photosystem ii ( ps ii ) plays a crucial 
 role in the photoinduced 
 water oxidation in oxygenic photosynthesis of plants , cyanobacteria , 
 and algae . the water oxidation catalyzed 
 by the mn ca cluster in ps ii results in emission of oxygen 
 molecules as residual , which most of aerobia on the earth rely on . the proteins constituting ps ii bind a large number and several kinds 
 of pigment molecules , such as , chlorophyll - a ( chl - a ) , pheophytin - a ( pheo - a ) , -carotene , and plastoquinone . most of the pigments are 
 bound in light - harvesting antennae , and only a minor number of a different 
 type , including chl - a , pheo - a , and 
 plastoquinones , are used as electron carriers along the electron - transfer 
 chain in the reaction center . during the past decade , the spatial 
 resolution of the crystallographically 
 determined structures of ps ii has been continuously improving . in 2011 , umena et al . have reported the crystal structure of ps 
 ii with 1.9  spatial resolution , which presently provides the most detailed structure , in particular 
 of the mn this study undoubtedly represents a great 
 step toward elucidating the water - oxidation mechanism in ps ii . the 
 improved structural information also offers greater opportunities 
 for a structure - based simulation of the light - harvesting dynamics 
 of the antenna system of ps ii . ps ii has been known to show a peculiar 
 temperature dependence of the fluorescence spectrum . figure 1a shows the temperature dependence 
 of the fluorescence spectrum of the ps ii core complex ( ps ii - cc ) 
 preparation from a thermophilic cyanobacterium , thermosynechococcus 
 vulcanus . wavelength 
 2-d images obtained by a streak scope setup . lowering temperature 
 down to 77 k results in an emergence of a sub - band at around 695 nm , 
 which hereafter we call f695 according to its peak wavelength . this 
 apparent red shift of the fluorescence peak upon cooling suggests 
 the existence of chl pool(s ) with lower excited - state energy(ies ) 
 than the primary donor ( pd ) on which the first photoinduced charge 
 separation takes place . figure 1a shows that 
 the relative intensity of f695 again decreases upon further cooling . 
 at 5 k , the main fluorescence band is located at around 685 nm ; hereafter 
 we call this band f685 . the blue shift of the fluorescence peak below 
 77 k is against the expectation because lowering temperature in general 
 suppresses thermal activations of uphill energy transfers , and so 
 one might expect a further enhancement of the fluorescence originating 
 from f695 . it is quite timely and pertinent to examine whether this 
 peculiar temperature dependence of the ps ii fluorescence spectrum 
 can be explained on the basis of the fine structural information now 
 available . ( a ) temperature dependence of the fluorescence spectrum of ps ii - cc 
 dimer from t. vulcanus . ( b ) schematic description that accounts for 
 the spectral change of the ps ii fluorescence spectrum upon lowering 
 temperature . thin horizontal lines within the gray box indicate the 
 exciton levels with higher excited - state energies than that of the 
 primary electron donor . those of the red - shifted chls , c685 , and c695 
 are indicated by the thick horizontal lines below the gray box . arrow 
 shows the excitation - energy flow , and its thickness indicates a rough 
 measure of the energy flux . a qualitative explanation for the experimental observation 
 shown 
 in figure 1a can be given by assuming two different 
 pigment pools , both having lower excited - state energies than that 
 of pd . we designate hereafter the two pigment pools as  c685  
 and  c695  according to their fluorescence peak wavelengths . 
 at 77 k , the energy gap between c695 and pd becomes inaccessible by 
 the thermal energy , while the uphill energy transfer from c685 to 
 pd is still active , and then an excited state is trapped on c695 resulting 
 in the enhancement of f695 . further lowering temperature suppresses 
 the uphill energy transfer from c685 to pd as well , and therefore 
 the relative intensity of f685 increases . as described above , 
 the overall tendency of the observation could 
 be explained on the basis of the qualitative model in figure 1b . it has been established that c695 resides in 
 the tightly bound proximal antenna protein , cp47 , according to the 
 similarity of the low - temperature fluorescence peak positions between 
 the isolated cp47 and ps ii - cc . c685 , on the other hand , has been considered to be bound to the 
 other proximal antenna , cp43 , although the involvement 
 of the pigments in cp47 can not be excluded . many research groups have 
 assigned c695 to chl29 ( chl numbering throughout this paper is according 
 to the nomenclature of loll et al . ) in 
 cp47 on the basis of the site - directed mutation study , the negative linear dichroism component in the red tail 
 of the absorption spectrum of cp47 . their study also suggested that 
 c685 , tentatively assigned to the lowest excited state in cp43 , is 
 contributed mainly from chl37 in cp43 . recent studies by the 
 jankowiak group , on the other hand , raised a question 
 on the above assignment of 
 c695 to chl29 . it was shown that storing of a diluted cp47 solution at 77 k considerably 
 degrades the protein and results in the blue shift of its fluorescence 
 peak at 5 k. they suggested that the above assignment of c695 to chl29 
 was based on the optical spectra of partly degraded cp47 samples . they also showed that the high efficiency of nonphotochemical hole 
 burning ( nphb ) at lower temperatures is partly a cause of the blue 
 shift of the fluorescence spectrum of cp47 upon lowering temperature . 
