Journal of Photochemistry and Photobiology B: Biology 104 (2011) 126–141

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Journal of Photochemistry and Photobiology B: Biology

journal homepage: www.elsevier .com/locate / jphotobiol

Short Review

Optical properties, excitation energy and primary charge transferin photosystem II: Theory meets experiment

Thomas Renger a,⇑, Eberhard Schlodder b,⇑a Institut für Theoretische Physik, Johannes Kepler Universität, Abteilung Theoretische Biophysik, Austriab Technische Universität Berlin, Max-Volmer-Laboratorium für Biophysikalische Chemie, Germany

a r t i c l e i n f o a b s t r a c t

Article history:Available online 7 April 2011

Keywords:ExcitonsCP43 complexCP47 complexD1D2cytb559 complexPhotosynthetic reaction center

1011-1344/$ - see front matter � 2011 Elsevier B.V. Adoi:10.1016/j.jphotobiol.2011.03.016

⇑ Corresponding authors.E-mail addresses: thomas.renger@jku.at (T. Re

tu-berlin.de (E. Schlodder).

In this review we discuss structure–function relationships of the core complex of photosystem II, asuncovered from analysis of optical spectra of the complex and its subunits. Based on descriptions of opti-cal difference spectra including site directed mutagenesis we propose a revision of the multimer model ofthe symmetrically arranged reaction center pigments, described by an asymmetric exciton Hamiltonian.Evidence is provided for the location of the triplet state, the identity of the primary electron donor, thelocalization of the cation and the secondary electron transfer pathway in the reaction center. We also dis-cuss the stationary and time-dependent optical properties of the CP43 and CP47 subunits and the exci-tation energy transfer and trapping-by-charge-transfer kinetics in the core complex.

� 2011 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1262. Optical properties of the PSII reaction center. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128

2.1. The multimer model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1282.2. The asymmetric exciton model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1292.3. The asymmetric exciton model with inclusion of a low-energy charge transfer state . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131

3. Electron transfer in the PSII RC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131

3.1. The primary electron donor and the time constant of primary charge separation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1313.2. Localization of the cation in the state Pþ680 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1323.3. Pathways of secondary electron transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133

4. Triplet localization and quenching mechanism in PSII RCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1345. Optical properties of the CP43 and CP47 core antennae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1346. Excitation energy transfer and trapping in PSII core complexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1387. Challenging questions and hypotheses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139

7.1. How fast is the primary charge transfer and what is the mechanism? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1397.2. Structure-based calculation of site energies and short-range effects in the ‘‘special pair’’. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139

8. Functional implications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140

1. Introduction

Photosystem II (PSII) captures solar energy by a large antennasystem consisting of chlorophylls (Chls) and carotenoids and trans-

ll rights reserved.

nger), eberhard.schlodder@

fers the energy to the reaction center of the complex, where theexcitation energy is used to create a charge separated state. Theoxidized donor of PSII is a very strong oxidant capable of drivingthe oxidation of water to O2. PSII of higher plants and algae consistsof the PSII core complex and peripheral light-harvesting com-plexes. Cyanobacteria contain only the PSII core which is composedof four large subunits (D1, D2, CP43, CP47), Cytb559 constitutedby subunits PsbE and PsbF, and several low molecular weight

T. Renger, E. Schlodder / Journal of Photochemistry and Photobiology B: Biology 104 (2011) 126–141 127

membrane-intrinsic subunits. At the lumenal side of the complexthree membrane extrinsic subunits are located. The structure ofPSII from Thermosynechococcus elongatus has been determined byX-ray crystallographic analysis to a resolution of 2.9–3.5 Å [1–3].Very recently, the resolution has been improved from 2.9 Å to1.9 Å as reported by Shen et al. on the 15th International Congressof Photosynthesis in Beijing. This improvement offers a significantamount of structural details that could not be reliably determinedbefore. The published X-ray structure of dimeric PSII at a resolutionof 2.9 Å [3], identifies 36 transmembrane helices and nearly 100cofactors which include: 35 Chla, 12 b-carotene, 2 pheophytin a(Pheoa), one b-type (Cytb559) and one c-type heme (Cytc550), 2plastoquinones (QA and QB), a non-heme-iron, the Mn4Ca-clusterresponsible for water oxidation and 25 integral lipids. CP43 andCP47 bind 13 and 16 Chla, respectively (see Fig. 1). The D1 andD2 protein subunits coordinate the cofactors involved in thelight-induced electron transfer reactions (see Figs. 1 and 2). Thecentral part of the RC contains four Chla and two Pheoa. Two trans-membrane branches of cofactors related by pseudo-C2 symmetryconnect the dimer PD1–PD2 and the plastoquinones (see Fig. 2).Each branch is composed of the so-called accessory chlorophyll(denoted as ChlD1 and ChlD2 in Ref. [3]), Pheoa (PheoD1 and PheoD2)and one plastoquinone (QA and QB, respectively). The dimer of twochlorophyll a molecules, PD1 and PD2, located on the lumenal side is

D1−D2 CP47CP43

Fig. 1. Chlorophyll containing subunits CP43, D1–D2, and CP47 of PSII corecomplexes in T. elongatus [3]. The transmembrane helices are shown as cylindersand colored in purple (CP43), yellow (D1), orange (D2) and red (CP47). The Chl’s(and the two Pheo’s) are shown in green, their phytyl chains have been truncatedfor clarity. A detailed arrangement of cofactors in the D1–D2 subunit containing theRC is shown in Fig. 2. (For interpretation of the references to color in this figurelegend, the reader is referred to the web version of this article.)

