. Photochem. Photobiol. A: Chem. 1993, 72, 61. (69) Gentemann, S.; Nelson, N. Y.; Jaquinod, L. A.; Nurco, D. J.; Leung, S. H.; Medforth, C. J.; Smith, K. M.; Fajer, J.; Holten, D. J. Phys. Chem. B 1997, 110, 1247. (70) Retsek, J. L.; Gentemann, S.; Medforth, C. J.; Smith, K. M.; Chirvony, V. S.; Fajer, J.; Holten, D. J. Phys. Chem. B 2000, 104, 6690. (71) Mullineaux, C. W.; Pascal, A. A.; Horton, P.; Holzwarth, A. R., Biochim. Biophys. Acta 1993, 1141, 23