 according to the fitting of the absorption , fluorescence , and hb spectra 
 of the  intact cp47 preparation , they concluded chl26 
 to be a more probable candidate for c695 rather than chl29 . their conclusion with respect to the lowest excited 
 state in cp43 was also somewhat different from that drawn from the latest theoretical study by mh 
 et al . as described above , controversial 
 opinions exist about the identity 
 of chl molecules responsible for f695 and f685 . more reliable assignments 
 of these red - shifted chl pools will offer better opportunities to 
 elucidate their functional roles . the major difficulty is in the determination 
 of the transition energy of each pigment molecule experiencing various 
 perturbations at its binding site , an energy which is therefore called 
  site energy  . another difficulty is the appropriate 
 description of the interplay between exciton - vibrational coupling 
 and excitonic coupling . whereas the latter tries to delocalize the 
 excited states , the former may lead to a dynamic localization . one 
 way to treat this problem is to implicitly take into account dynamic 
 localization by grouping the pigment molecules into several coherent 
 domains . the local modulation 
 of transition energies by the protein dynamics is described on the 
 basis of the spectral density that can be extracted from fitting of 
 fluorescence line narrowing data . as 
 discussed above , a critical part of the simulation of exciton transfer 
 and optical spectra is the determination of parameters , in particular 
 the site energies of the pigments . one way , used by raszewski and 
 renger , is to determine them from a fit 
 of optical spectra of the cp43 , cp47 , and rc- subunits . of course , 
 a direct structure - based simulation of site energies would be preferable 
 but is highly nontrivial , because of the complexity of the problem . here , we stress 
 that the qualitative explanation based on the schematic 
 model in figure 1b requires the following conditions 
 to be fulfilled : ( 1 ) the energy transfer between c695 and c685 is 
 very slow or practically inhibited below 77 k , and ( 2 ) the majority 
 of the excitation energy is funneled into c685 , while only a minor 
 part flows into c695 . if one of the above two conditions is not fulfilled , 
 then the fluorescence peak below 77 k should be fixed at around 695 
 nm . this means that the light - harvesting dynamics in ps ii at cryogenic 
 temperatures is highly sensitive to the actual locations of c685 and 
 c695 within ps ii - cc . the simulation of cryogenic light - harvesting 
 dynamics of ps ii - cc using several possible assignments of c695 will 
 thus enable us to evaluate these assignments . this work is organized 
 in the following way : we start with a characterization 
 of the materials and experimental and theoretical methods used . next , 
 the optical spectra of the cp43 , cp47 , and rc subunits are investigated 
 and used for the determination of site energies . these site energies 
 are applied next , together with the excitonic couplings obtained for 
 the latest structure of ps ii - cc determined by umena et al . , to model stationary and time - resolved optical 
 spectra of the core complex and compare with experimental data obtained 
 in this study . in addition , we investigate the predictive power of 
 different sets of site energies from the literature . finally , we discuss 
 the results and relate them to the physiological function of photosystem 
 ii . ps ii - cc 
 dimer was purified from cells of a thermophilic cyanobacterium , t. vulcanus , grown at 5254 c as described 
 previously . briefly , thylakoid membranes 
 were isolated from cells and solubilized with n - dodecyl - n , n - dimethylamine - n - oxide 
 ( ldao ) to obtain crude ps ii particles . the crude ps ii particles 
 were then solubilized with 1% ( w / v ) sodium n - dodecyl--d - maltoside 
 ( ddm ) , and the solubilized samples were passed through an anion - exchange 
 chromatography column to isolate the ps ii dimer complex . the obtained 
 ps ii dimer complex was suspended in 20 mm mes - naoh ( ph 6.0 ) , 20 mm 
 nacl , 3 mm cacl2 , and 25% ( w / v ) glycerol and stored in 
 liquid n2 until use . a typical optical density of the stock 
 solution was about 110 cm at the peak of the qy band . storing the solution with this high 
 protein concentration is important to maintain the intactness of the 
 sample which was judged from the strong f695 emission at 77 k shown 
 in figure 1 a. for measurements at cryogenic 
 temperatures , a stock solution of 
 the sample was diluted with a 33 mm mes - naoh buffer ( ph 6.0 ) containing 
 5 mm cacl2 , 33 mm nacl , and 0.05% dm . the diluted solution 
 was mixed with twice the volume of glycerol to maintain its transparency 
 at cryogenic temperatures . the final optical density of the sample 
 was 0.1 mm at the peak of the qy band . the solution was contained in a 1-mm light - path 
 cuvette and dark adapted for at least 10 min for complete relaxation 
 of the sample to the s1qa configuration . the solution was then inserted into the sample room of a liquid - helium 
 flow - type cryostat ( cf1204 , oxford instruments , inc . , precooled down to 80 k. the streak camera set - ups 
 for the time - resolved fluorescence measurements 
 are basically the same as those described in the previous studies . the sample was excited by the frequency - doubled light at 430 nm 
 from a ti : sapphire laser ( maitai ; spectra - physics , mountain view ) 
 with a pulse duration of 110 fs and a repetition rate of 80 mhz . a 
 relatively high excitation power of 100 w ( 1.3 pj / pulse ) 
 was needed to obtain a high enough s / n ratio in the present study . the excitation laser was focused on 
 the sample by using a 150-mm focal length lens . the fluorescence emitted 
 to the direction perpendicular to the incidence of the excitation 
 light was collected by two lenses and focused into the entrance slit 
 of the polychromator . the beam diameter at the focal plane was roughly 
 estimated to be 130 m by using the numerical aperture of 0.5/150 . then , the excitation power per unit area was 0.5 w / cm ( we took into account the 45 angle between the sample 
 window normal and the incidence of the excitation light ) . a global multiexponential fitting 
 analysis was performed as described in previous papers to obtain the fluorescence decay - associated spectra ( das ) . as indicated by neupane et al . , this 
 irradiation dose should result in the saturated nphb at 5 k. the measurement 
 was done in the order of the lowest to highest temperatures . thus , 
 the measurement at 5 k was done first , then those at higher temperatures 
 followed . since the relative f695 intensity at 77 k did not change 
 in spite of the high irradiation dose in the precedent measurements 
 at 5 and 36 k , we consider that the lowest excitonic state in cp47 
 was maintained intact after the several data accumulations . suggested that the rather intense 
 irradiation of ps ii - cc at cryogenic temperature results in the formation 
 of a stable qa ( closed rc ) state . therefore , 
 the excitation intensity used here results in a complete conversion 
 of the sample to the stable qa state . 
