† These authors contributed equally to this work. Previous work on intact thylakoid membranes showed that transient formation of a zeaxanthin radical cation was correlated with regulation of photosynthetic light harvesting via energy-dependent quenching. A molecular mechanism for such quenching was proposed to involve charge transfer within a chlorophyllzeaxanthin heterodimer. Using near infrared (880-1100 nm) transient absorption spectroscopy, we demonstrate that carotenoid (mainly zeaxanthin) radical cation generation occurs solely in isolated minor light-harvesting complexes that bind zeaxanthin, consistent with the engagement of charge transfer quenching therein. We estimated that less than 0.5% of the isolated minor complexes undergo charge transfer quenching in vitro, whereas the fraction of minor complexes estimated to be engaged in charge transfer quenching in isolated thylakoids was more than 80 times higher. We conclude that minor complexes which bind zeaxanthin are sites of charge transfer quenching in vivo and that they can assume Non-quenching and Quenching conformations, the equilibrium LHC(N)↔LHC(Q) of which is modulated by the transthylakoid pH gradient, the PsbS protein, and protein-protein interactions. INTRODUCTION Higher plant photosynthesis is initiated by absorption of light in pigment-binding (antenna) proteins that transfer absorbed solar energy to the reaction centers of photosystems (PS) II and I where energy conversion begins (1). The PSIIassociated light-harvesting complexes (LHCs) bind chlorophylls and carotenoids that are involved in both the harvesting and transfer of energy to the reaction center, and the harmless dissipation of excitation energy in excess of photosynthetic capacity (2). Thus, the PSII LHCs are critical 'branch-points' for energy partitioning during photosynthesis. The peripheral antenna consists of trimeric complexes composed of LHCII proteins, the major LHC of higher plant antennae. In between the peripheral LHCII and the reaction center there are three 'minor' LHCs referred to as CP29, CP26, and CP24 (1). Dissipation of excess light energy during photosynthesis involves several photoprotective mechanisms which are collectively referred to as non-photochemical quenching (NPQ) (2,3). The predominant component of NPQ is referred to as energy dependent quenching, or qE, and it is rapidly reversible and correlated with zeaxanthin (Z) formation (4). Mutants of A. thaliana have been instrumental in confirming the involvement of Z (5) and identifying a role for PsbS in qE (6). The npq4 mutant lacks a functional PsbS protein and exhibits very little qE (6). The PsbS protein has been subsequently proposed to be involved in controlling qE by sensing thylakoid lumen pH (7). The xanthophyll cycle consists of the enzymatic and reversible conversion of the thylakoidassociated pigment violaxanthin (V) to antheraxanthin (A) to Z (8). Very little qE is exhibited in the A. thaliana mutant referred to as npq1 which is impaired in its ability to convert V to Z as a result of a lesion in the gene encoding the thylakoid lumen-localized enzyme violaxanthin de-epoxidase (5). Conversely, the npq2 mutant of A. thaliana constitutively accumulates Z and lacks V and neoxanthin due to a lesion in the gene encoding Z epoxidase (5). 2 Elucidating the molecular details of qE has proved to be a major challenge. An approach involving computational modeling (9-11), molecular genetics (5,6), biochemistry (12) and ultrafast laser spectroscopy •+ in an qE-dependent manner (13). The NIR TA kinetics from isolated thylakoids of various mutants of A. thaliana specifically impaired in qE, including the npq1 and the npq4 mutants, did not show transient Car formation (13). Transient Car •+ signals in npq2 and npq2lut2, a mutant that constitutively accumulates Z but also lacks lutein, were very similar to each other (13). These results, along with the spectral signature (13), imply that the Car •+ transiently formed in the wild type thylakoids is a Z •+ species. The quenching of bulk chlorophyll by transfer of energy to a [Chl-Z] quenching complex that undergoes charge separation and subsequent recombination to the ground state provides a simple model for qE (13). Initiation of qE invokes a