Structure-Based Modeling of Fluorescence Kinetics of Photosystem II

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Structure–Based Modeling of Fluorescence Kinetics of Photosystem II: Relation between Its Dimeric Form and Photoregulation Ahmed Ibrahim Ali Mohamed, Ryo Nagao, Takumi Noguchi, Hiroshi Fukumura, and Yutaka Shibata J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.5b09103 • Publication Date (Web): 29 Dec 2015 Downloaded from http://pubs.acs.org on January 6, 2016

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The Journal of Physical Chemistry

Structure–Based Modeling of Fluorescence Kinetics of Photosystem II: Relation between Its Dimeric Form and Photoregulation Ahmed Mohamed,a Ryo Nagao,b Takumi Noguchi,b Hiroshi Fukumura,a Yutaka Shibataa a

Department of Chemistry, Graduate School of Science, Tohoku University, Aramaki Aza

Aoba, Aoba-Ku, Sendai 980-8578, Japan. b

Division of Material Science (Physics), Graduate School of Science, Nagoya University, Furo-

Cho, Chikusa-Ku, Nagoya 464-8602, Japan.

1 ACS Paragon Plus Environment


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Abstract Photosystem II- enriched membrane (PSII-em) consists of PSII core complex (PSII-cc) which is surrounded by peripheral antenna complexes. PSII-cc consists of two core antenna (CP43 and CP47) and the reaction center (RC) complex. Time-resolved fluorescence spectra of a PSII-em were measured at 77 K. The data were globally analyzed with a new compartment model, which has a minimum number of compartments and is consistent with the structure of PSII-cc. The reliability of the model was investigated by fitting the data of different experimental conditions. From the analysis, the energy-transfer time constants from the peripheral antenna to CP47 and CP43 were estimated to be 20 ps and 35 ps, respectively. With an exponential time constant of 320 ps, the excitation energy was estimated to accumulate in the reddest chlorophyll (Red Chl), giving a 692-nm fluorescence peak. The excited state on the Red Chl was confirmed to be quenched upon the addition of an oxidant, as reported previously. The calculations based on the Förster theory predicted that the excitation energy on Chl29 is quenched by ChlZD1+, which is a redox active but not involved in the electron-transfer chain, located in the D1 subunit of RC, in the other monomer with an exponential time constant of 75 ps. This quenching pathway is consistent with our structurebased simulation of PSII-cc, which assigned Chl29 as the Red Chl. On the other hand, the alternative interpretation assigning Chl26 as the Red Chl was not excluded. The excited Chl26 was predicted to be quenched by another redox active ChlZD2+ in the D2 subunit of RC in the same monomer unit with an exponential time constant of 88 ps.


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Introduction In oxygenic photosynthesis, light energy is transformed into chemical energy in the thylakoid membrane, which includes both photosystem II (PSII) and photosystem I (PSI). PSII is a large pigment-protein complex that uses the light energy as a driving force of water oxidation.1 The water splitting is catalyzed by the Mn-Ca cluster, where molecular oxygen is produced. The structure of the cyanobacterial PSII-core complex (PSII-cc) has been resolved using X-ray crystallography with a resolution of around 1.9 Å.2 It was found that PSII-cc consists of more than 20 subunits. Proteins PsbA and PsbD represent the D1 and D2 branches, respectively. They form a heterodimer, which binds six chlorophyll a’s (Chla), two pheophytins (Pheo), and two plastoquinones (QA and QB). Two other proteins, PsbB and PsbC, constitute the two inner antenna complexes, called CP43 and CP47, respectively. CP47 and CP43 contain 16 and 13 Chla, respectively.3 Though the structure of PSII-cc from higher plants is not available at present, it is assumed to be similar to that of cyanobacteria. The composition of the proteins involved in the stabilization of the water-splitting cluster is the main difference in PSII between higher plants and cyanobacteria.4 PSII from higher plants is surrounded by the peripheral antenna complexes, whereas that of cyanobacteria does not bind the membranal peripheral antenna. Light ̶ harvesting complex associated with PSII (LHCII) is the main



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component of the peripheral antenna in plants and algae. The structure of the trimeric LHCII was resolved up to a 2.5-Å resolution.5,

6

Each monomer is

composed of two amphipathic helices and three transmembrane helices, which bind eight Chla, six Chlb, and some other cofactors. The distance between Chl pigments was estimated to be around 10 Å, which is short enough for fast energy transfer in the LHCII monomeric units. Time-resolved fluorescence measurements at cryogenic temperature (77 K) can give us detailed information about some of the fast energy-transfer processes that cannot be resolved at room temperature. The red-shifted fluorescence peak around 695 nm, which is afterward called F695, is one example of the emissive species that has been distinguished at 77 K. This peak suggests that there is a pigment pool with lower excited-state energy than the primary donor in which the primary photoinduced charge separation occurs. It has been proposed that the emissive species of F695 is placed in the core antenna, CP47, based on the similarity between the fluorescence peak at 695 nm in the isolated CP47 and that in PSII-cc at a low temperature.7-11 Many research groups have proposed that a monomeric Chl, namely Chl29 (Chl numbering throughout this paper is according to the nomenclature of Loll et al.3) in CP47, is responsible for F695 according to the site-directed mutation study12 and the absorption and linear dichroism (LD) spectra of the CP47


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subunit.10 In agreement with these studies, from the fit of linear optical spectra, Raszewski and Renger suggested that the lowest excited state in CP47 is localized on Chl29.13 Their calculations showed that the second lowest excited state in CP47 at 683 nm comes from more than one exciton transition. Chl11 and Chl24, which are rather distant from each other, contribute to different transitions at this wavelength. Shibata et al.14 also supported these calculations. They measured the time-resolved fluorescence spectra of cyanobacterial PSII-cc at different temperatures, from 5 to 180 K, and compared the results with the simulated spectra. A good agreement between the experimental and simulated spectra was obtained for the absorption spectra. In addition, they confirmed that the site energies obtained by Raszewski and Renger13 can well reproduce the temperature dependence of the time-resolved fluorescence spectra of PSII-cc. The lowest excited states in the CP43 complex were assigned to be on Chl43 and Chl45 in the same domain and Chl37 in the other domain.14 Recently, Jankowiak and co-workers have raised a question about the above assignments of site energies. They proposed another assignment of Chl26, not Chl29, to be the lowest excited state.15 Thus, the assignment of the Red Chl is still under debate. They also suggested that the low-temperature fluorescence spectra of PSII reported by various groups so far are affected by



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the degradation of the sample by repeated freeze–thaw cycles and/or intense laser irradiations. We confirmed that the repeated freeze–thaw cycles actually have only a limited influence on the fluorescence spectra of PSII. We found that the fluorescence kinetics of PSII at 77 K is highly reproducible under our excitation laser-power range. Reppert et al.16 proposed that the lowest energy pigments in CP43 were localized on Chl44 and Chl37 characterized by nonphotochemical hole burning (NPHB) spectroscopy. Very recently Hall et al.17 conducted a circular polarized luminescence (CPL) spectroscopy to identify the lowest-energy pigments in the CP43 complex. They confirmed that there are strong excitonic interactions for at least one of the two well-known emitting states of CP43 named “A” and “B”. They evaluated the CP43 spectra of the previous structure-based models in light of the new CPL data. Their analysis supported the assignments of the lowest excited states by Shibata at al.14 Schweitzer et al. have reported a fluorescence-quenching effect in PSII induced by the addition of an oxidizing agent.18 The quencher has been assigned to the oxidized form of chlorophyll Z (ChlZ) according to the electronparamagnetic resonance (EPR) measurements. They found that the fluorescence quenching of PSII is mainly described by the shortening of the lifetime of the slowest decay-associated spectrum (DAS), which is assigned to