 by using the molar extinction coefficient of chl - a at 430 nm of 1.15 m cm and 
 the above estimated excitation power , we can estimate the averaged 
 frequency of excitation for an individual molecule to be 1.7  
 10 s. it was reported that , on the 
 other hand , in the closed ps ii - cc the primary charge - separated state 
 is converted with a high quantum efficiency to the charge - recombined 
 triplet state . this triplet state has been traditionally 
 called the p680 triplet or p680 , while it is now known 
 to reside on the accessory chl of the d1 branch . the half - life 
 of the p680 triplet was reported to be a mixture of 1.5 s ( 50% ) 
 and 15 s ( 50% ) . these half - life 
 times are considerably shorter than the averaged excitation cycle 
 of the present study . thus , in the present excitation configuration , 
 the molecules are considered to be populated dominantly in the qa state , while only a limited part of the 
 molecules are trapped in p680 state . the absorption 
 spectra of the sample at various temperatures were 
 measured by using a conventional spectrometer ( uv-3100 , shimazu , kyoto ) 
 equipped with the helium flow - type cryostat . the cd spectrum at a 
 liquid nitrogen temperature was measured by using a conventional cd 
 spectrometer ( j700 , jasco , tokyo ) equipped with a liquid nitrogen 
 bath - type cryostat ( dn2 , oxford instruments , inc . , the solution was contained in a 10-mm light - path acrylic cuvette 
 in both cases . baseline of the cd spectrum was corrected by subtracting 
 the signal obtained at the same temperature with the same cuvette 
 containing the solvent only . the spectral profiles and 
 the exciton - transfer dynamics were simulated 
 on the basis of the dynamical theory of optical spectra , which was 
 detailed in previous papers . the calculations were based on the latest ps ii crystal structure 
 by umena et al . since the dimeric form 
 of ps ii is considered to be the intact state under physiological 
 conditions , we evaluated the importance of 
 energy transfer between the two monomers within a dimer . in order 
 to examine this effect , all the 74 pigments ( 70 chl - a s and 4 pheo - a s ) contained in the 
 ps ii - cc dimer the pigment 
 molecules in the ps ii - cc dimer were divided into several 
 exciton domains . a pigment having an excitonic coupling higher than 
 a certain threshold with any pigment of an exciton domain is assumed 
 to belong to the same domain , and the delocalization of the excited 
 states was restricted to the pigments in the same domain . the dynamic localization of the exciton was 
 thus accounted for in an implicit way . the threshold was set to 30 
 cm , which is a typical value for the reorganization 
 energy of the local pigment protein coupling . since the excitonic 
 couplings between pigments in different ps ii - cc monomers are weaker 
 than this threshold , the pigments of the two monomers are in different 
 domains , and the stationary optical spectra of ps ii - cc monomers are 
 identical to those of ps ii - cc dimers . exciton dynamics was calculated 
 by the combined application of redfield theory and generalized frster 
 theory for the transfer 
 between exciton states in the same and in different domains , respectively , 
 using non - markovian time - local theory for the optical transitions 
 in the exciton domains . input parameters of the simulation are 
 the excitonic couplings 
 between the pigments , the site energies , the electron - vibrational 
 coupling strength ( huang  rhys factor s ) , the 
 primary charge - separation rate , and the spectral density j( ) characterizing the frequency dependence of the exciton - vibrational 
 coupling . the excitonic couplings were calculated with the poisson - tresp 
 method established earlier by using the pigment coordinates from the crystal structure . the electrostatic calculations of poisson tresp 
 were performed before using the program mead with the transition charges determined from the fit of the electrostatic 
 potential of the quantum chemically determined transition density 
 of the s0  s1 transition energies of 
 chl - a and pheo - a . according to the 
 previous study , the excitonic coupling 
 between the special pair chls pd1 and pd2 was 
 assumed to be 158 cm instead of the value calculated 
 from the poisson - tresp method in order to take into account the electron 
 exchange interactions between the two chls . the electron phonon 
 coupling s was determined to be 0.6 from a simulation 
 of the experimentally observed spectral broadening upon temperature 
 rise . in order to include static disorder , the spectra were averaged 
 with respect to a gaussian distribution function for the site energies 
 of the pigments by using a monte carlo method . the primary charge - separation 
 was assumed to start at the accessory 
 chl in the d1 branch ( chld1 ) . the primary charge - separation rate constant kmrp from an exciton state |m > to the primary radical 
 pair state |rp > is described by |cchld1(m)|kintr , where |cchld1(m)| is the probability of finding chld1 excited 
 in the mth exciton state and kintr is 
 the intrinsic rate constant for electron transfer . according to the 
 previous study , kintr was taken as ( 100 fs ) and ( 6 ps ) for open and closed rcs , respectively . since the 
 experimental data considered were obtained at cryogenic temperatures , 
 the charge - recombination reactions were not included in the present 
 study . we used an approximate form of the spectral density j( ) , which was extracted from 
 fluorescence line - narrowing ( fln ) spectra of the b777 complex , as in the previous paper:1with s1 = 0.8 , s2 = 0.5 , 1 = 0.069 
 mev , and 2 = 0.24 mev . protein complexes 
 and found to be very similar in shape to the j( ) 
 extracted from fln spectra of a different complex . in general it is found that the shape of j( ) is very similar , but the amplitude varies somewhat between 
 different complexes . independent evidence for the validity of this 
 finding is due to the fact that in the case of pigment - containing 
 glass forming materials , the functional profile of the spectral density 
 shows no significant dependence on the material hosting the pigment , 
 while the value s is host dependent . we assumed that the present experimental condition using 
 the excitation at 430 nm results in a nonselective excitation of chls . accordingly , we assumed a uniform initial excited population among 
 all the pigments in the simulation of the fluorescence dynamics . the excitonic couplings vmn calculated 
 using the latest ps ii structure by umena et al . are plotted against those calculated using the structure 
 by guskov et al . in figure 2 . the numerical values of these couplings are compiled in 
 a file  coupling_si.xls  in the supporting information ( si ) . alterations in the excitonic 
 couplings were only minor except for the pair chl11chl12 in 
 cp47 . a large change in the excitonic coupling for this pair was due 
 to the modified configuration of chl11 . in the latest structure , its 