conformational change of at least one of the LHCs, triggered by contributions from ∆pH, PsbS, and Z (15). This implies that an equilibrium exists between nonquenching (N) and quenching (Q) forms: LHC(N) ↔ LHC(Q). Indeed the presence of distinct N and Q conformations in detergent solution has been reported on the basis of spectroscopic measurements •+ signal as a diagnostic that this is indeed the case for the minor LHCs (CP24, CP26, and CP29). Using the Z •+ signature to infer CT quenching, we estimate that ~80 times more of the minor complexes undergo CT quenching in thylakoids engaged in steady-state qE in comparison to isolated complexes. Taken together our results are suggestive of the minor complexes being sites of CT quenching in vivo. MATERIALS AND METHODS Isolation of antenna LHCs with specific xanthophylls Unstacked thylakoids were isolated from leaves of dark-adapted wild type, conditions under which V accumulates, and npq2 strains of A. thaliana as previously described (19). Solubilized samples were then fractionated by ultracentrifugation in a 0.1 to 1 M sucrose gradient containing 0.06% α-DM and 10 mM HEPES at a pH of 7.5 (22 h at 280,000 g, 4°C). This procedure yields distinct bands from which monomeric (Band 2) and trimeric LHCII (Band 3) complexes can be separated (18). SDS-PAGE analysis of Bands 2 and 3 from dark-adapted wild type and npq2 mutant strains was performed with a TrisTricine buffer system as described in (20). Pigments were extracted from the isolated antenna complexes with 80% acetone, then separated and quantified by HPLC as described in 3 NIR transient absorbance The NIR TA laser system has been previously described (13). Briefly, the repetition rate was 250 kHz and the pump pulses were tuned to ~650 nm (i.e. the chlorophyll b Q y transition). The maximum pump energy and FWHM of the pulse auto-correlation trace were ~48 nJ/pulse and ~40 fs, respectively. We chose 650 nm as our excitation wavelength because the output power of our OPA was higher than that at 680 nm, yielding higher signal:noise ratios. Chlorophyll b to a energy transfer occurs on the 100-200 fs and tens of ps timescale Measurements of fluorescence lifetimes Time-resolved fluorescence was detected using a time-correlated single photon counting (TCSPC) technique. A Ti:sapphire oscillator (1.1 W at 910 nm, 76 MHz Coherent MIRA 900) pumped an optical parametric oscillator (Coherent MIRA OPO). Output pulses with 110 mW average power at 1300 nm were frequency doubled in a 1 mm BBO crystal to generate 650 nm pulses with an average power of ~7 mW. Using a home-made pulse picker driven by a RF frequency generator (CAMAC CD 1000), the repetition rate of the excitation laser beam was reduced to 3.8 MHz. Fluorescence signals were detected by a temperature controlled microchannel plate photomultiplier (Hamamatsu, R2809U-01) and amplified by a preamplifier (Becker & Hickl GmbH, HFAC-26dB). Triggering pulses were obtained by partial reflection of the excitation beam with a silicon photodiode (Newport, 818-BB-20) and a 1 GHz preamplifier (EG&G ORTEC, 9306). A personal computer with a TCSPC module (Becker and Hickl GmbH, SPC-600) was used for data collection and processing. The resulting instrument function had a FWHM of ~40 ps. The timing card for our TCSPC set up possessed 6.1 ps/channel resolution. The maximum amplitude of the signals was greater than 15,000-20,000 counts. PAM chlorophyll a fluorescence of isolated thylakoids A pulsed amplitude modulated (PAM) chlorophyll a fluorimeter, as described in RESULTS Isolation of PSII LHCs containing specific xanthophyll species Our aim was to determine whether CT quenching could be supported in isolated LHCs with specific xanthophyll compositions. The LHCs isolated from dark-adapted wild type and npq2 are expected to be specifically enriched in V and Z, respectively. We performed HPLC to analyze the xanthophyll content of Bands 2 and 3 of npq2 and wild type strains Evidence for transient Z • • • •+ formation solely in minor complexes To explore whether or not Car •+ formation could be supported in isolated LHCII and MLHC complexes, NIR TA kinetic traces were measured by photoexcitation of the monomeric •+ formation. Similarly, the LHCII-V and LHCII-Z TA traces at 1000 