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the fluorescence peak around 695 nm. We consider that revisiting Schweitzer’s work about the fluorescence quenching of PSII may be useful to shed light on the origin of the Red Chl as well as to reveal the probable quenching pathway in PSII based on the structure. Extracting information from time-resolved fluorescence spectra requires constructing a detailed compartment model describing the processes that affect the observed fluorescence. Target analysis is an important method to specify the microscopic rate constants that describe the decay of each compartment as well as the energy transfers between them. Snellenburg et al. succeeded in building a new compartment model with eight compartments to fit the time-resolved spectra of the PSII-enriched membrane (PSII-em) at 77 K.19 Though their model could reproduce the observed fluorescence dynamics, it was not based on the known structure of the PSII complex. Moreover, the large number (eight) of compartments made the analysis too complicated. In the present study, we conducted time-resolved fluorescence studies of PSII-em at 77 K. PSII-em consists of a core complex (PSII-cc) surrounded by several peripheral light-harvesting pigment-protein complexes (LHCII). We tried to construct a new compartment model that has a minimum number of compartments and is consistent with results of the microscopic model based on the structure of PSII-cc.14 The reliability of the compartment model has been



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further investigated by fitting the time-resolved fluorescence data at different experimental conditions. We found that this compartment model was successful in fitting the data in different conditions. The aim of this model is to identify the probable excitation-energy-transfer pathway from LHCII to PSII-cc. We also used it to study the fluorescence-quenching effect induced by the addition of an oxidizing agent. We will calculate the energy-transfer rates between the quencher tentatively assigned to (ChlZ+) and the emitting pigments in PSII-cc based on the Fo rster theory.

Materials and Methods (A)

Sample preparation A PSII-enriched membrane (PSII-em) was prepared from spinach following

the previous method20 and suspended in a buffer containing 40 mM MES-NaOH (pH 6.5), 0.4 M Sucrose, 15 mM NaCl, and 5 mM MgCl2. The sample solution with an optical density (OD) of around 140/mm at 678 nm was stored in a liquid N2 storage tank until use. The Chla/Chlb ratio was estimated by measuring the absorption spectrum of the 80% acetone solution extract by using

a

spectrophotometer

(UV-3100PC,

SHIMADZU,

Kyoto).

The

concentrations of Chla and Chlb are estimated according to the equations in ref. 21. The Chla/Chlb ratio was calculated to be around 2.2. The unknown 8 ACS Paragon Plus Environment

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number of n LHCII trimers bound to one PSII reaction center can be estimated from the following equation: Chla/Chlb = (53+24n) / (10+18n).22 It was found that there were around 1.98 LHCII trimers per reaction center. The stock sample was diluted to an optical density at 678 nm of 0.51/mm with the same buffer. For the time-resolved fluorescence measurements at low temperature, 66% (v/v) glycerol was added to the sample solution to keep it transparent. The final optical density of the sample was around 0.17/mm. In the oxidation experiments, we added 7 mM ferricyanide, (K3[Fe(CN)6]), to the sample along with a short illumination for around 5 minutes by the excitation laser source just before measurement. It has been known that this treatment oxidizes Cyt b559 and induces the accumulation of oxidized ChlZ.23,

24

The

purpose of these experiments is to study the role of an oxidized ChlZ as a fluorescence quencher. The sample solution was contained in the copper sample holder with two acrylic windows and cooled to 77 K in a liquid-nitrogen glass dewar. The sample was cooled by the thermal conduction through the copper and kept always over the liquid N2 surface. The sample temperature was measured by a chromel–gold/iron alloy thermocouple. We confirmed that the repeated freeze-thaw cycles have practically no effect on the fluorescence spectra and their decays of PSII-em. (Fig. S1 in supporting information)



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(B)

Time-resolved fluorescence spectroscopy Time-resolved fluorescence measurements were performed using a

picosecond streak camera system (C10627, Hamamatsu Photonics Inc., Hamamatsu). The sample was excited by the frequency-doubled light at 430 nm and 460 nm from a Ti-sapphire laser (MaiTai, Spectra-Physics, Mountain View) with a repetition rate of 80 MHz and a pulse width of 110 fs. An excitation power of 20 µW (0.25 pJ/pulse) was enough for an appropriate S/N ratio. The excitation spot diameter was typically 27 µm, and the excitation power per unit area was around 4 W/cm2, which is low enough to avoid singlet–singlet annihilation.25 The excitation beam was blocked after each measurement by an automatic shutter. The fluorescence emitted in the same direction of an excitation light was collected by two lenses and focused into the entrance slit of the polychromator with a slit width of 50 µm. The excitation wavelength was cut by a long-pass filter. The FWHMs of the instrumental response function (IRF) were 10.7 ps and 42.6 ps for 1-ns and 5-ns time ranges, respectively. The time-resolved spectra were measured at the magic angle configuration to avoid the rotational relaxation effect. Fluorescence light was diffracted in the polychromator, which was set at a central wavelength of 680 nm, with a spectral resolution of 3.5 nm at the slit width of 50 µm. Streak images were recorded over a spectral range of approximately 255 nm, with


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intervals of 0.4 nm. The wavelength-dependent sensitivity of the system was corrected by measuring the emission spectrum of a standard lamp with the same setup.

(C)

Global and target analyses Time-resolved fluorescence data obtained with the streak camera system

provide a two-dimensional image of fluorescence intensity depending on both wavelength and time. In the global analysis, the wavelength region from 655 nm to 715 in the streak images was divided into 26 wavelength slices and summed along the wavelength axis to obtain the fluorescence time profile at each wavelength region. The data were globally fitted to a sum of exponential decays, and the Gaussian shape is assumed for the IRF for the convolution procedure. The amplitude of each decay component was plotted against the wavelength, called the decay-associated spectrum (DAS). This analysis reflects the decay (positive amplitude) and rise (negative amplitude) components with their corresponding time constants. A DAS with a positive peak at a short wavelength and a negative peak at a long wavelength describes an energytransfer process, whereas a DAS with only positive amplitudes describes the excitation-energy trapping either by charge separation or by a natural fluorescence lifetime of the pigments.



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In the target analysis, a kinetic model (compartment model) that consists of many pigment pools can be used for fitting the time-resolved fluorescence data. The spectrum of each compartment, called the species-associated spectrum (SAS), is estimated by plotting the amplitude of each compartment as a function of wavelength. Energy and electron-transfer rate constants were estimated by solving the differential rate equation for each compartment. (For further information about global and target analyses, see references.26-28)

Results and Discussion (A)

Effect of excitation wavelengths on the fluorescence kinetics of

non-oxidized PSII-em at 77 K We have measured the time-resolved fluorescence spectra at 77 K of PSII-em with closed RCs containing the reduced form of quinone A upon excitation at 430 nm and 460 nm.14, 23, 29, 30 The redox status of the electrontransfer chain under a dark condition was typically neutral (P680 QA), where P680 and QA are the special pair and the secondary electron acceptor, respectively. During the laser excitation, (P680+ QA−) with a long lifetime (2-5 ms) will be accumulated in average. In this state, the primary electron donor, the accessory (Acc) Chl, and the primary acceptor, pheophytin, are still active. A closed RC at room temperature shows much slower fluorescence decay than an 12 ACS Paragon Plus Environment

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open RCs.23, 31 On the other hand, at 77 K, The charge recombination hardly repopulates the excited state of Chl to generate the delayed fluorescence. At room temperature, the excited state of Chl will be repopulated by the recombination of the charge-separated state (P680+ QA−) and this repopulation will induce the delayed fluorescence. The excitation at 430 nm mainly excites Chla pigments, whereas the excitation at 460 nm leads to the excitation of Chlb pigments, which are located only in the peripheral antenna. Figure 1 shows the fluorescence-decay curves of both 430-nm and 460-nm excitations. The 430nm excitation results in a slightly faster decay than that observed with the 460nm excitation. This may be because the 460-nm excitation induces a more initial excitation population in the peripheral antenna, from which the migration of the excitation to the trap takes longer time.22 To obtain more information about the energy transfers, we fitted the fluorescence-decay curves of PSII-em excited at 430 nm and 460 nm globally to the sum of five exponential decays. The fitting quality by the five exponential decays is good, as shown in Fig. 2. (The amplitude of each component plotted against the wavelength is called the decay-associated spectrum (DAS).). The residual plots of this fitting are shown in Fig. S2 in the supporting information. The estimated DAS from the global analysis is shown in Fig. 3. The 8.2-ps DAS in Fig. 3A shows a major negative peak at 678 nm and a small positive peak at 668