 molecular plane was flipped along the axis connecting the 5- and 15-carbon 
 to give a 90 rotation of its qy transition 
 dipole moment from that in the previous structure . correlation 
 between the excitonic couplings between pigments in 
 the ps ii - cc dimer calculated based on the latest structure by umena 
 et al . and on that by guskov et al . we simulated the static spectra of the isolated subunits 
 using 
 the excitonic couplings obtained here , treating the site energies 
 as fit parameters with initial values taken from the literature . for 
 this purpose , we used the site energies reported by raszewski and 
 renger for the spectral simulations of 
 the cp47 and rc subunits , while we applied those reported by mh 
 et al . for the cp43 subunit the site 
 energies for cp43 were obtained from a combination of structure - based 
 quantum chemical / electrostatic calculations with a subsequent refinement 
 fit and comparison with the experimental data . the inhomogeneous width 
 of the distribution function for the site energies was set at similar 
 values as those employed in the previous studies , with inh = 180 cm for the rc pigments and inh = 150 
 cm in cp43 and cp47 with the following exceptions : 
 120 cm for the accessory chl in the d1-branch 
 of the rc and inh = 90 cm for chl43 and chl45 in cp43 . these exceptive values for the two 
 chls in cp43 were necessary to fit the prominent peaks observed in 
 the absorption spectra . revealed that the spectra of the isolated cp47 in the early 
 reports were somewhat different from those obtained for more intact 
 preparations . however , so far no ld and cd spectra were reported for 
 the latter . the calculations using the previously determined 
 site energies 
 well reproduced the experimental spectra . however , the fittings were 
 not as good as in the previous studies because of the utilization 
 of the slightly different structure . in order to improve the fitting 
 quality , we refined the site energies through an optimization procedure , 
 in which the site energies were adjusted to give the local minimum 
 of the  value defined as2here , abs(t)/ld(t)/cd(t)/fl(t ) are the absorption / linear 
 dichroism / circular dichroism / fluorescence spectra at temperature t. the superscript  obs / sim  indicates the 
 experimental / simulated spectrum . since the fitting quality for the 
 cd , ld , and fluorescence spectra was not as good as that for the absorption 
 spectra in general , the a factors were introduced 
 to balance the contribution from each spectrum to the  value . here , we set aabs = 1 , ald = afl = 0.2 , 
 and acd = 0.04 . comparisons of the 
 experimental and simulated subunit spectra are 
 given in figures s1 ( rc ) , s2 ( cp47 ) , and s3 ( cp43 ) . for example , figure s3 shows various static spectra of the 
 isolated cp43 at 4 and 77 k reported in the literature in comparison 
 to the simulations . quantitative agreements are obtained between the 
 experimental and simulated curves except for the cd spectrum . the 
 experimental cd spectrum of cp43 has a nonconservative profile , which 
 can not be explained by the present exciton theory , which gives a conservative 
 cd signal . despite a large deviation of the cd spectrum , its overall 
 features , e.g. , the negative and positive peak positions , could still 
 be reproduced in the simulation . using the cp43 site energies by mh 
 et al . resulted in a better fitting of 
 the cd spectrum to the experimental one than the results by raszewski 
 and renger . the obtained site energies 
 for rc , cp47 , and cp43 are listed in tables 1 , 2 , and 3 , respectively . the coherent domains that each pigment belongs to are listed in the 
 third rows of these tables . the 4 nm red - shifted value in 
 parentheses for chld1 is the value in the ps ii - cc . figure 3 shows the comparison 
 of the experimental 
 absorption spectrum ( solid line ) of the isolated subunits at 4 k ( panel 
 a ) and of the ps ii - cc dimer at 5 k ( panel b ) with the simulated curves 
 ( open circles ) . the simulated spectrum of ps ii - cc was calculated 
 using the site energies determined through the refinement process 
 described above and corresponds to the sum of the three simulated 
 subunit spectra . the experimental subunit spectra in panel a are normalized 
 so that the ratio of the spectral area , rc : cp43:cp47 , gives the value 
 8:13:16 that follows from the pigment stoichiometry . although quantitative 
 fittings were obtained for the subunit spectra , the simulated spectrum 
 of ps ii - cc looks qualitatively different from the experimental one . the most conspicuous discrepancy is the position of the shoulder peak , 
 occurring at around 684 nm in the experimental spectrum and at around 
 682 nm in the calculations . comparison of the experimental absorption spectra 
 ( solid lines ) 
 of the ( a ) isolated cp47 , cp43 , and rc at 4 k and ( b ) ps ii - cc dimer 
 from t. vulcanus at 5 k with the calculated 
 curves ( circles ) using the site energies in tables 1 , 2 , and 3 . one problem could arise from the 
 fact that the subunit spectra 
 had to be taken from higher plants , whereas the ps ii - cc spectra are 
 those of t. vulcanus .. recently , boehm 
 et al . have reported the low - temperature 
 absorption and fluorescence spectra of the isolated cyanobacterial 
 cp43 and cp47 complexes , which are only slightly different from the 
 isolated cp43 and cp47 complexes from higher plants . the sum of the 
 subunit absorption spectra of the cyanobacterial cp43 , cp47 , and rc 
 complexes still significantly deviates from the experimental ps ii - cc 
 spectrum in figure 3b ( data not shown ) . most 
 likely , alterations in the protein conformations during the isolation 
 procedure cause modifications of the site energies and result in the 
 poor fitting in figure 3b . further adjustments 
 of the site energies were needed to describe these spectra . nevertheless , we found that the simulations using the site energies 
 from the fits of subunit spectra gave qualitatively similar time - resolved 
 spectra of the ps ii - cc as the experiment ( data not shown ) , indicating 
 that these site energies are already close to the optimal values . we carried out a further refinement of the site energies in ps ii - cc 
 to improve the fitting . here , we paid attention to the peak in the 
 experimental absorption spectrum of ps ii - cc at around 684 nm , which 
 was located at around 682 nm in the simulation . this band is obviously 
 assigned to the peak in the cp43 subunit spectrum at around 682 nm . actually , boehm et al . have reported 
 a red shift of this band in the cyanobacterial cp43 complex from that 
 in the higher plant cp43 . thus , the shifted peak position of this 
 peak indicates that at least the site energies in cp43 have to be 
 refined for a better description of the experimental spectra . we note that we included already the 4 nm red shift of the accessory 
 chl of the d1-branch occurring between rc and ps ii - cc preparations 
 as inferred before from light - induced difference spectra of the cyanobacterial 