PheoD1

ChlD1

PD1 PD2

QA BQ

PheoD2

ChlzD1ChlzD2

TyrZ

Car

Chl

OEC

CarD2

D1

D2

Fe

Fig. 2. Cofactor arrangement in the central D1–D2 heterodimer of PSII corecomplexes of T. elongatus [3]. The Chl’s are colored in green, Car’s in red, Pheo’s inyellow, plastoquinones in black, TyrZ in blue. The Mn4Ca cluster of the oxygenevolving complex (OEC) is shown as purple spheres and the non-heme iron (Fe) asorange sphere. The substituents of the macrocycles of the Chl’s and Pheo’s havebeen truncated for clarity. (For interpretation of the references to color in this figurelegend, the reader is referred to the web version of this article.)

often referred to as P680. Originally, P stood for ‘‘pigment’’ and 680is the wavelength of the maximum bleaching in the red uponoxidation of this pigment at room temperature as a result of thelight-induced charge separation in the reaction center [4]. The rea-sonable agreement between the absorbance difference spectrum forthe oxidation of P680 and that for the oxidation of monomeric Chlahas been taken as evidence that P680 is a chlorophyll a molecule [4].A word of caution is necessary concerning the term P680. The spec-troscopic term P680 that is generally used in the literature no longerindicates the same entity in its cationic and singlet and triplet ex-cited form but different subsets of the six innermost pigments ofthe RC [5]. In this review, the lumenal dimer of Chla molecules willbe termed PD1–PD2, or ‘‘special pair’’, solely based on the structuralmodel in reference to the special pair PA–PB of purple bacteria(Figs. 3 and 4). The structural identities of the primary donor (spec-troscopic term P�680), the oxidized donor (spectroscopic term Pþ680)and the reaction center triplet (spectroscopic term 3P680) of PSIIreaction centers will be discussed in detail in this review. The PSIIreaction center is of the same type as that of purple bacteria.Figs. 2–4 exhibit the high structural similarity between thearrangements of pigments. There is an almost perfect overlay ofChlD1–PheoD1 with its counterpart BA–HA in purple bacteria (leftpart of Fig. 4). A remarkable difference exists between the two spe-cial-pair structures (right part of Fig. 4). Whereas the ring systemsof the two bacteriochlorophylls constituting the dimer PA–PB in thecase of purple bacteria overlap with their pyrrole rings I, the ‘‘spe-cial pair’’ of the PSII RC is in a staggered rather than an eclipsedconformation. It seems that a slight tilt of the two ‘‘special pair’’Chls with respect to each other has disrupted the wavefunctionoverlap in PSII. This small tilt might have had large consequencesfor the optical and redox properties of the ‘‘special pair’’, as will

BB

QAQ B

HAHB

BA

PA PB

Car

Fe

Fig. 3. Cofactor arrangement in the purple bacteria reaction center of R. sphaeroides[6]. The bacteriochlorophylls are colored in green, the carotenoid in red, bacteri-opheophytins in yellow, ubiquinones in black. The non-heme iron (Fe) is shown asorange sphere. The substituents of the macrocycles of the bacteriochlorophylls andbacteriopheophytins have been truncated for clarity. (For interpretation of thereferences to color in this figure legend, the reader is referred to the web version ofthis article.)

HA

BA

Chl D1

PheoD1

D1P D2P

PBPA

Fig. 4. Left part: overlay of accessory (bacterio-) chlorophylls and (bacterio-)pheophytins of the electron transfer active branch of PSII (grey) and the RC ofpurple bacteria (black). Right part: overlay of the special pair (bacterio-) chloro-phylls of PSII (grey) and the bacterial RC (black) [7].

128 T. Renger, E. Schlodder / Journal of Photochemistry and Photobiology B: Biology 104 (2011) 126–141

be discussed in detail further below (Section 7.1). Whereas theabsorbance spectrum of the RC of purple bacteria (bRC) consistsof separate bands that reflect the absorbance of the special pairPA–PB, the two accessory bacteriochlorophylls BA and BB and ofthe two bacteriopheophytins HA and HB of the two branches Aand B (e.g. [8]), the spectrum of D1D2cytb559 complexes contain-ing the RC of PSII [9,10] shows almost no structure even at 4 K (e.g.[11,12]). All optical bands strongly overlap. In this review we willdiscuss approaches to decipher the optical properties of PSII reac-tion centers despite this ‘‘spectral congestion’’ problem.