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nm. An overall feature of this DAS describes an internal conversion from higher-lying electronic states of Chls to lower-lying ones. The small positive peak at 668 nm may describe a small contribution from the energy transfer from the blue Chla to the redder Chla within LHCII. The 2.3-ps DAS in Fig. 3B has a larger positive amplitude than the 8.2-ps DAS in Fig. 3A. This 2.3-ps component might reflect the energy transfer from Chlb and blue Chla to Chla in LHCII. These results are consistent with the previous transient absorption (TA)32-35 and the time-resolved fluorescence studies36 of isolated LHCII, which revealed that the energy transfer from Chlb to Chla takes place with two major time constants of 300–600 fs and 3–7 ps at 77 K. An energy transfer between Chla molecules in LHCII was reported to take place on a 1-ps time scale or longer.37-39 The second fastest DAS components, with time constants of 17 ps (430nm excitation) and 22 ps (460-nm excitation), represent mixtures of the energy transfer to the primary donor (trapping) and the energy transfer from the pigments emitting at 675 nm in LHCII to those emitting at 683 nm in PSII-cc. The larger negative peak for the 460-nm excitation can be explained by a larger energy-transfer contribution. Moreover, it should be noted that the migration times from LHCII to PSII-cc in the present study were estimated to be 17 ps (430-nm excitation) and 22 ps (460-nm excitation), which are faster than those


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calculated by a simple course-grained model.22 This model calculated the migration time at room temperature to be 35 and 39 ps upon 430-nm and 460nm excitations, respectively. The difference in migration time between the present data and those in ref. 22 is due to the different temperatures. At room temperature, the uphill energy transfer competes with the downhill energy transfer and increases the effective migration time. It is very important to compare the time constants shown above with those described in a previous study on the singlet–singlet annihilation of isolated LHCII.40 The study predicted that the average migration time in PSII would be close to N×~32 ps, N being the number of LHCII trimers per reaction center and 32 ps being the equilibration time of an excitation over the LHCII trimer in a random aggregate of trimers. The migration time from the outer antenna to the core complex in the present study is much faster than the reported equilibration time (32 ps) of an excitation over LHCII trimers. We can conclude that the arrangement and organization of LHCII in PSII-em have been optimized to yield a specific pathway in which an efficient excitation-energy transfer is realized. It is very interesting to know the favorable excitationenergy-transfer pathway from LHCII to the core complex. Therefore, we tried to construct a compartment target model to obtain more information, as will be discussed in section (C).



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The decay components with time constants of 84 ps (430-nm excitation) and 79 ps (460-nm excitation) have small negative peaks at 692 nm, indicating that a small amount of excitation energy is transferred to the Red Chl, which is considered to be the emitting pigment in CP47.41-43 Our structure-based simulation in the previous paper14 was in good agreement with the timeresolved fluorescence data of PSII-cc purified from a thermophilic cyanobacterium, Thermosynechococcus vulcanus. It was suggested that Chl29 in CP47 is responsible for the fluorescence peak at 692 nm. The 84-ps and 79ps DAS components are similar to the value of 87 ps estimated in our previous study on PSII-cc. In PSII-em, the positive peak position of the third DAS components is about 4 nm blue-shifted as compared with that in PSII-cc, probably because it contains many LHCII binding several Chlb and blue Chla. In an early study,29 a DAS with a time constant of 132 ps, similar to the present 84-ps and 79-ps components, did not show an energy-transfer pattern to the emitting state at 692 nm. On the other hand, 84-ps (430-nm excitation) and 79ps (460-nm excitation) DAS components have a mixture of energy transfers to the 692-nm emitting pigment and the trapping by the charge separation process. There have been several reports about sub-picosecond transientabsorption measurements of isolated PSII-RC.44, 45 The time constants of the 84ps and 79-ps DAS components are similar to those of the second decay


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component of the excited state in PSII-RC (30 ps to 100 ps). They have been attributed to charge separation, after a direct excitation, of a relatively longlived state in the PSII-RC which is degenerate with the primary donor (P680) and subsequent slow energy-transfer from this state to the primary donor. The fourth DAS components, with time constants of 250 and 260 ps in Fig. 3A and B, respectively, have an emission maximum at 683 nm. They describe the energy trapping by the charge-separation process in the primary donor. The origin of this decay phase is assumed to be Chl45 or Chl43 in CP43.13 The slowest decay components with the time constants of 4.7 and 5.1 ns show the natural lifetime of Chla. We estimated the contribution from each DAS component to the steady-state emission spectrum as shown in Fig. S3 in the supporting information. In Fig. S3, we show DASs multiplied by their lifetimes.8 Fig. S3 confirms that the two major components at 683 nm and 692 nm in the steady-state fluorescence spectrum of PSII-em at 77 K are mainly contributed by the 250-ps decay component and the 4.7-ns one, respectively. The time constants of the slowest DASs are somewhat faster than the slowest DASs of PSII-cc.23

(B)

Effect of oxidant on the fluorescence kinetics of PSII-em at 77 K



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In this section, we discuss the results obtained for the PSII-em sample with an oxidative agent, K3[Fe(CN)6], along with a short illumination at 77 K to oxidize Cyt b559 and produce a high yield of oxidized ChlZ.23,

24

We have

measured the time-resolved fluorescence spectra of the oxidized PSII-em at different excitation wavelengths, 430 and 460 nm, at 77 K. Figure 4A and B compares the fluorescence-decay profiles of the oxidized (black) and nonoxidized (gray) PSII-em for the 430-nm and 460-nm excitations, respectively. Figure 4 shows more drastic acceleration of the fluorescence decay for the 430nm excitation than for the 460-nm excitation. Figure 4 insets show the timeintegrated fluorescence spectra. The fluorescence intensity of the oxidized sample excited at 430 nm showed a more significant reduction than that for the 460-nm excitation. Thus, we could confirm that the fluorescence emission of PSII-em is quenched when the sample is treated with [Fe(CN)3]3− as the oxidant, probably due to the presence of a specific quencher(s) in the complex. Schweitzer et al. suggested that the quencher was a ChlZ cation located in PSIIcc.18 Our data in Fig. 4 showed that the excitation energy can be quenched effectively when PSII-cc is mainly excited at 430 nm, whereas the quenching process is less effective when LHCII is mainly excited at 460 nm. This clearly confirmed that the quencher is located within PSII-cc. In the next section (C),


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we will explain the less-effective quenching of the 460-nm excitation by using the compartment model analysis. The time-resolved fluorescence data from the oxidized sample were globally fitted by five exponential decay components. The estimated DASs are shown in Fig. 5. By comparing the DASs in Fig. 3A and Fig. 5, we found that the time constants of all the components were a little shortened upon addition of the oxidant. The 70-ps DAS in Fig. 5 has a larger negative peak at 692 nm as compared with the counterpart DAS in the non-oxidizing condition with a time constant of around 80 ps in Fig. 3. Interestingly, this seems to indicate that the excitation-energy trapping by charge separation is limited in the oxidizing condition as compared with the case of the non-oxidizing condition. However, we will not discuss this effect more in detail because the reduced positive peak of the 70-ps DAS might be due to the interference by the DAS with a quite similar time constant of 130 ps. The decay of the slowest component, which is the main target of this work, was clearly accelerated from around 4.7 ns in the non-oxidized sample to around 0.8 ns in the oxidized sample. Thus, we can infer that the quenching of the steady-state fluorescence is associated with the nanosecond DAS component centered at 692 nm, as shown in the Fig. 4A inset. According to previous studies,18, 46, 47 here we tentatively assign ChlZ+ as the potential quencher in PSII-cc at 77 K. In general, at a low temperature, the