 ps ii - cc . here , we investigated whether the ps ii - cc 
 spectra can be simulated if , in addition , only the site energies of 
 cp43 are allowed to vary . figure 4 shows the 
 comparison of the simulated and experimental cd ( present study ) , absorption , 
 and ld spectra from the literature of 
 ps ii - cc . the fitting was drastically improved by the optimization 
 procedure , in which we searched for the local minimum of the  value defined as3adjustments of the site energies were restricted 
 to cp43 . the cd spectrum is not included in the  calculation , because the experimental cd spectrum of ps ii - cc is 
 highly nonconservative nevertheless , the simulation could roughly 
 reproduce the overall peak positions of the cd spectrum as shown in 
 figure 4a . the obtained optimized site energies , 
 which are listed in table 4 , were used to calculate 
 the temperature dependence of the absorption spectrum of ps ii - cc 
 as shown in figure 5 . a qualitatively correct 
 description of the temperature dependence of the experimental data 
 is obtained . the deviations are slightly larger at temperatures above 
 157 k. comparison of the experimental ( a ) circular dichroism ( present 
 study),(b ) linear dichroism , and ( c ) 
 absorption ( present study ) spectra of the cyanobacterial ps ii - cc 
 dimer from thermosynechococcus sp . 
 with the calculated spectra using the site energies in tables 1 and 2 ( rc and cp47 , respectively ) 
 and modified ones in cp43 listed in table 4 . the solid lines show the experimental data at 5 and 77 k. the triangles 
 and circles show the calculated spectra at 5 and 77 k , respectively . the temperature dependence of the experimental 
 absorption spectrum 
 ( solid lines ) of the ps ii - cc dimer from t. vulcanus , compared with the calculated ones ( open circles ) using the site 
 energies listed in tables 1 , 2 , and 4 . we confirmed that the obtained set of site energies 
 actually gave 
 the local minimum of the  value defined in eqs 2 and 3 by plotting its dependences 
 on slight perturbations of the site energies . in figures s4 and s5 , it is shown that the  value takes its local minimum at the optimized site energies . the 
 curvature of the  vs site - energy perturbation plot 
 can be a rough measure of the relative reliability of the determined 
 site energy . chls with relatively low site energies tended to show 
 high curvatures of the plot . thus , we can say that the site energies 
 of chls with low site energies were determined with higher reliabilities 
 as compared with those with high site energies . using the refined 
 site energies , we carried out the simulation 
 of the time - resolved fluorescence spectrum of ps ii - cc at 5 , 36 , 77 , 
 and 180 k. under the present experimental condition , rc is expected 
 to be in the closed form , where the intrinsic rate constant of the 
 primary charge separation , kintr , was 
 inferred to be ( 6 ps ) . in addition , we took into account the electrochromic shift induced 
 by the reduction of qa as described previously . the shift of the site energy of pheod1 amounts to 1.5 nm to the red . the shifts of the other pigments were 
 calculated to be < 1 nm and then omitted in the present study . figure 6 shows the comparison of the experimental 
 time  wavelength 2d fluorescence images ( left column ) with the 
 simulated ones ( right column ) . the simulated 2d images were convoluted 
 with the instrumental response function of the experiment with fwhm 
 of 89 ps . the simulations reveal the emergence of the 695-nm peak 
 with a slow decay rate upon lowering temperature down to 77 k and 
 its relative suppression upon further cooling . the simulated and experimental 
 temperature dependencies of the time - integrated spectra are shown 
 in figure 7 . although the peak wavelengths 
 in the simulated spectra are not exactly the same as those in the 
 experimental ones , the spectral features and their temperature dependences 
 are qualitatively well reproduced . it should be noted that in figure 6 a slow fluorescence decay component at around 683 
 nm in the experimental dynamics at 77 and 180 k is missing in the 
 simulated dynamics . the slight discrepancy in the peak position of 
 the time - integrated spectrum at 180 k in figure 7 might be due to the lack of the slow decay component in the simulated 
 dynamics . we further tried to refine the site energies by adjusting 
 those in cp47 , in addition to those in cp43 . however , fitting of cp47 
 site energies did not improve the overall fit of experimental data 
 ( data not shown ) . if the site energies of cp47 proposed recently by 
 reppert et al . are used , a less satisfactorily 
 description is obtained ( dashed lines in figure 7 ) . d ) 
 time - resolved fluorescence spectrum of the ps ii - cc dimer from t. vulcanus , compared with the calculated ( right , 
 e  h ) ones using the site energies in tables 1 , 2 , and 4 . the calculated time - resolved fluorescence spectra were convoluted 
 with the instrumental response functions , which were approximated 
 by a gaussian function with fwhm of 89 ps . ( a ) temperature dependence of the experimental static fluorescence 
 spectra of the ps ii - cc dimer from t. vulcanus . ( b ) the calculated static fluorescence spectra of ps ii - cc dimer 
 ( circles ) from t. vulcanus , using the 
 site energies in tables 1 , 2 , and 4 . the dashed lines show the 
 spectra calculated by replacing the site energies in table 2 ( cp47 ) by the values reported by reppert et al . in the present study , a refined set of the site 
 energies for the 
 pigments in isolated cp43 , cp47 , and rc preparations was obtained 
 from a fit of optical spectra of these subunits . it was found that 
 further adjustment of site energies in particular of the cp43 pigments , 
 is necessary in order to explain the optical spectra of ps ii - cc . this change most likely reflects conformational changes that occur 
 during the isolation procedure of the subunits . with these site energies 
 an explanation is obtained for the inverse temperature dependence 
 of the fluorescence spectrum of ps ii - cc between 77 and 5 k. this 
 explanation is closely related to the identity and nature of low - energy 
 excited states in the antenna . the temperature dependence can be explained 
 by two different red - shifted chl pools , c695 responsible for f695 
 and c685 responsible for f685 . the blue shift of the fluorescence 
 occurring below 77 k reflects the fact that the majority of the excitation 
 energy is funneled into c685 , while only a minor part is transferred 
 to c695 . in the present study chl29 is an isolated chl which does not constitute 
 a coherent domain with other chls , mainly because of an unfavorable 
 orientation of its q  y transition 
 dipole moment with respect to those of the other nearby chls . this 
 orientation of the qy transition dipole 
 moment is consistent with the ld spectrum of the isolated cp47 complex 
 showing a slight negative sign in its long - wavelength tail region . 