The following scheme of primary reactions was established forPSII by various spectroscopic techniques. After absorption of lightan electronically excited state of the reaction center chlorines isconverted into a short-lived charge separated state. The formationof the radical pair Pþ680Pheo�D1 occurs within about 50 ps. The kinet-ics of excitation energy transfer and light-induced primary chargeseparation in D1D2cytb559 complexes and intact PSII complexeshave been studied intensively by time-resolved absorption andfluorescence spectroscopy [13–17]. However, it is still a matter ofdebate which step is rate limiting: the transfer of the excitation en-ergy to the reaction center or the charge separation [7,13–15,18].Subsequent stabilization of charge separation is achieved by elec-tron transfer from PheoD1

� to QA in 200–500 ps. Pþ680 is reduced pri-marily in the nanosecond time range by the donor TyrZ, the redoxactive tyrosine 161 of the D1-polypeptide. The oxidized TyrZ is inturn reduced by the Mn4Ca-cluster. The manganese cluster storesthe oxidizing equivalents required for the oxidation of two watermolecules to molecular oxygen. In this way, the one-electron pho-tochemistry is connected to the overall four-electron process ofwater oxidation. At low temperatures (T < 100 K), electron transferfrom QA to QB and from TyrZ to P680+ is blocked and charge recom-bination from Pþ680Q�A occurs with a half-life time between 2 and5 ms [19,20]. The extent of Pþ680Q�A formation decreases progres-sively with successive flashes. This decrease can be explained byan electron transfer from secondary electron donors (Cytb559,Car, ChlZ) to Pþ680 that occurs with a low quantum yield in compe-tition with charge recombination of Pþ680Q�A. Such secondary elec-tron transfer leads to a progressive accumulation of long-livedcharge separated states (e.g. Car+QA

�) with increasing flash num-ber. The electric field caused by the negative charge located onQA� and the positive charge located on the secondary donor leads

to shifts of the transition energies of nearby pigments (Stark ef-fect). The formation of these long-lived states has been investi-gated in detail by absorbance difference spectroscopy andmagnetic resonance techniques (for review see [21]). The quantumefficiency of photo-induced oxidation of secondary electron donorsvary widely [22].

A characteristic electrochromic blue shift of the QX band ofPheoD1 is observed around 550 nm (the so-called C550 band shift[23]) and is an indicator of the formation of Q�A [24]. In the QY re-gion the assignment of the observed electrochromic band shifts iscomplicated by the strong overlap of the electronic transitions ofthe pigments in the PSII reaction center and by the stronger excitoncoupling in the QY region. Only recently, the absorbance differencespectra associated with the light-induced formation of these long-lived states in PSII core complexes could be described quantita-tively in the framework of an exciton model that we termasymmetric exciton model. The latter assumes that the mean siteenergies of the pigments in the two symmetric branches of theRC are different (see below and [7,5]). When the electron transferto the quinone acceptors is blocked by pre-reduction or by removalof the primary quinone acceptor QA, then the reaction center tripletstate 3P is formed, in high yield at low temperatures by chargerecombination of Pþ680Pheo�D1 following singlet–triplet mixing inthe radical pair state [25,26]. EPR studies with oriented PSII sam-ples have shown that the plane of the Chl which carries the triplet

state is oriented like that of the accessory chlorophylls [27], i.e., thetriplet state is localized on one of the accessory chlorophylls, mostprobably on ChlD1 [28]. Although the overall reaction scheme isclear, the molecular identities of some of the functional statesand the mechanistic and kinetic details are not. This review dis-cusses recent work combining optical spectroscopy, site-directedmutagenesis and theory that enables to elucidate the electronic(excitonic) states of the PSII reaction center chlorines, in particular,the nature of the excited state that drives the primary charge sep-aration and to answer the question where the cation radical of theprimary donor and the triplet state formed by charge recombina-tion of the primary radical pair are localized. The next part of thisreview describes the current knowledge of optical spectra of theantenna subunits CP43 and CP47 and PSII core complexes. The lat-ter will be termed intact PSII complexes because they represent theminimal preparation unit retaining oxygen evolving capacity.

Finally, the structure-based deciphering of the spectral proper-ties offers the opportunity to calculate the excited state dynamicsbased on a minimal set of common parameters and to relate the re-sults with experimental data obtained by ultra-fast optical spec-troscopy. We include a discussion of PSII of the Chld-containingcyanobacterium Acaryochloris marina, since its RC most likely con-tains both Chla and Chld, which makes the interpretation of opticalspectra easier because the spectral overlap of the different bands isless than in the usual PSII that contains only Chla.

2. Optical properties of the PSII reaction center

2.1. The multimer model

To explain the marked difference in the optical properties of theRCs of purple bacteria and PSII, discussed in the introduction, itwas concluded [29,30] that the ‘‘special pair’’ in PSII has a weakerexcitonic coupling than in bRC such that all nearest neighbor cou-plings are of the same magnitude of about 100 cm�1, assuming alsoa similar overall arrangement of the pigments in the two RCs. Inthese so-called multimer models ([29,31–33]) the excited stateswhere found to be delocalized over several pigments, wherebyoscillator strength is redistributed towards the low-energy excitonstates. The assumption of high structural similarity between thearrangement of pigments in the two types of RC is supported byan early homology modeling computational study [30] and bythe early [34] and recent X-ray crystallographic studies [1–3](compare Figs. 2 and 3).