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primary electron-transfer pathway is nearly blocked, and the secondary electron-donation pathway, consisting of Cyt b559, Chl, and Car, is activated. Tracewell et al. measured the near-IR absorption spectra of a pre-oxidized Mndepleted spinach PSII membrane sample after illumination at temperatures from 6 to 120 K. They performed Gaussian decomposition of the near-IR spectra to estimate the number of contributing components and their relative intensities. At low temperatures, they found two peaks at 817 and 850 nm representing ChlZ+ and one more peak at 895 nm representing Car+. When the sample was warmed to 77 K, the intensities of the ChlZ+ peaks were almost unchanged, whereas the Car+ peak was significantly reduced. These results indicated that Car+ QA− is a less stable charge-separated state than ChlZ+ QA−.24, 47

Schweitzer and Brudvig carried out EPR spectroscopy and fluorescence

measurements on the PSII membrane sample.48 They prepared many PSII samples with different, well-defined stable redox states to study the relationship between the redox states of the cofactor pigments and the fluorescence emission. They illuminated the untreated PSII sample at 77 K for 90 s to induce the ChlZ oxidized form. They concluded that the fluorescence intensity was quenched due to the formation of ChlZ+. The actual fluorescence quenching by ChlZ+ is considered to be induced by excitation-energy transfers from Chl molecules to ChlZ+. According to the


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Förster theory, a reasonable overlap between the fluorescence emission spectrum of a Chl pigment (donor) and the absorption spectrum of ChlZ+ (acceptor) is required for an efficient energy transfer. The previous study49 about the absorption spectrum of the Chl cation in solution indicated that there is a reasonable overlap between the emission spectrum of a Chl molecule and the tail in the shorter-wavelength side of the absorption band of the lowest excited state of ChlZ+ at around 800 nm. Excitation-energy flows from Chl pigments to the quencher then will be quickly relaxed to its lowest electronic-excited state at around 810 nm by the nonradiative internal conversion process. The reverse energy transfer is practically inhibited because it is steeply uphill. It was also suggested that ChlZ+ can play a role in nonphotochemical quenching in PSII,50,

51

as the xanthophyll cycle does in the PSII peripheral

antenna.52 This implies that the ChlZ+ quenching mechanism would be considered one of the photoprotection mechanisms in plants under steadystate, high-light conditions. However, the fluorescence-quenching pathway by ChlZ+ is still unknown. Generally, ChlZ+ should accept excitation energy from another Chl pigment in PSII-cc. This Chl pigment might be located either in the reaction center or in the core antenna complexes (CP43 and CP47).



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(C)

Construction of a compartment model consistent with the PSII

structure In this section, a new compartment target model with the minimum number of compartments was constructed. This model is based on the results of structure-based simulation of PSII-cc.14 Briefly, in ref. 14, the authors considered the orientation, position, and site energy of each Chl molecule based on the PSII structure2 to calculate the fluorescence kinetics at different temperatures and compared them with the experimental ones. The main finding of the research was that the lowest excited-state pigment in the PSII complex was Chl29 (Red Chl) located in CP47. Using this model, we fitted the time-resolved fluorescence data of PSII-em under different experimental conditions. In the first part of this section, we used this model to fit the timeresolved fluorescence data of the 430-nm and 460-nm excitations without an oxidant. The newly constructed model for PSII-em consists of four emitting compartments and one non-emitting compartment, as shown in Fig. 6. The compartments are as follows: LHCII (black), CP43+RC (blue), CP47 bulk (CP47b) (green), the Red Chl of CP47 (red), and the first radical pair (orange). Here, CP47b is assumed to contain Chls in CP47 except for the Red Chl. As shown in Fig. 6, we tentatively combine RC with CP43 in the same compartment. This is


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consistent with our previous structure-based simulation, which predicted a much more efficient energy transfer between RC and CP43 than that between RC and CP47. At 77 K, it is impossible to keep RC open. We expressed the electrontransfer processes by only one compartment (RP1), which represents the first charge-separated state ChlD+ Pheo−. We can omit the second radical pair compartment (RP2), which represents the second charge-separated state, P680+ Pheo−, because P680+ QA− is already accumulated in our experimental conditions. All the free kinetic parameters of 430-nm and 460-nm excitations were globally fitted except for the initial population parameters. In this model, we assumed an irreversible excitation-energy flow from LHCII to PSII-cc. This assumption was given according to the compartment analysis results. We found that the reverse rate constants from PSII-cc to LHCII were very small when we performed the analysis without the constraint. For the actual fitting process, we neglected these reverse rate constants and set their values equal to 0. Snellenburg et al.19 assumed in their target analysis that the spectral areas of all SASs are equal to each other based on an assumption that all the Chl pigments possess the same oscillator strength. This assumption is, however, not always valid because of the situation in a photosynthetic proteins, as



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described below. In this type of analysis, each compartment often contains many pigments connected through strong excitonic couplings. This leads to the formation of delocalized excited states that absorb light at different energies from the localized excited states and also leads to a redistribution of the oscillator strengths among different delocalized excited states.13, 53 Thus, the area of the SAS may depend on the different delocalized excited states in each compartment. A good fitting quality was obtained, as shown in Fig. 7A and B, for the 430-nm and 460-nm excitations, respectively. The residual plots of this fitting are shown in Fig. S4 in the supporting information. The obtained population kinetic profiles of the 430-nm and the 460-nm excitations and the corresponding SAS are shown in Fig. 7C, D, and E, respectively. The lesseffective quenching upon the 460-nm excitation can be explained by the initial population of the CP47 compartment, as shown in Fig. 6. It is around 0.1 in the case of 460-nm excitation, whereas it is around 0.2 in the case of 430-nm excitation. The difference in the initial population of CP47 leads to the change of the apparent excitation-energy quenching rate. The SAS of the LHCII compartment shows a blue-shifted emission peak at 675 nm because it contains Chlb and blue Chla pigments, which emit at shorter wavelengths. The red-pigment compartment of CP47 has an SAS with a peak at


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692 nm, which is consistent with an emission peak at 692 nm in the timeintegrated fluorescence spectra of PSII-em at 77 K. The CP47b compartment emits at 682 nm. This emission probably comes from the second lowest optical band in CP47, according to anisotropy measurements10 and the theoretical calculations.13 It was found that the two low-energy pigments, Chl11 and 24 (Chl numbering is again according to Loll et al.), in CP47 are responsible for different exciton transitions at 682 nm. The SAS of the (CP43+RC) compartment has an emission peak at 680 nm. This peak is 2–3 nm blue-shifted from the known emission wavelength of the CP43 complex at 682 nm. This shift is due to the combination of CP43 with RC. (Three pigments are observed to contribute to the lowest exciton state at 682 nm in CP43. Two of them, Chl43 and 45, are in the same domain, whereas the third one, Chl37, is in another dimer domain with Chl34.53-56) The estimated time constants of the energy transfers, the inverse of the rate constants, in PSII-em are given in Fig. 6. The standard deviations of these rate constants are shown in table S1 in the supporting information. The energytransfer time constants from LHCII to CP47b and to (CP43+RC) are around 20 ps and 35 ps, respectively. The time constant of the energy transfer from (CP43+RC) to CP47b is around 240 ps, whereas that for the reverse direction is around 360 ps. Then, the excitation energy transfers from CP47b to the Red Chl



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with the time constant of around 320 ps. From these results, we can suggest that most of the excitation energy in PSII-em flows to CP47 and then accumulates in the Red Chl. The second part of this section includes a new target model for the fitting of the time-resolved fluorescence data of the oxidized sample excited at 430 nm and 460 nm. Basically, the model is similar to that in Fig. 6 with the exception of four quenching paths being introduced to the emitting compartments, as shown by dotted red arrows in Fig. 6. We assumed that the electron and energy-transfer time constants are not altered by the oxidation, whereas SASs will be modified. We also assumed that the SAS of the Red Chl compartment should be the same for the 430-nm and 460-nm excitations in the new model because it contains only one pigment. Thus, the SAS of the Red Chl is globally fitted, whereas those of the other compartments are not globally fitted. In addition, the kinetic parameters of the quenching paths are globally fitted and set free. The fitting quality of this target model is good, as shown in Fig. 8A and B for the 430-nm and 460-nm excitations, respectively. The residual plots of this fitting are shown in Fig. S5 in the supporting information. The resulting population kinetic profiles of 430-nm and 460-nm excitations are depicted in Fig. 8C and D, respectively. The corresponding SAS of 430-nm and 460-nm


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excitations are depicted in Fig. 8E and F, respectively. In this fitting, the quenching rates were set free to vary, and their initial values were set as 0. We found that after the fitting, only the quenching paths of the Red Chl and CP47b have significant values, whereas the two other quenching paths do not have any appreciable values. The time constants of the quenching path of CP47 and the Red Chl are around 1.4 ns and 490 ps, respectively. These results suggest that upon addition of the oxidizing agent, the Red Chl is mostly quenched, while CP47b is partially quenched.