 because of its position at the periphery and relatively weak excitonic 
 coupling to the nearby chls , only a minor part of the excitation energy 
 flows into chl29 . these features of chl29 are important for the temperature 
 dependence of the fluorescence spectrum , as will be discussed in more 
 detail below . have proposed an alternative assignment of c695 to chl26 based on 
 their fitting of the absorption and the nphb spectra . we carried out 
 the calculations by using the site energies reported by reppert et 
 al . as shown in figures 
 s2 and s6 , the calculated cd and ld spectra of the isolated 
 cp47 as well as the time - resolved fluorescence spectra of ps ii - cc 
 are qualitatively different from the experimental ones . the latter 
 simulations overestimate the relative intensity of f695 at 5 k. the 
 simulated time - resolved fluorescence shows a separate peak at around 
 695 nm even at 5 and 36 k , in contrast to the experimental data . we note that there are still some systematic deviations between the 
 experimental ld spectrum and the one calculated for the present site 
 energies concerning the contribution of c695 occurring at the low - energy 
 side of the spectrum that need to be explained in future work . according to the previous work by 
 mh et al . and by raszewski and 
 renger the low - energy pigments in cp43 
 are chl37 , chl43 , and chl45 . the present refinement fits of optical 
 spectra of the cp43 subunit ( table 3 ) roughly 
 agree with this assignment . a slight alteration in the present study 
 is that chl37 belongs to another coherent domain and does not contribute 
 to the low - energy exciton state in cp43 . importantly chl43 and chl45 
 are part of a large exciton domain , and the present fit of optical 
 spectra of ps ii - cc ( table 4 ) suggests that 
 in ps ii - cc , in addition , two other chls ( 41 and 47 ) of the same exciton 
 domain get low site energies . therefore , there is a lot more oscillator 
 strength in c685 of cp43 as compared to c695 in cp47 . this difference 
 explains why at very low t ( 5 k ) the fluorescence 
 maximum is around 685 nm rather than 695 nm . at this low temperature , 
 energy transfer and nonradiative trapping by the rc are frozen out , 
 and the higher oscillator strength of f685 determines the position 
 of the maximum . if temperature is increased , fast thermal activation 
 of low - energy excitons in cp43 sets in , and the subsequent trapping 
 by the rc diminishes the fluorescence yield of f685 . since energy 
 transfer from c695 in cp47 to the rc is still blocked , f695 now has 
 the largest fluorescence yield and determines the maximum of the fluorescence . this effect explains the inverse temperature shift of the fluorescence 
 maximum observed between 5 and 77 k in ps ii - cc . reported that saturated 
 nphb induces a blue shift of the 695 nm fluorescence of cp47 to 692 
 nm . to exclude interference of this effect with our interpretation , 
 we measured the 5 k fluorescence before the 77 k fluorescence and 
 still found the 77 k fluorescence peak at 695 nm . hence nphb induced 
 bleaching of the low - energy state of cp47 can be excluded in the present 
 experiments . it is interesting to note that the common picture 
 of a light - harvesting 
 antenna as providing an excitation energy funnel for guiding the excitons 
 to the rc is not met by cp47 , which exhibits a low - energy trap state 
 far away from the reaction center . of course , at physiological temperature 
 thermal energy is sufficient to allow for an easy escape of excitons 
 from this trap . however , the existence of such states shows that the 
 rc in ps ii is a very shallow trap . in fact , because of the more or 
 less isoenergetic site energy landscape throughout ps ii - cc , the entropic 
 factor ( larger number of antenna than reaction center pigments ) gives 
 rise to an about three times larger rate for exciton transfer from 
 the rc to the core antennae than vise versa . nevertheless , the ultrafast electron transfer in open rcs still 
 catches nearly every exciton that reaches the rc . if , however , the 
 rc is closed , then the slower electron transfer allows the excitons 
 to escape back in the antennae . the low site energy of chl29 in cp47 
 will give the population of this pigment a large weight in ( quasi ) 
 thermal equilibrium , and hence the question arises about the functional 
 role of chl29 being responsible for c695 . by assuming fast 
 intradomain exciton relaxations , we can calculate an effective energy - transfer 
 rate from an equilibrated domain a to a domain b as4here , the boltzmann factor f(ma ) of an exciton state ma in domain a is given by5where kb is the 
 boltzmann constant and ma is the energy of the exciton state ma . the individual rate constants kmamb for exciton transfer from exciton state ma in domain a to exciton state mb in domain b are obtained from generalized frster 
 theory , as described previously . the 
 green arrows in figure 8 show important energy - transfer 
 paths between domains with rate constants calculated according to 
 eqs 4 and 5 . here , we 
 show only the transfer paths whose rate constants at 77 k were estimated 
 to exceed 10 ns . the thickness of an arrow in 
 figure 8 is proportional to the square root 
 of the interdomain rate constant at 77 k , which was calculated by 
 disorder averaging the rate constant given by eq 4 . the disorder averaged rate constants in unit of ns are compiled in a file  interdomainrate_si.xls  available 
 in the si . arrows show important interdomain energy - transfer 
 paths , whose 
 rate constants at 77 k exceed 10 ns . the thickness 
 of an arrow is proportional to the square root of the transfer rate 
 constant at 77 k between domains connected by the arrow . two examples 
 of highly temperature - dependent transfer paths are indicated with 
 their disorder averaged inverse rate constants in unit of picoseconds 
 at 77 k ( red ) and 5 k ( black ) . chls ( and pheos ) in blue , orange , green , 
 cyan , yellow , and red belong to the domains 16 , respectively . some of the transfer paths shown in figure 8 are highly sensitive to lowering temperature below 77 k. 