Originally [29,30] it was assumed that the smaller excitoniccoupling in the special pair of PSII is due to a larger interpigmentdistance between the two special pair chlorophylls. However, asshown in the right part of Fig. 4 it is rather a tilt than a translationthat distinguishes the two special pairs. A quantum chemical anal-ysis of the Coulomb and short-range couplings and site energyshifts revealed that the large excitonic coupling and the low siteenergy of the special pair in purple bacteria are caused by short-range electron exchange effects [35]. Therefore, most likely the dis-rupted overlap of p-electron wavefunctions in particular of onering of the macrocycles of the pigments, caused by the mutual inplane rotation of the pigments, is responsible for the smaller exci-tonic coupling and higher QY transition energy of the special pair inPSII. Except for the slight tilt in the special pair, there is a strikingsimilarity between the arrangement of cofactors in the two RCs.For example, there is perfect overlay between the macrocycles ofthe accessory (bacterio) chlorophylls and the (bacterio) pheophy-tins in the two RCs, as shown for the electron transfer active branchin the left part of Fig. 4. The functional implications of this similar-ity and the difference in the special pair will be discussed later indetail.

0

0.2

0.4

0.6

0.8

d M/m

(ω)

0

0.2

0.4

0.6

0.8

d M/m

(ω)

650 660 670 680 690

wavelength / nm

0

0.2

0.4

0.6

0.8

d M/m

( ω)

M = 1

M = 2

m = ChlD1m = Pheo

D2

M = 6M = 3

m = PD1

m = PD2

m = PheoD1

M = 4

m = ChlD2

M = 5

Fig. 5. The densities of exciton states dM(x), M = 1, . . ., 6 (black lines, Eq. (1)) arecompared with the exciton state pigment distribution functions dm(x) for thedifferent RC pigments (red lines, Eq. (2)). The exciton Hamiltonian suggested in ref.[40] was used. (For interpretation of the references to color in this figure legend, thereader is referred to the web version of this article.)

T. Renger, E. Schlodder / Journal of Photochemistry and Photobiology B: Biology 104 (2011) 126–141 129

The multimer models of PSII assume the same mean local tran-sition energies of the RC pigments. These so-called site energies aredefined as the transition energies at which the pigments would ab-sorb light in their local environment defined by the protein bindingsite for the hypothetical case of absent resonance energy transfer(excitonic) coupling between the different pigments. Roughlyspeaking, the positions and intensities of optical bands of the cou-pled pigments are obtained from the eigenenergies and eigenvec-tors of the exciton matrix that contains in the diagonal the siteenergies and in the off-diagonal the excitonic couplings. To takeinto account disorder introduced by slow (compared to the excitedstate lifetime of the pigments) conformational motion of the pro-tein, one has to diagonalize this matrix many times for differentrealizations of disorder in site energies.

Usually a Gaussian distribution function of a certain width is as-sumed for the site energies, correlations in this static disorder atdifferent sites are neglected and the spectra are averaged by aMonte Carlo procedure (e.g. [36]). Protein dynamics which are fastcompared to the excited state life times of the pigments lead tohomogeneous broadening and to additional shifts of the opticalbands. These effects are taken into account by dynamical theoriesas reviewed elsewhere (e.g. [36,37]). If the same mean site energiesare assumed, as in the multimer models, the degree of delocaliza-tion and the contribution of the pigments in the different delocal-ized (exciton) states depends strongly on the particular realizationof disorder. In all multimer models a large contribution of the ‘‘spe-cial pair’’ in the lowest exciton state was found [29,32,33,38,39].We note that this result also holds for the pentamer model intro-duced by Jankowiak et al. [38,39]. In this model, which considereddifferent site energies of the pigments, the pheophytin of the D2-branch of the reaction center is assumed (i) to be decoupled fromthe other pigments and (ii) to absorb at 668 nm [38,39]. These con-clusions were based on optical difference spectra involving sodiumdithionite treatment that was believed to reduce the pheophytin ofthe D2-branch. However, the assumptions of the pentamer modelare in conflict with the crystal structure of PSII [3] and the ex-change experiments by Germano et al. [12].

2.2. The asymmetric exciton model

Raszewski et al. [40] revised the multimer model by assumingdifferent mean site energies of the pigments and determining theseenergies from a fit of the linear absorbance, linear dichroism, fluo-rescence and circular dichroism spectra of D1D2cytb559 com-plexes. Afterwards the site energies were tested in the simulationof various optical difference spectra of D1D2cytb559 complexesinvolving chemically modified, oxidized and reduced pigmentsand pigments in the triplet state [40]. Blue-shifted site energieswere obtained for the ‘‘special pair’’ and a red-shifted site energyfor the accessory chlorophyll of the D1-branch (ChlD1). In Fig. 5the resulting density of exciton states (probability that the transi-tion into the exciton state M occurs at the indicated wavelength)

dMðxÞ ¼ hdðx�xMÞidis ð1Þ

where �hxM is the transition energy between the ground state andexciton state M and h. . .idis denotes an average over disorder in siteenergies, is compared with the exciton state pigment distributionfunction