(D)

Validity estimation of models In this section, we will discuss in detail the molecular origins of the Red

Chl and the quencher to understand our experimental results described in the above sections. According to the experimental results and target analysis in the present study, we confirmed that the Red Chl is quenched upon addition of the oxidizing agent. The quenching of the fluorescence peak at 692 nm, as shown in the Fig. 4A inset, corresponds to the quenching of the Red Chl compartment in Fig. 6. The molecular origin of the 692-nm fluorescence band in PSII-cc is still under debate. According to the studies of the low-energy states of CP47,10 it was concluded that the low-energy state emitting around 692 nm is due to a monomeric Chl whose Qy transition dipole moment has an angle larger than



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35° with the membrane plane. This finding is in agreement with the fit of linear optical spectra made by Raszewski and Renger.13 They suggested that the lowest excited state of CP47 is localized on a monomeric Chl, namely Chl29. We, in the previous paper, showed that a model assuming Chl29 as the Red Chl can give a very nice reproduction of the experimental fluorescence dynamics of PSII-cc, including its temperature dependence.14 Thus, here we consider Chl29 as the primary candidate for the Red Chl. On the other hand, Jankowiak and co-workers have raised a question about the above assignment. They proposed another assignment of Chl26 to be the lowest excited state.15 As they pointed out, we could confirm the alteration of the fluorescence spectrum by the repeated freeze–thaw cycles, although the effect is limited. Here, we will test the above two assignments and examine the validity of each interpretation by comparing the present experimental results. Actually, in PSII-cc, there are two ChlZ pigments, ChlZD1 and ChlZD2, located at the peripheral edge of RC, close to CP43 and CP47, respectively. It is still under debate which ChlZ of these two is photo-oxidized; ChlZD1 was proposed to be photo-oxidized based on a study using site-directed mutants at D1-H118 and D2-H117 in the cyanobacterium Synechocystis PCC 6803,57 whereas ChlZD2 was assigned to be photo-oxidized in PSII from Chlamydomonas reinhardtii.58 There are another reports showing that both ChlZD1 and ChlZD2 are photo-oxidized


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based on analysis of the near-infrared absorption bands of ChlZ.47, 59 We will use the above two assignments (ChlZD2+ and ChlZD1+) in the following discussion because we do not have enough information to evaluate which is more likely at present. We calculated the excitation-energy transfer time constant between the hypothetical donor pigments (Chl29 or Chl26) and the acceptors (ChlZD2+ or ChlZD1+) in the dimer structure of PSII-cc based on the Fo rster theory to tentatively identify the proper energy-quenching pathway upon addition of the oxidizing agent. To do this, we should calculate the overlap integral of the normalized absorption of the acceptor and the fluorescence spectra of the donor,60 as well as the excitonic couplings. The transfer rate is expressed as in the following equation61:

1

= = 1.18 and = d,

(1)

where has a unit of ps ! , is the excitonic coupling in cm ! , and is the spectral overlap integral between the fluorescence spectrum of the donor and the absorption spectrum of the acceptor. and are normalized so that the integrated area gives unity.



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Excitonic couplings between the pigments were calculated14 with the Poisson-TrEsp method established earlier62-64 by using PSII crystal structure.2 The second parameter in Eq. 1, the spectral overlap integral, was calculated by using spectral profiles modeled by Gaussian functions, as shown in Eq. 2:61

or =

!

√& '

exp *− ,

- -. / '/

01,

(2)

where σ and ν4 are the width and peak position of the spectrum in a unit of cm−1, respectively. The peak position and width of the fluorescence spectrum of Chl donor molecules and the absorption spectrum of the ChlZ acceptor are determined by the Gaussian fitting of SAS of Red Chl shown in Fig. 8 and the reported absorption spectrum of the Chl cation radical in solution, respectively. The absorption spectrum of Chl cation radical was obtained by pulse radiolysis of 2% Triton X100-water N2O saturated solutions of 7.5 × 10-5 M Chla containing 5 × 10-2 M SCN− at room temperature.49 The parameters for the simulated absorption spectrum of ChlZ+ were estimated to be ν4 = 12500 cm−1 (800 nm) and σ = 1000 cm−1, whereas the simulated fluorescence spectra of a neutral Chl ν4 and σ were estimated to be 14390 cm−1 (694 nm) and 272 cm−1, respectively. The simulated fluorescence and absorption spectra according to Eq. 2 are shown in Fig. S6 in the supporting information. The integration of the 30 ACS Paragon Plus Environment

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product of these spectra results in the value of 6.74×10−3 cm-1 for the spectral overlap between the donor and acceptor. Table 1 lists the calculated excitonic coupling values and the energytransfer time constants of eight donor–acceptor combinations in the dimer structure of PSII-cc. We found that the excitonic coupling between Chl29 and ChlZD1+ in the other monomer unit was the highest one, ca. 1.3 cm−1, while the couplings of Chl29 with the other hypothetical quenchers were negligible. On the other hand, the coupling between the alternative Red Chl candidates, Chl26, and ChlZD2+ in the same monomer was ca. 1.2 cm−1, which is dominant as compared with the other coupling values. The calculations predicted that the excitation energy in Chl29 is efficiently quenched within 75 ps by ChlZD1+ in the other monomer unit, whereas the quenching of Chl26 by ChlZD2+ in the same monomer has a time constant of 88 ps. According to our calculations, there are many other pigments with faster energy-transfer time constants than those listed in Table 1 (not shown). However, according to the structure-based simulation14 and the hole-burning study,15 they have been assigned to have significantly higher site energies as compared with Chl29 and Chl26, making the assignment of the Red Chl to these pigments unlikely. Based on the above calculations, we proposed a model shown in Fig. 9, in which the Red Chl is assigned to Chl29 and the excitation energy on it is



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efficiently quenched by ChlZD1+ in the other monomer unit upon addition of the oxidizing agent. This model is consistent with the assignment that Chl29 is the lowest excited state according to the structure-based simulation.14 This qualitative model provides a proper excitation-energy-quenching pathway, as shown. Thus, we can suggest that Chl29 has a potential function in the photoprotective role in PSII-cc. It is also interesting to point out the importance of the dimeric structure of PSII-cc. The quenching pathway from Chl29 to ChlZD2 proposed here suggests that the dimer structure plays an important role in the photoregulation processes in the PSII complex. A direct proof of this model is still missing and will be a novel future goal. On the other hand, the present estimations of the energy-transfer rates do not exclude assignment of the Red Chl to Chl26. The distance between these two Chls seemed to be larger than that between ChlZD1 and Chl29 in the two-dimension image in Fig. 9. The two distances are not so different actually in the three-dimension space. The quenching pathway from Chl26 to ChlZD2 in the same monomer unit is a possible alternative interpretation. One method to verify the above two possibilities is to measure the effect of an oxidizing agent on the fluorescence kinetics of an isolated PSII monomer. If only the quenching pathway from Chl29 to ChlZD1 actually functions, the addition of an oxidizing agent will have little effect on the fluorescence dynamics of the monomeric PSII.