 two important 
 examples of such temperature - sensitive transfer paths are indicated 
 by the numbers in red ( 77 k ) and black ( 5 k ) beside arrows , which 
 are the inverse rate constants in unit of picoseconds between the 
 domains connected by the arrows . the inverse rate constant in cp47 
 from domain 4 to chl29 , the c695 primary candidate , is 80 ps at 77 
 k , while it slows down to 550 ps at 5 k. the reverse transfer also 
 shows a similar temperature dependence . hence , at 77 k the equilibration 
 between the large domain 4 and chl29 is still fast compared to the 
 radiative lifetime . since the transfer from domain 4 to the rc at 
 this temperature is already very slow ( the inverse rate constant is 
 150 ps ) , domain 4 effectively functions as an antenna for 
 chl29 . upon lowering the temperature further to 5 k , this antenna 
 practically becomes decoupled from chl29 , and therefore , the intensity 
 of emission from this state goes down . another important temperature - dependent 
 path is that from domain 6 in cp43 to domain 1 including the pd , showing 
 an inverse rate constant of 52 ps at 77 k and 330 ps at 5 k. the reverse 
 transfer rate constant through this path does not change so much upon 
 lowering temperature . thus , the equilibrium between cp43 and the rc 
 drastically shifts toward the former at 5 k. since domain 6 is responsible 
 for f685 , the much slower transfer through this path at 5 k is relevant 
 to the longer lifetime and the increased intensity of f685 below 77 
 k. figure 9 shows das obtained by a global - fitting 
 analysis of the present experimental time - resolved fluorescence data . 
 at 77 k , a das component with a time constant of 87 ps shows a negative 
 peak at around 695 nm , indicating an energy - transfer process to f695 . 
 at 5 k , the only component with a negative amplitude at 695 nm in 
 the das has a lifetime of 630 ps . this behavior is well reflected 
 by the transfer times between domain 4 and chl29 in cp47 ( figure 8) , which slows down from 80 ps at 77 k to 550 ps 
 at 5 k. the maximum of the das component with the largest time constant 
 ( ranging from 1.2 ns at 180 k to 2.9 ns at 5 k ) between 180 and 77 
 k shifts to longer wavelengths and , when temperature is further decreased , 
 gradually shifts back to shorter wavelengths . whereas the red shift 
 between 180 and 77 k reflects the effective decoupling of cp47 from 
 the rc , the subsequent blue shift results from the decoupling of cp43 
 from the rc and the decoupling of chl29 from the remaining pigments 
 in cp47 . das calculated by a global multiexponential analysis of the fluorescence 
 decay curves of ps ii - cc at ( a ) 5 k , ( b ) 36 k , ( c ) 77 k , and ( d ) 180 
 k. the estimated time constant of each das is specified in the figure . as shown in figure 8 , c695 is 
 located at the peripheral region of ps 
 ii - cc . this might suggest a function of c695 as an energy sink at 
 the interfacial region between the peripheral and core antennae . at 
 first glance it seems not to be the optimal solution to place an energy 
 sink at the periphery of a light - harvesting complex far away from 
 the rc . however , when considering the whole photosystem ii including 
 also the peripheral light - harvesting complexes cp26 , cp29 , and lhc - ii , it becomes obvious that chl29 might act as an 
 entrance for excitation energy from cp29 . hence , excitation energy 
 from the peripheral antennae might be efficiently focused on c695 
 due to its low - lying energy . the focusing function of c695 is effective 
 especially for higher - plant ps ii supercomplexes , in which the core 
 and peripheral antennae are rather isoenergetic . caffari et al . , based on their single - particle electron microscopy 
 study , concluded that chl11 in domain 2 of cp47 might act as a linker 
 between cp29 and cp47 . the close proximity between this pigment and 
 chl29 would indeed allow for efficient energy transfer between the 
 two complexes . another reason for the location of the energy 
 sink of cp47 at chl29 might be a possible photoprotective function 
 of this pigment . in 1998 , schweitzer et al . have reported a fluorescence quenching effect in ps ii induced by 
 an addition of potassium hexachloroiridate . the primary candidate 
 for the quencher was assigned to the oxidized form of chlz . it is well - known that chl cations exhibit a red - shifted absorbance 
 compared to the singlet ground state of chl . most likely , a -carotene ( card2 ) in the d2-branch 
 of the rc mediates electron transfer between chlzd2 and 
 the oxidized special pair in the rc . it is interesting 
 to note that , based on an analysis of optical difference spectra involving 
 reduced quinone qa and oxidized -carotene 
 in the rc , it can be excluded that the -carotene of the d1-branch 
 is involved in secondary electron transfer . the exclusive use of card2 , therefore , generates cationic 
 states predominantly in the cp47 subunit , which is connected to the 
 d2-branch of the rc . it seems not unlikely that the low - energy excitons 
 that accumulate at chl29 in cp47 are quenched by those cationic states : so far , photoprotective quenching within the core complex of ps 
 ii has been considered not to be a major contributor of the nonphotochemical 
 quenching ( npq ) . this conclusion is based on early experiments using 
 chl - b less mutant , which revealed that substantial 
 suppression of lhc ii resulted in suppression of npq . on the other hand , miyake et al . recently suggested a possibility that in a lichen - symbiotic green 
 alga under drought stress , an exciton quencher emerges and functions 
 within the ps ii core antenna in addition to those within peripheral 
 antennae to avoid unexpected charge separations at pd . the present 
 study implies a potential ability of chl29 to play a role as a core - antenna 
 exciton quencher in emergency , although we admit that a direct proof 
 of this hypothesis is still missing and will be an important future 
 goal . as shown in figure 6 , the experimental fluorescence 
 dynamics at 77 and 180 k contains a slowly decaying component at around 
 685 nm , which is absent in the simulated dynamics . there have been 
 several papers that reported slow fluorescence decay components in 