nm exhibit bi-exponential decays without any rise component ( formation, even when LHCII binds Z. To explore the slight differences between the NIR TA traces of the LHCII-V and LHCII-Z complexes, we generated a difference kinetic trace (blue curve) and found differences which were of the same amplitude as the noise level before time zero. Furthermore, the convoluted rise time constant (~50 ps) seems to be equal to the fastest decay components of the LHCII-V or -Z kinetics, which likely originates from a slight difference in chlorophyll ESA, or singlet-singlet annihilation, between the two types of complexes. In contrast , the 1000 nm TA kinetic of the MLHC-Z sample The MLHC-Z sample contains several monomeric complexes •+ signal could originate. •+ formation in these complexes. These combined results imply that the transient Car •+ absorption. However, the peak of the Z •+ spectrum was recently shown to be centered at ~980 nm (24). Therefore, these results imply that the observed transient Car •+ signal in the MLHC-Z sample represents Z •+ formation. Nonetheless, since the MLHC-V and MLHC-Z samples contain 11.5 lutein molecules/100 chlorophylls •+ signal is a Lut •+ . The spectrum of the Lut •+ peaks at ~920 and ~950 nm depending upon the solvent used (25-27), and has recently also been reported to peak at 880 nm (24), significantly blue-shifted relative to the peak of the spectrum in •+ signal is present on the blue shoulder of the Z •+ spectrum. Therefore, we conclude that the observed transient Car •+ signal is mainly due to Z •+ formation specifically in minor complexes that bind Z, consistent with CT quenching of chlorophyll excited states therein. Z-dependent quenching of chlorophyll excited states in isolated antennae LHCs To obtain direct information about the quenching of chlorophyll excited states, chlorophyll a fluorescence quantum yields were estimated using time-correlated single photon counting (TCSPC). The fluorescence kinetics of the LHCII trimeric complexes ( Comparable NIR TA and PAM fluorescence analyses of thylakoids In order to explore CT quenching in an intact system, NIR TA kinetics were obtained in thylakoids engaged in qE. 6 To compare the NIR TA kinetics from thylakoids with estimates of qE that also take into consideration non-zero values for k P , we adapted a recently introduced convention (33,34) for expressing the fraction of photons dissipated by qE, or quantum yield of qE (Φ qE ). where the modulated fluorescence parameters are defined in terms of rate constants as Fm'=k f /(k f + k ISC + k IC + k qE + k qT + k qI ), Fs= k f /(k f + k ISC + k IC + k qE + k qT + k qI + k P ), and Fm"= k f /(k f + k ISC + k IC + k qI + k qT ). Rate constants other than k qE , k qT , and k qI (i.e. the rate constants for qE, state transitions, and the slowly recovering component of NPQ referred to as qI, respectively) are defined according to Comparison of CT quenching in isolated LHCs and thylakoids To compare the extent of CT quenching within the isolated MLHC-Z complexes with that in thylakoids engaged in qE, we determined the concentration of excited minor complexes (MIcomp An estimate of 1.13 x 10 10 MIcomp * was obtained for the MLHC-Z sample. Since the minor LHCs isolated from the npq2 mutant bind 1 Z per complex (18), the fraction of MIcomp* undergoing CT quenching was estimated as: Using an OD of ~1.2 x 10 -5 for the Z •+ in the npq2 monomeric sample, 4.7 x 10 7 Z •+ species were estimated within the RV. According to Eqn. 2, ~0.42% of the MIcomp* were approximated to be undergoing CT quenching, an analysis that is consistent with the model in which the equilibrium MLHC(N) ↔ MLHC(Q) is shifted predominantly, although not completely, to the left. We suggest that the slight shift of the equilibrium to the right is mediated simply by binding of Z to the isolated complexes. Similarly, we obtained a value of 1.09 x 10 10 MIcomp * within the RV for the isolated thylakoids. Since each minor complex in isolated thylakoids binds on average 0.33 Z per monomer (37) (i.e. fewer than that of the minor complexes isolated from the npq2 mutant) we estimated, using an OD of 5.85 x 10 -4 for Z •+ formation, 2.33 x 10 9 Z •+ within the RV. In order to take into consideration the dynamics of