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Conclusion We measured the time-resolved fluorescence spectra of PSII-em at 77 K excited at 430 nm and 460 nm. The fluorescence-decay profiles were fitted by the global and target analyses. We succeeded in globally fitting the time-resolved fluorescence data of non-oxidized and oxidized samples excited at 430 nm and 460 nm by using a new compartment model. The model has a minimum number of compartments and is consistent with the microscopic model based on the structure of PSII-cc. According to the results of the target analysis, we found that the excitation-energy transfer from LHCII to the Red Chl through CP47 is the favorable excitation-energy pathway. We also confirmed that the Red Chl is drastically quenched upon addition of the oxidizing agent. Based on the energy-transfer rate calculations, we suggested a qualitative model that describes possible excitation-energy-quenching pathways in a PSII-cc dimer. One of the possible quenching pathways consists of Chl29 as the energy donor and ChlZD1+ in the other monomer as the energy quencher. At the present stage, we cannot deny another possible pathway that consists of Chl26 as the energy donor and ChlZD2+ in the same monomer unit as the energy quencher. If we can prove experimentally that the dimeric structure of PSII-cc has an



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essential role in the quenching process, then we would be able to give a clear answer for the reason the PSII naturally forms a dimer.

References (1) Van grondelle, R.; Dekker, J. P.; Gillbro, T.; Sundström, V. Energy-Transfer and Trapping in Photosynthesis. Biochim. Biophys. Acta. 1994, 1187, 1-65. (2) Umena, Y.; Kawakami, K.; Shen, J. R.; Kamiya, N. Crystal Structure of Oxygen-Evolving Photosystem II at a Resolution of 1.9 Angstrom. Nature 2011, 473, 55-65. (3) Loll, B.; Kern, J.; Saenger, W.; Zouni, A.; Biesiadka, J. Towards Complete Cofactor Arrangement in the 3.0 Angstrom Resolution Structure of Photosystem II. Nature 2005, 438, 1040-1044. (4) Nield, J.; Kruse, O.; Ruprecht, J.; da Fonseca, P.; Buchel, C.; Barber, J. ThreeDimensional Structure of Chlamydomonas Reinhardtii and Synechococcus Elongatus Photosystem II Complexes Allows for Comparison of their OxygenEvolving Complex Organization. J Biol. Chem. 2000, 275, 27940-27946. (5) Liu, Z. F.; Yan, H. C.; Wang, K. B.; Kuang, T. Y.; Zhang, J. P.; Gui, L. L.; An, X. M.; Chang, W. R. Crystal Structure of Spinach Major Light-Harvesting Complex at 2.72 Angstrom Resolution. Nature 2004, 428, 287-292. (6) Standfuss, R.; van Scheltinga, A. C. T.; Lamborghini, M.; Kuhlbrandt, W. Mechanisms of Photoprotection and Nonphotochemical Quenching in Pea Light-Harvesting Complex at 2.5 Angstrom Resolution. Embo J. 2005, 24, 919928.


Page 34 of 56

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(7) Van dorssen, R. J.; Plijter, J. J.; Dekker, J. P.; Denouden, A.; Amesz, J.; Vangorkom, H. J. Spectroscopic Properties of Chloroplast Grana Membranes and of the Core of Photosystem-II. Biochim. Biophys. Acta. 1987, 890, 134-143. (8) Andrizhiyevskaya, E. G.; Chojnicka, A.; Bautista, J. A.; Diner, B. A.; van Grondelle, R.; Dekker, J. P. Origin of the F685 and F695 Fluorescence in Photosystem II. Photosynth. Res. 2005, 84, 173-180. (9) Reppert, M.; Acharya, K.; Neupane, B.; Jankowiak, R. Lowest Electronic States of the CP47 Antenna Protein Complex of Photosystem II: Simulation of Optical Spectra and Revised Structural Assignmentst. J. Phys. Chem. B 2010, 114, 11884-11898. (10) de Weerd, F. L.; Palacios, M. A.; Andrizhiyevskaya, E. G.; Dekker, J. P.; van Grondelle, R. Identifying the Lowest Electronic States of the Chlorophylls in the CP47 core Antenna Protein of Photosystem II. Biochemistry 2002, 41, 1522415233. (11) Komura, M.; Shibata, Y.; Itoh, S. A New Fluorescence Band F689 in Photosystem II Revealed by Picosecond Analysis at 4-77 K: Function of Two Terminal Energy Sinks F689 and F695 in PSII. Biochim. Biophys. Acta 2006, 1757, 1657-1668. (12) Shen, G. Z.; Vermaas, W. F. J. Chlorophyll in a Synechocystis Sp Pcc-6803 Mutant without Photosystem-I and Photosystem-II Core Complexes - Evidence for Peripheral Antenna Chlorophylls in Cyanobacteria. J. Biol. Chem. 1994, 269, 13904-13910. (13)

Raszewski, G.; Renger, T. Light Harvesting in Photosystem II Core

Complexes Is Limited by the Transfer to the Trap: Can the Core Complex Turn into a Photoprotective Mode?. J. Am. Chem. Soc. 2008, 130, 4431-4446. 35 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(14) Shibata, Y.; Nishi, S.; Kawakami, K.; Shen, J. R.; Renger, T. Photosystem II Does Not Possess a Simple Excitation Energy Funnel: Time-Resolved Fluorescence Spectroscopy Meets Theory. J. Am. Chem. Soc. 2013, 135, 69036914. (15) Chen, J. H.; Kell, A.; Acharya, K.; Kupitz, C.; Fromme, P.; Jankowiak, R. Critical Assessment of The Emission Spectra of Various Photosystem II Core Complexes. Photosynth. Res. 2015, 124, 253-265. (16) Reppert, M.; Zazubovich, V.; Dang, N. C.; Seibert, M.; Jankowiak, R. LowEnergy Chlorophyll States in the CP43 Antenna Protein Complex: Simulation of Various Optical Spectra. II. J. Phys. Chem. B 2008, 112, 9934-9947. (17) Hall, J.; Renger, T.; Picorel, R.; Krausz, E. Circularly Polarized Luminescence Spectroscopy Reveals Low-Energy Excited States and Dynamic localization of Vibronic Transitions in CP43. Biochim. Biophys. Acta (in press) (18) Schweitzer, R. H.; Melkozernov, A. N.; Blankenship, R. E.; Brudvig, G. W. Time-Resolved Fluorescence Measurements of Photosystem-II: The Effect of Quenching by Oxidized Chlorophyll Z. J. Phys. Chem. B 1998, 102, 8320-8326. (19) Snellenburg, J. J.; Dekker, J. P.; van Grondelle, R.; van Stokkum, I. H. M. Functional Compartmental Modeling of the Photosystems in the Thylakoid Membrane at 77 K. J. Phys. Chem. B 2013, 117, 11363-11371. (20) Ono, T.; Inoue, Y. Effects of Removal and Reconstitution of the Extrinsic 33-Kda, 24-Kda and 16-Kda Proteins on Flash Oxygen Yield in Photosystem-II Particles. Biochim. Biophys. Acta. 1986, 850, 380-389. (21)

Porra, R. J.; Thompson, W. A.; Kriedemann, P. E. Determination of

Accurate Extinction Coefficients and Simultaneous-Equations for Assaying


Page 36 of 56

Page 37 of 56

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Chlorophyll-a and Chlorophyll-B Extracted with 4 Different Solvents Verification of the Concentration of Chlorophyll Standards by AtomicAbsorption Spectroscopy. Biochim. Biophys. Acta. 1989, 975, 384-394. (22) Broess, K.; Trinkunas, G.; van Hoek, A.; Croce, R.; van Amerongen, H. Determination of the Excitation Migration Time in Photosystem II Consequences for the Membrane Organization and Charge Separation Parameters. Biochim. Biophys. Acta. 2008, 1777, 404-409. (23) van der Weij-de Wit, C. D.; Dekker, J. P.; van Grondelle, R.; van Stokkum, I. H. M. Charge Separation Is Virtually Irreversible in Photosystem II Core Complexes with Oxidized Primary Quinone Acceptor. J. Phys. Chem. A 2011, 115, 3947-3956. (24)