dmðxÞ ¼X

M

cðMÞm

�� ��2dðx�xMÞ* +

dis

ð2Þ

that contains the probabilities cðMÞm

��� ���2 to find pigment m excited inexciton state M. As seen in Fig. 5, the lowest exciton state (M = 1)has a large contribution from ChlD1, the second lowest exciton state(M = 2) is dominated by the pheophytin of the D2-branch (PheoD2)

and only the third lowest exciton state (M = 3) contains the excitedstates of the ‘‘special pair’’ PD1–PD2 which are delocalized over thetwo ‘‘special pair’’ pigments. Because of the large difference be-tween the site energy of ChlD1 and its symmetry partner ChlD2 ofthe other branch, we term the present model asymmetric excitonmodel. The difference in site energies leads to partial localizationof the exciton states that is seen in Fig. 5 by the dominant contribu-tion of the different pigments m to only one exciton state M, exceptfor the special pair. Nevertheless, shoulders of dm(x) appearing atadditional exciton states M show that the excited states are notcompletely localized, i.e., exciton effects are present.

The rather harsh biochemical treatment of PSII that is necessaryto isolate D1D2cytb559 complexes from the PSII core complex hasraised the question about the influence of the isolation procedureon the optical and redox properties of the RC pigments (e.g.[41,42]). A difficulty in the interpretation of experiments on PSIIcore complexes is the overlap of optical bands of the RC pigmentswith those of the antenna subunits. One way to overcome thisproblem is to measure optical difference spectra of core complexeswhere the primary and secondary electron transfer reactions wereused to convert particular RC pigments into a different electronicstate. Due to the difference, these spectra display the propertiesof the pigment that has undergone a change in electronic stateand of those pigments which are coupled to it. As the couplings be-tween RC pigments and the pigments bound to CP43 and CP47 areweak, the difference spectra reveal direct information about the RCpigments without interference from the antenna pigments. Thereis, of course, still the problem of overlapping optical bands of thepigments (exciton transitions) in the RC. The latter can be solved

wavelength / nm

-2

-1

0

1

2

Δ O

D /

a.u.

wildtypeP

D1 666 -> 658 nm

wavelength / nm

-2

-1

0

1

2

ΔOD

/ a.

u.

wildtypeChl

D1 680 -> 683 nm

ChlD1

680 -> 678 nm

660 670 680 690-1.5

-1

-0.5

0

0.5

1

ΔO

D /

a.u.

wildtypeD1-His198Gln

660 670 680 690660 670 680 690

660 670 680 690-1.5

-1

-0.5

0

0.5

1

ΔOD

/ a.

u.

wildtypeD1-Thr179HisD1-Thr179Glu

experiments

calculations

Fig. 6. Pþ680Q�A � P680QA spectra of wild-type and mutant PSII core complexes from Synechocystis sp. PCC 6803 measured at 80 K [43,28] (upper part) are compared withcalculated spectra [7] (lower part). In the calculations of the mutant spectra, the site energy of the pigment affected by the mutation was shifted with respect to its wild-typevalue as indicated in the legend, and the cation was assumed to be localized at PD1.

130 T. Renger, E. Schlodder / Journal of Photochemistry and Photobiology B: Biology 104 (2011) 126–141

by combining low-temperature optical difference spectroscopywith site-directed mutagenesis, in which the amino acid residuesin the local environment of certain RC pigments are replaced. If thisreplacement leads only to changes in the local transition energy ofthe nearest pigment, valuable information about the optical prop-erties of this pigment can be obtained.

Such experiments were performed by Diner et al. [43] on mu-tants of D1-His198, the axial ligand of PD1 and by Schlodder et al.[28] on mutants of D1-Thr179, an amino acid residue nearest toChlD1. The experiments were carried out on PSII core complexesfrom Synechocystis sp. PCC 6803. Important conclusions could bedrawn about the identity of functional states at low temperatures,as discussed in detail below.

The P+QA� � PQA absorbance difference spectra of the wild-type

and the mutants are compared in Fig. 6 with calculated spectra,using the exciton Hamiltonian of Raszewski et al. [40] discussedabove. The only adjustment of the D1D2cytb559 Hamiltoniannecessary to reach agreement with the experimental data on thewild-type is a 4 nm shift of the site energy of ChlD1 to even longerwavelengths, i.e. lower energies. In addition, the static disorder ofthis pigment had to be reduced from 180 cm�1 (D1D2cytb559) to120 cm�1 (core complex) for a quantitative fit of the experimentalspectra of core complexes [7].

In the calculations of the mutant spectra the site energy of thepigment nearest to the amino acid replaced by the site-directedmutation was allowed to vary. The very nice agreement betweenthe calculations and the experiments provides evidence (i) thatthe mutation investigated in the experiments induced indeed onlychanges in the site energy of the nearest pigment, and (ii) thatD1D2cytb559 complexes provide a valid model system of the RCin PSII core complexes, as concerns the prominent optical proper-ties. An independent check of the model parameters was obtainedfrom triplet-minus-singlet (T–S) difference spectra of the sametype of mutants, which could be explained theoretically by usingthe shifts in site energy values for the mutants deduced from theP+QA

� � PQA difference spectra discussed above [7].