 ps ii similar to those observed in the present 
 study . these earlier studies concluded that the slow decay components 
 reflect the emission from the excited states induced by the charge 
 recombination at pd . such a charge - recombination - induced fluorescence 
 seems unlikely because the large energy gap of the charge recombination 
 is unsurpassable at low temperatures . this apparent inconsistency 
 was solved by inferring a suppression of the relaxation of the charge - separated 
 state , which results in much reduced free energy gap at low temperature . in the present simulations we have not considered these effects , 
 which would , however , not change our explanation of the inverse temperature 
 dependence of fluorescence . the introduction 
 of exciton domains in the present study is based on qualitative arguments 
 concerning the relative strength of excitonic and exciton - vibrational 
 coupling . an explicit treatment of dynamic localization effects of 
 excitons by the exciton - vibrational coupling would be desirable for 
 a more accurate description of exciton transfer . nonperturbative theories 
 have been developed in recent years ( as discussed , eg . , in renger 
 and mh ) that , in principle , could 
 be used for this purpose . however , so far these approaches are numerically 
 too expensive for a system as large as ps ii - cc . an important future 
 goal will be to develop more efficient nonperturbative calculation 
 schemes . we believe that the present treatment will still be valid 
 to describe the overall behavior of the fluorescence dynamics . another 
 open point concerns the temperature dependence of site energies , which 
 was neglected , or in a very few cases inferred from the fit of optical 
 spectra . close inspection of the absorbance 
 spectrum of ps ii - cc in figure 5 suggests that 
 the deviation between theory and experiment is somewhat larger above 
 150 k. one reason could be a temperature - dependent shift of site energies , 
 e.g. , due to temperature - dependent protonation patterns in the protein 
 interior . the enhanced conformational fluctuation above the glass 
 transition point might also contribute to such a shift . further development concerning the structure - based calculation of 
 site energies are required to clarify this point . in the present work , time- and temperature - dependent fluorescence 
 spectroscopy has been combined with theory to investigate the nature 
 and connectivity of low - energy excited states in ps ii - cc . the inverse 
 temperature shift of the fluorescence band observed between 5 and 
 77 k reflects the different nature of low - energy excited states in 
 the cp43 and cp47 core antennae . whereas in cp43 these states are 
 part of a large exciton domain that is well connected to the rc , in 
 cp47 a single pigment far away from the rc is responsible for the 
 fluorescence at low temperature . whereas only small thermal activation 
 is required to connect the low - energy excited states of cp43 with 
 the rc giving rise to fluorescence quenching by primary electron transfer , 
 the low - energy excited state , localized on chl29 in cp47 , stays disconnected 
 up to 77 k , thus determining the fluorescence maximum of ps ii - cc 
 at this temperature . the existence of such low - energy excited states 
 which are not well connected with the rc seems to be in obvious contradiction 
 to an efficient excitation energy funnel that is commonly expected 
 in photosynthesis . obviously getting enough excitons into the rc is 
 not the only demand of nature in ps ii . for closed rcs , because of 
 the much diminished primary electron - transfer rate , excitons have 
 a high probability to leave the rc back to the antennae . unwanted 
 secondary electron transfer is known to proceed along the d2-branch , 
 most likely involving card2 , chlzd2 , and cytb559 , 
 on the cp47 side of the rc . we suggest that singlet excited states 
 on chl29 in cp47 may be quenched by these cationic states , thus protecting 
 the rc from being overloaded with excitation energy . hence , the requirement 
 for photoprotection might be one reason that has led to the present 
 unconventional excitation energy landscape in ps ii - cc . in addition , 
 chl29 most likely acts as a focusing point for excitation energy arriving 
 from the peripheral antenna .
the experimentally 
 obtained time - resolved fluorescence spectra 
 of photosystem ii ( ps ii ) core complexes , purified from a thermophilic 
 cyanobacterium thermosynechococcus vulcanus , at 5180 k are compared with simulations . 
 dynamic localization 
 effects of excitons are treated implicitly by introducing exciton 
 domains of strongly coupled pigments . 
 exciton relaxations within a 
 domain and exciton transfers between domains are treated on the basis 
 of redfield theory and generalized frster theory , respectively . 
 the excitonic couplings between the pigments are calculated by a quantum 
 chemical / electrostatic method ( poisson - tresp ) . 
 starting with previously 
 published values , a refined set of site energies of the pigments is 
 obtained through optimization cycles of the fits of stationary optical 
 spectra of ps ii . 
 satisfactorily agreement between the experimental 
 and simulated spectra is obtained for the absorption spectrum including 
 its temperature dependence and the linear dichroism spectrum of ps 
 ii core complexes ( ps ii - cc ) . 
 furthermore , the refined site energies 
 well reproduce the temperature dependence of the time - resolved fluorescence 
 spectrum of ps ii - cc , which is characterized by the emergence of a 
 695 nm fluorescence peak upon cooling down to 77 k and the decrease 
 of its relative intensity upon further cooling below 77 k. the blue 
 shift of the fluorescence band upon cooling below 77 k is explained 
 by the existence of two red - shifted chlorophyll pools emitting at 
 around 685 and 695 nm . 
 the former pool is assigned to chl45 or chl43 
 in cp43 ( chl numbering according to the nomenclature of loll et al . 
 nature2005 , 438 , 1040 ) while 
 the latter is assigned to chl29 in cp47 . 
 the 695 nm emitting chlorophyll 
 is suggested to attract excitations from the peripheral light - harvesting 
 complexes and might also be involved in photoprotection .