Depaula, J. C.; Innes, J. B.; Brudvig, G. W. Electron-Transfer in

Photosystem-II at Cryogenic Temperatures. Biochemistry 1985, 24, 8114-8120. (25) Deinum, G.; Aartsma, T. J.; Van grondelle, R.; Amesz, J. Singlet-Singlet Excitation Annihilation Measurements on the Antenna of RhodospirillumRubrum between 300 and 4-K. Biochim. Biophys. Acta. 1989, 976, 63-69. (26) van Stokkum, I. H. M.; Larsen, D. S.; van Grondelle, R. Global and Target Analysis of Time-Resolved Spectra. Biochim. Biophys. Acta. 2004, 1657, 82-104. (27)

Holzwarth, A.; Amesz, J.; Hoff, A. Data Analysis of Time-Resolved

Measurements. In Biophysical Techniques in Photosynthesis; Kluwer Academic Press: Dordrecht, 1996. (28) van Stokkum, I. H. M.; van Oort, B.; van Mourik, F.; Gobets, B.; van Amerongen, H. (Sub)-Picosecond Spectral Evolution of Fluorescence Studied



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

with a Synchroscan Streak-Camera System and Target Analysis. In Biophysical Techniques in Photosynthesis; Springer: Dordrecht, 2008. (29) van der Weij-de Wit, C. D.; Ihalainen, J. A.; van Grondelle, R.; Dekker, J. P. Excitation Energy Transfer in Native and Unstacked Thylakoid Membranes Studied by Low Temperature and Ultrafast Fluorescence Spectroscopy. Photosynth. Res. 2007, 93, 173-182. (30)

Hughes, J. L.; Smith, P.; Pace, R.; Krausz, E. Charge Separation in

Photosystem II Core Complex Induced by 690-730 nm Excitation at 1.7 K. Biochim. Biophys. Acta 2006, 1757, 841-851. (31) Vassiliev, S.; I Lee, C.; Brudvig, G. W.; Bruce, D. Structure-Based Kinetic Modeling of Excited-State Transfer and Trapping in Histidine-Tagged Photosystem II Core Complexes from Synechocystis. Biochemistry 2002, 41, 12236-12243. (32) Connelly, J. P.; Müller, M. G.; Hucke, M.; Gatzen, G.; Mullineaux, C. W.; Ruban, A. V.; Horton, P.; Holzwarth, A. R. Ultrafast Spectroscopy of Trimeric Light-Harvesting Complex II from Higher Plants. J. Phys. Chem. B 1997, 101, 1902-1909. (33) Trinkunas, G.; Connelly, J. P.; Müller, M. G.; Valkunas, L.; Holzwarth, A. R. Model for the Excitation Dynamics in the Light-Harvesting Complex II from Higher Plants. J. Phys. Chem. B 1997, 101, 7313-7320. (34) Kleima, F. J.; Gradinaru, C. C.; Calkoen, F.; van Stokkum, I. H. M.; van Grondelle, R.; van Amerongen, H. Energy Transfer in LHCII Monomers at 77 K Studied by Sub-Picosecond Transient Absorption Spectroscopy. Biochemistry 1997, 36, 15262-15268.


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(35) Gradinaru, C. C.; Özdemir, S.; Gulen, D.; van Stokkum, I. H. M.; van Grondelle, R.; van Amerongen, H. The Flow of Excitation Energy in LHCII Monomers: Implications for the Structural Model of the Major Plant Antenna. Biophys. J. 1998, 75, 3064-3077. (36) Zhang, S. J.; Wang, S. C.; He, J. F.; Chen, H. Energy Transfer among Chlorophylls in Trimeric Light-Harvesting Complex II of Bryopsis Corticulans. Acta Biochim. Biophys. Sin. 2006, 38, 310-317. (37) Du, M.; Xie, X. L.; Mets, L.; Fleming, G. R. Direct Observation of Ultrafast Energy-Transfer Processes in Light-Harvesting Complex-II. J. Phys. Chem. 1994, 98, 4736-4741. (38) Bittner, T.; Wiederrecht, G. P.; Irrgang, K. D.; Renger, G.; Wasielewski, M. R. Femtosecond Transient Absorption-Spectroscopy on the Light-Harvesting Chl a/b Protein Complex of Photosystem-II at Room-Temperature and 12 K. J. Chem. Phys. 1995, 194, 311-322. (39) Visser, H. M.; Kleima, F. J.; van Stokkum, I. H. M.; van Grondelle, R.; van Amerongen, H. Probing the Many Energy-Transfer Processes in the Photosynthetic Light-Harvesting Complex II at 77 K Using Energy-Selective SubPicosecond Transient Absorption Spectroscopy. J. Chem Phys. 1996, 210, 297312. (40) Barzda, V.; Gulbinas, V.; Kananavicius, R.; Cervinskas, V.; van Amerongen, H.; van Grondelle, R.; Valkunas, L. Singlet-Singlet Annihilation Kinetics in Aggregates and Trimers of LHCII. Biophys. J. 2001, 80, 2409-2421. (41) Caffarri, S.; Broess, K.; Croce, R.; van Amerongen, H. Excitation Energy Transfer and Trapping in Higher Plant Photosystem II Complexes with Different Antenna Sizes. Biophys. J. 2011, 100, 2094-2103. 39 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(42)

Page 40 of 56

Van Amerongen, H; Dekker, J. P. Light Harvesting in Photosystem II. In

Light-Harvesting

Antennas

In

Photosynthesis;

Kluwer

Academic,

the

Netherlands, 2003. (43) Groot, M. L.; Breton, J.; van Wilderen, L. J. G. W.; Dekker, J. P.; van Grondelle, R. Femtosecond Visible/Visible and Visible/Mid-IR Pump-Probe Study of the Photosystem II Core Antenna Complex CP47. J. Phys. Chem. B 2004, 108, 8001-8006. (44) Groot, M. L.; van Mourik, F.; Eijckelhoff, C.; van Stokkum, I. H. M.; Dekker, J. P.; van Grondelle, R. Charge Separation in the Reaction Center of Photosystem II Studied as a Function of Temperature. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 4389-4394. (45) Visser, H. M.; Groot, M. L.; van Mourik, F.; van Stokkum, I. H. M.; Dekker, J. P.; van Grondelle, R. Subpicosecond Transient Absorption Difference Spectroscopy on the Reaction Center of Photosystem II: Radical Pair Formation at 77 K. J. Phys. Chem. 1995, 99, 15304-15309. (46) Kitajima, Y.; Noguchi, T. Photooxidation Pathway of Chlorophyll Z in Photosystem II as Studied by Fourier Transform Infrared Spectroscopy. Biochemistry 2006, 45, 1938-1945. (47) Tracewell, C. A.; Cua, A.; Stewart, D. H.; Bocian, D. F.; Brudvig, G. W. Characterization of Carotenoid and Chlorophyll Photooxidation in Photosystem II. Biochemistry 2001, 40, 193-203. (48) Schweitzer, R. H.; Brudvig, G. W. Fluorescence Quenching by Chlorophyll Cations in Photosystem II. Biochemistry 1997, 36, 11351-11359.