Another point we want to raise about the optical properties ofRC pigments is about the relevance of low-temperature experi-ments for obtaining physiologically relevant information. There isa remarkable change of several difference spectra with increasingtemperature [44]. These spectra, which contain a multiple bandstructure at low temperature , at room temperature all exhibit justa single bleaching at 680 nm. The overlap of different bands atroom temperature, of course, is an obstacle in the interpretation ofexperiments. So the question arises, whether the primary reactionsdetected at low temperature are the same as those at high T.As shown in Fig. 7, the temperature dependence of thePþ680Pheo� � P680Pheo and the Pþ680Q�A � P680Q A spectra can be

modelled by assuming the same identity of functional states at dif-ferent temperatures and taking into account the temperaturedependence of the dielectric constant leading to a stronger screen-ing of the Coulomb interaction at higher temperature and therebyto smaller electrochromic shifts in site energies. The increase of �eff

reflects the higher flexibility of polar side chains above the glasstransition temperature (200 K). Similar room temperature valuesof �eff were inferred from Stark shift measurements on photosys-tem I [45] and on an a-helix [46]. In addition, a slight shift in siteenergies of PD1 and ChlD1 had to be assumed for a quantitative fitof the data. This shift, however, does not alter the exciton modelin that still the lowest exciton state is localized at ChlD1. In the caseof the T–S spectrum, more than one triplet state contributes at hightemperature, as discussed in detail below. The origin of the site en-ergy shift could be a change in the protonation pattern of the pro-tein, or a temperature-dependent mixing of the excited states withcharge transfer states, as in the reaction center of purple bacteria[47].

The set of site energies inferred in the asymmetric exciton mod-el by Raszewski et al. [40,7] was confirmed by calculations of linearoptical spectra and Stark spectra and comparison with experimen-tal data by Novoderezhkin et al. [48]. The latter model explicitly in-cluded mixing of exciton states with a low energy charge transferstate as will be discussed in more detail in the following.

-1

-0.5

0

Δ O

D /

a.u.

PD1

+Pheo

D1

--P

D1Pheo

D1

-0.5

0

0.5

PD1

+Q

A

--P

D1Q

A

-0.5

0

Δ O

D /

a.u.

-0.5

0

670 680 690

wavelength / nm

-0.5

0

Δ O

D /

a.u.

670 680 690

wavelength / nm

-0.5

0

25 K 5 K

77 K 170 K

666/682 ε

eff = 2

666/679ε

eff = 3666/682

εeff

= 3

666/682ε

eff = 2

300 K300 K

670/679ε

eff = 8

670/679ε

eff = 8

Fig. 7. Temperature dependence of the Pþ680 Pheo� � P680Pheo (left half) andPþ680 Q�A � P680QA (right half) spectra of T. elongatus. The calculations [7] (solidlines) are compared with experimental data [44] (solid circles). The temperature-dependent wavelengths corresponding to the site energies of PD1 and ChlD1 ( PD1/ChlD1) and the dielectric constant �eff used in the calculation of electrochromic shiftsare shown as well at each temperature.

T. Renger, E. Schlodder / Journal of Photochemistry and Photobiology B: Biology 104 (2011) 126–141 131

2.3. The asymmetric exciton model with inclusion of a low-energycharge transfer state

From the 2 nm redshift of the Stark spectrum of theD1D2cytb559 complex with respect to the second derivative ofits absorbance spectrum, Frese et al. [49] concluded that the low-energy exciton state of PSII RC is mixed with a charge transfer(CT) state and suggested the state ChlD1

+PheoD1� as a possible can-

didate. It is, however, not obvious, whether the relatively large spa-tial separation between ChlD1 and PheoD1 allows for sufficientwavefunction overlap for such a mixing.

The first quantitative simulation of the Stark spectrum was pro-vided by Novoderezhkin et al. [48], who included a low-energycharge transfer state explicitly in the exciton Hamiltonian and cal-culated the spectra in the framework of modified Redfield theory.The calculations showed that the 2 nm redshift discussed aboveis in fact an exciton effect, not related to the CT state. The influencereported of the latter is an enhancement of the red part of the Starkspectrum. By varying local transition energies including that of theCT state and also the identity of the CT state and comparison withexperimental data (eight different types of linear spectra and theStark spectrum) they arrived at similar site energies for the RC pig-ments as Raszewski et al. [40] and concluded that the most likelycandidate for the CT state is the intra special pair state PD1

+PD2�

[48]. Based on this finding, it was suggested that primary charge

transfer in photosystem II may start either from the special pairor from ChlD1, depending on the specific realization of disorder thatdetermines the nature of the low-energy exciton states from whereelectron transfer starts. As will be discussed in Section 7, it is nottrivial to include a charge transfer state explicitly in the excitontheory. Therefore, the approach by Novoderezhkin et al. [48] repre-sents an important first step but an extension of the theory, includ-ing dynamic localization effects, is needed to draw definiteconclusions about the nature of the CT states in the RC of PSII.The idea of a low-energy charge transfer state in PSII is in line withthe finding of Krausz and coworkers [50,51] who detected a long-wavelength homogeneously broadened excited state of the RC thatis capable of charge separation. The nature of this state is, however,different from that of the CT state proposed by Novoderezhkinet al. [48]. Krausz et al. [42] reported the absence of CT spectral fea-tures in D1D2cytb559 complexes and suggested that the absorp-tion feature in the red region, interpreted as due to CT intensity,is in fact pigment hot band absorption in a 77 K spectrum utilizedfor the model. We will discuss this unresolved topic further inSection 7 and concentrate in the following on models of primaryelectron transfer in photosystem II that were inferred from time-resolved experiments.