energy transfer within the thylakoid membrane, and to express the limits of CT quenching in the minor complexes (as estimated according to Eqn. 2), the approximations for CT quenching in thylakoids took into account singlet-singlet annihilation (i.e. by assuming a range of percentages of the complexes were doubly excited and subtracting off this fraction from the total number of MIcomp*) in the minor complexes and excitation energy transfer (EET) from LHCII to a CT quenching site within the minor complexes. DISCUSSION Our results and those of our earlier work demonstrate that Z •+ formation in isolated thylakoid membranes correlates positively with all phenomena (i.e. Z, PsbS, ∆pH etc.) that distinguish qE from the other components (i.e. qI, etc.) of NPQ (13). It might, therefore, seem unlikely that this key signature of qE would be observable in isolated, Z-bound LHCs where no pH gradient or PsbS is present. The absence of a Car •+ signal in isolated, LHCII trimeric complexes that were shown to bind Z ( •+ formation •+ . The Z •+ signal putatively originates within one or more of the minor complexes (CP29, CP26 and CP24), suggesting that the CT mechanism of quenching might occur within these sites in vivo. Without experiments on pure samples of the individual minor complexes, it is not yet possible to say whether the Z •+ signal is associated specifically with only one of the minor complexes or with more than one of them. Analysis of A. thaliana mutants suggest that none of the minor complexes is a unique site of qE in vivo. Antisense plants that specifically lack CP29 or CP26 exhibit little effect on qE (38), whereas depletion of CP24 was recently shown to decrease (but not eliminate) qE due to a perturbation of the PSII antenna structure (39). The minor complexes are believed to occupy a position in the bulk antennae between the LHCII periphery and the reaction center (40,41). Dekker and Boekema (42) have suggested that energy transfer from LHCIIs in the PSII supercomplex may flow through the minor complexes to reach the PSII core, and that excitation energy from CP24 may flow through CP29 to reach the core. The placement of three minor complexes between the moderately/strongly bound LHCII trimers and the PSII core (see We estimate that less than ~1% of the excited minor complexes in the MLHC-Z sample undergo CT quenching. These results are consistent with the need for protein-protein interactions in the native membrane (15), combined with the ∆pH and protonated PsbS (2,13,44), to shift the equilibrium MLHC-Z(N)↔MLHC-Z(Q) significantly to the right, a notion that is supported by the observed ~80-fold increase in the fraction of minor complexes that were estimated to be undergoing CT quenching in thylakoids engaged in steady-state qE The very small fraction of isolated MLHC-Z complexes able to mediate CT quenching is consistent with our fluorescence decay measurements. Tests showed that we could detect a 5 ps component, e.g. the timescale of Z •+ formation that is indicative of CT quenching One of the implications of this work is that NPQ is highly heterogeneous, both in its molecular mechanisms and its sites in the antenna of PSII, issues currently under debate in the literature (2,13,44). Our results suggest that the minor complexes serve as sites of CT quenching during qE. Experiments in which individual chlorophyll molecules are successively removed from the MLHC-Z complex should be valuable in pinning down the molecular mechanism of the transition between non-quenching and quenching states since the energy of the CT state of [Chl-Z] dimers depends sensitively on the separation and orientation of the two molecules (10,11). We found no evidence for CT quenching CONCLUSIONS Our results strongly suggest that minor LHCs (CP24, CP26 and CP29) provide sites for CT quenching, a mechanism for dissipating chlorophyll singlet excited states that was previously shown to correlate specifically with qE (13). The minor complexes are well positioned to dissipate excess absorbed energy in PSII. Experiments using individual complexes may further pinpoint the precise location of CT quenching in PSII, as well as the nature of the presumed conformational transition required to turn 'on' or 'off' the CT mechanism