Page 41 of 56

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(49) Chauvet, J. P.; Viovy, R.; Santus, R.; Land, E. J. One-Electron Oxidation of Photosynthetic Pigments in Micelles - Bacteriochlorophyll-a, Chlorophyll-a, Chlorophyll-B, and Pheophytin-A. J. Phys. Chem. 1981, 85, 3449-3456. (50) Buser, C. A.; Diner, B. A.; Brudvig, G. W. Photooxidation of Cytochromeb(559) in Oxygen-Evolving Photosystem-II. Biochemistry 1992, 31, 1144911459. (51) Horton, P.; Ruban, A. V.; Walters, R. G. Regulation of Light Harvesting in Green Plants. Annu. Rev. Plant Physiol. 1996, 47, 655-684. (52) Ruban, A. V.; Johnson, M. P.; Duffy, C. D. P. The Photoprotective Molecular Switch in the Photosystem II Antenna. Biochim. Biophys. Acta. 2012, 1817, 167181. (53)

Renger, T. Theory of Excitation Energy Transfer: From Structure to

Function. Photosynth. Res. 2009, 102, 471-485. (54)

Hughes, J. L.; Smith, P.; Pace, R.; Krausz, E. Charge Separation in

Photosystem II Core Complexes Induced by 690-730 nm Excitation at 1.7 K. Biochim. Biophys. Acta. 2006, 1757, 841-851. (55) Groot, M. L.; Frese, R. N.; de Weerd, F. L.; Bromek, K.; Pettersson, A.; Peterman, E. J. G.; van Stokkum, I. H. M.; van Grondelle, R.; Dekker, J. P. Spectroscopic Properties of the CP43 Core Antenna Protein of Photosystem II. Biophys. J. 1999, 77, 3328-3340. (56) Jankowiak, R.; Zazubovich, V.; Ratsep, M.; Matsuzaki, S.; Alfonso, M.; Picorel, R.; Seibert, M.; Small, G. J. The CP43 Core Antenna Complex of Photosystem II Possesses Two Quasi-Degenerate and Weakly Coupled Q(Y)Trap States. J. Phys. Chem. B 2000, 104, 11805-11815.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(57) Stewart, D. H.; Cua, A.; Chisholm, D. A.; Diner, B. A.; Bocian, D. F.; Brudvig, G. W. Identification of Histidine 118 in the D1 Polypeptide of Photosystem II as the Axial Ligand to Chlorophyll Z. Biochemistry 1998, 37, 10040-10046. (58) Wang, J.; Gosztola, D.; Ruffle, S. V.; Hemann, C.; Seibert, M.; Wasielewski, M. R.; Hille, R.; Gustafson, T. L.; Sayre, R. T. Functional Asymmetry of Photosystem II D1 and D2 Peripheral Chlorophyll Mutants of Chlamydomonas Reinhardtii. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 4091-4096. (59) Tracewell, C. A.; Brudvig, G. W. Multiple Redox-Active Chlorophylls in the Secondary Electron-Transfer Pathways of Oxygen-Evolving Photosystem II. Biochemistry 2008, 47, 11559-11572. (60) Pullerits, T.; Hess, S.; Herek, J. L.; Sundström, V. Temperature Dependence of Excitation Transfer in LH2 of Rhodobacter Sphaeroides. J. Phys. Chem. B 1997, 101, 10560-10567. (61) Shibata, Y.; Yamagishi, A.; Kawamoto, S.; Noji, T.; Itoh, S. Kinetically Distinct Three Red Chlorophylls in Photosystem I of Thermosynechococcus Elongatus Revealed by Femtosecond Time-Resolved Fluorescence Spectroscopy at 15 K. J. Phys. Chem. B 2010, 114, 2954-2963. (62) Müh, F.; Madjet, M. E.; Renger, T. Structure-Based Simulation of Linear Optical Spectra of the CP43 Core Antenna of Photosystem II. Photosynth. Res. 2012, 111, 87-101. (63)

Adolphs, J.; Müh, F.; Madjet, M. E. A.; Busch, M. S. A.; Renger, T.

Structure-Based Calculations of Optical Spectra of Photosystem I Suggest an Asymmetric Light-Harvesting Process. J. Am. Chem. Soc. 2010, 132, 3331-3343.


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(64) Adolphs, J.; Müh, F.; Madjet, M. E. A.; Renger, T. Calculation of Pigment Transition Energies in the FMO Protein. Photosynth. Res. 2008, 95, 197-209.

Figure captions Fig. 1 Fluorescence time profiles of non-oxidized PSII-em measured by streak camera setup at 77 K. Excitation wavelengths were 430 nm (black) and 460 nm (gray). The profiles were processed by averaging the decay over the whole spectral range from 670 nm to 700 nm, and they were normalized to their maximum.

Fig. 2 Fluorescence time profiles of non-oxidized PSII-em (red) and their fittings (blue) by five exponential functions. Excitation wavelengths were 430 nm (A) and 460 nm (B). The monitoring wavelengths of the curves are 659 nm, 671 nm, 685 nm, and 700 nm from bottom to top.

Fig. 3 Estimated DASs of the non-oxidized PSII-em result from global analysis using five exponential decays. Excitation wavelengths were 430 nm (A) and 460 nm (B).

Fig. 4 Effect of the addition of [Fe(CN)6]3− as an oxidant on the fluorescence time profiles of PSII-em upon the 430-nm (A) and 460-nm (B) excitations. All 43 ACS Paragon Plus Environment


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the profiles were processed by averaging the decay over the whole spectral range from 670 nm to 700 nm, and they were normalized to their maximum. The inset shows the time-integrated fluorescence spectra of non-oxidized and oxidized PSII-em excited at 430 nm.

Fig. 5 Estimated DASs of an oxidized PSII-em result from global analysis using five exponentials. Excitation wavelength was 430 nm.

Fig. 6 Compartment model used for the target analysis of PSII-em, consisting of five compartments: four of them, LHCII (black), CP47b (green), CP43+RC (blue), and Red Chl (red), represent the fluorescent compartments. The fifth one, RP1, is non-fluorescent and represents the primary radical pair. The vertical axis shows the relative energy of the fluorescent compartments. The values in the boxes are the initial populations of the components for the 430-nm (left) and 460-nm (right) excitations. Blue and black arrows represent excitation-energy and electron transfers, respectively. Red arrows represent the quenching paths.

Fig. 7 Results of the target analysis of the fluorescence-decay kinetics of nonoxidized PSII-em. Panels (A) and (B) show the fitting quality of the 430-nm and


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460-nm excitations, respectively. The monitoring wavelengths of the curves in (A) and (B) are 658 nm, 674 nm, 686 nm, and 698 nm from bottom to top. Panels (C) and (D) show the population kinetics at different excitation wavelengths, 430 nm and 460 nm, respectively. Panel (E) shows the estimated SASs, which are determined globally for the 430-nm and 460-nm excitations.

Fig. 8 Results of the target analysis of the fluorescence-decay kinetics of oxidized PSII-em with [Fe(CN)6]3−. Panels (A) and (B) show the fitting quality of the 430-nm and 460-nm excitations, respectively. The monitoring wavelengths of the curves in (A) and (B) are 658 nm, 674 nm, 686 nm, and 698 nm from bottom to top. Panels (C) and (D) show the population kinetics at different excitation wavelengths, 430 nm and 460 nm, respectively. Panels (E) and (F) show the estimated SASs of the 430-nm and 460-nm excitations, respectively.

Fig. 9 Schematic diagram shows pigment contribution to the PSII-cc dimer structure. Two thick arrows indicate the excitation-energy transfer from Chl29 (donor) in one monomer unit to ChlZD1+ (quencher) in the other monomer unit. Two thin arrows represent the energy transfer from Chl26 to ChlZD2+ in the same monomer.



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Table 1 Estimated excitonic couplings and the energy-transfer time constants between red Chl pigments in CP47 (donor) and ChlZ+ pigments.


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Fig. 1



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Fig. 2


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Fig. 3



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Fig. 4


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Fig. 5



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Fig. 6


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Fig. 7



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Fig. 8


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Fig. 9

donor / acceptor

location

Chl29 to ChlZD2 Chl29 to ChlZD1 Chl29 to ChlZD2 Chl29 to ChlZD1 Chl26 to ChlZD2 Chl26 to ChlZD1 Chl26 to ChlZD2 Chl26 to ChlZD1

same monomer same monomer different monomer different monomer same monomer same monomer different monomer different monomer

excitonic coupling (cm-1) 0.10 -0.10 0 -1.3 1.2 0 -0.20 -0.20

Table 1


energy transfer time constant (ps) 13×103 13×103 --75 88 --3.0×103 3.0×103


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TOC image


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Structure-Based Modeling of Fluorescence Kinetics of Photosystem II

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