3. Electron transfer in the PSII RC

3.1. The primary electron donor and the time constant of primarycharge separation

van Grondelle and collaborators [13] and Holzwarth and collab-orators [15] inferred independently from femtosecond VIS-pump/IR-probe studies and VIS-pump/VIS-probe experiments, respec-tively, that the primary electron transfer at room temperature oc-curs between ChlD1 and PheoD1. However, the reported timescalefor Pheo reduction differs by a factor of 4–5. Whereas Groot et al.[13] found a 600–800 fs time constant, that of Holzwarth et al.[15] is 3 ps. Taking into account the 30% equilibrium populationof ChlD1 at T = 300 K (Fig. 8), these times correspond to intrinsictime constants for primary electron transfer of 200 fs and 1 ps,respectively. The faster of the two time constants is supportedfrom calculations of excitation energy transfer and trapping, as willdiscussed in detail below.

It is interesting to note also that a fast subpicosecond time con-stant for electron transfer was found by van Brederode and vanGrondelle et al. [52,53] for electron transfer between the accessorybacteriochlorophyll BA and the accessory pheophytin HA in the RCof purple bacteria, which is an order of magnitude faster than the3 ps time constant for electron transfer starting at the special pair.However the strong red shift of the low-energy special pair excitedstate in the bacterial RC makes it the primary electron donor, sincethe excited state of BA could not accept the excitation energy fromthe LH1 core antenna which absorbs resonant with the special pair.The fact that the excited states in PSII RC are much closer in energyled van Grondelle and van Brederode [54] to suggest that the fastside pathway in bacterial RCs might be the dominant one in PSII.This idea became more and more alive during the last ten years.But a final proof is still missing. The PSII RC of Chld containing A.marina, discussed below, seems to be a nice model system to solvethis problem.

As noted already above, an alternative suggestion of van Gron-delle and coworkers [48,16] is the existence of two parallel elec-tron transfer pathways, one starting at ChlD1 and one at PD1,where the relative weight is determined by the particular realiza-tion of disorder that determines the equilibrium populations of ex-cited pigment states. Recent time-resolved pump-probe data [16],interpreted within this model, were used to infer the respective

PD1

PD2

ChlD1

ChlD2

PheoD1

PheoD2

0

20

40

60

80

P m

(eq)

/ %

PD1

PD2 Chl

D1Chl

D2Pheo

D1Pheo

D2

0

10

20

30

Pm

(eq)

/ %

5 K

300 K

Fig. 8. Equilibrated population of local excited states PðeqÞm (Eq. (5)) at T = 5 K (upper

part) and T = 300 K (lower part). The exciton Hamiltonian of Raszewski et al. [40,7]was used for the calculations.

132 T. Renger, E. Schlodder / Journal of Photochemistry and Photobiology B: Biology 104 (2011) 126–141

time constants for electron transfer along the two paths. Electrontransfer with ChlD1 as primary donor was reported to occur withan apparent time constant of 3 ps as suggested before by Holz-warth et al. [15] (see above), whereas the faster charge transfertime of 1 ps is attributed to electron transfer starting at the specialpair. However, a detailed theoretical analysis of the data withmodified Redfield theory assigns the faster time constant againto electron transfer starting at ChlD1 [55]. Myers et al. [17]measured two-dimensional electronic spectra of D1D2cytb559complexes at 77 K and concluded that the primary charge transfertime is distributed in the 1–3 ps time range. From femtosecondpump-probe spectra of D1D2cytb559 complexes measured at roomtemperature, Shelaev et al. [56] concluded that primary chargetransfer starts at the special pair with a time constant of 0.9 ps.However, their interpretation includes the assumption that ChlD1

absorbs at 670 nm, in contradiction to the asymmetric excitonmodel discussed above.

The obvious confusion about the mechanism and kinetic detailsof primary electron transfer in PSII is caused by the strong overlapof optical bands. In the following, we want to investigate whetherthe energetics of excited states predicted by the asymmetric exci-ton model can give hints to solve this issue. The question of the pri-mary electron donor will be related to the energetics of the excitedstates giving rise to different equilibrium populations at differenttemperatures. Since exciton relaxation between the six core pig-ments in the RC is ultrafast [17], electron transfer starts from anequilibrated manifold of exciton states. In this case, the rate con-stant for primary electron transfer starting at pigment m can bewritten as [40,7]

kðETÞm ¼

XM

f ðMÞ cðMÞm

�� ��2kð intrÞm

* +dis

ð3Þ

where f(M) is the Boltzmann factor that expresses the equilibratedpopulation of exciton states, cðMÞm

��� ���2 is the probability to find the pri-mary electron donor m excited in exciton state M, and kðintrÞ

m is theintrinsic rate constant for electron transfer starting at the putativeprimary donor m. If, for simplicity, we assume that kðintrÞ

m does notdepend critically on disorder in site energies, the rate constantbecomes

kðETÞm ¼ PðeqÞ

m kðintrÞm ð4Þ

where the equilibrium population of the excited state of pigment mreads

PðeqÞm ¼

XM

f ðMÞ cðMÞm

�� ��2* +dis

: ð5Þ

In Fig. 8 this quantity is plotted for all the six core pigments m in theRC. The manifold of equilibrium populations PðeqÞ

m contains a contri-bution from the particular m of the primary electron donor of theRC.

We identify the whole manifold of equilibrium populations PðeqÞm

of the RC with the spectroscopic state PH

680, since it characterizesthe excited state of the RC prior to electron transfer. We see thatat low temperature , PH

680 is almost entirely formed by the excitedstate of ChlD1, whereas at room temperature the excited states ofall six pigments contribute to PH

680. Based on the above PðeqÞm it seems

clear that at low temperature primary electron transfer starts atChlD1, as it was concluded from mutant difference spectra [43]and from a comparison of the experimental and calculated temper-ature dependence of primary electron transfer [40]. However, atroom temperature electron transfer could in principle also startat the ‘‘special pair’’, as in the RC of purple bacteria, since thereis significant population of the excited states of PD1 and PD2 (Fig. 8).

Below, we will discuss a PSII RC that even at room temperatureexhibits strongly asymmetric equilibrium populations of excitedstates with a dominant contribution from ChlD1. This system,therefore, would be ideally suited to investigate the kinetic detailsof the electron transfer reaction starting at ChlD1.

Finally, we would like to mention that the charge separationmechanism and rate may be different in PSII core complexes andisolated D1D2cytb559 complexes containing only the reaction cen-ter pigments. Although the exciton Hamiltonians were found to bevery similar [40,7], the electron transfer couplings could be differ-ent because they depend exponentially on interpigment distancesand therefore are more sensitive to slight conformational differ-ences than the site energies and excitonic couplings. As discussedabove, a low lying CT state was only detected for core complexes[42]. Recently, evidence was reported for a low-energy charge sep-arating state in PSII at physiological temperatures [57].

3.2. Localization of the cation in the state Pþ680

After primary electron transfer, by subsequent hole transfer thecation may localize on a different pigment than the primary elec-tron donor. The low-temperature mutant spectra of Diner et al.[43] provided evidence for hole stabilization at the ‘‘special pair’’chlorophyll PD1. In agreement with this conclusion, calculationsof the wild-type P+QA

� � PQA spectra by Raszewski et al. [7] givebest agreement with the experimental data if the cation is assumedto be localized on PD1 (Fig. 9).

Is there any evidence about the identity of Pþ680 at physiologicaltemperatures? The calculation of the temperature dependence ofthe P+QA

� � PQA difference spectrum (Fig. 7) suggests that theidentity of the state Pþ680 ¼ PþD1 is the same at all temperatures.EPR studies at room temperature by Zech et al. [58] determineda distance of 27.4 ± 0.3 Å between the negative charge on QA

�

and the positive charge on Pþ680. Based on this distance it is not pos-

650 660 670 680 690 700 710 720 730 740 750wavelength / nm

-0.5

0

0.5

Δ O

D /

a.u.

experimentP

D1/P

D2 = Chla/Chld

PD1

/PD2

= Chla/Chla

Fig. 10. The experimental P+QA� � PQA difference spectrum of A. marina at 77 K

[65] is compared with calculations [66] assuming a Chla homodimer or a Chla/Chldhetero dimer in the ‘‘special pair’’.

PheoD1

(Pheoa)

ChlD1

(Chld)

PD1

(Chla)

PD2

(Chld)Chl

D2 (Chld)

PheoD2

(Pheoa)

0

0.2

0.4

0.6

0.8

prob

abili

ty

T = 300 K

Fig. 11. Equilibrium populations (Eq. (5)) of local excited states of RC pigments in A.marina

650 660 670 680 690 700

wavelength / nm

-0.5

0

0.5

ΔOD

/ a.

u.ExperimentP

D1P

D2Chl

D1

5 K

QA

D1P

Fig. 9. Calculation [7] of Pþ680Q�A � P680Q A difference spectrum using differentassumptions for the localization of the cation in the Pþ680 state in comparison withexperimental data [44]. The inset shows the pigments of the RC, where the Chlswere colored in accordance with the cation localization assumed in the calculations.(For interpretation of the references to color in this figure legend, the reader isreferred to the web version of this article.)

T. Renger, E. Schlodder / Journal of Photochemistry and Photobiology B: Biology 104 (2011) 126–141 133

sible to distinguish cation localization on PD1 from that on PD2.However, cation localization on ChlD1 or ChlD2 can be excluded aswell as a distribution of the cation over all four Chls. ENDOR[59,60] and FTIR [61] studies show a very asymmetric charge dis-tribution between PD1–PD2 indicating that the cation is stabilizedmainly on PD1, the chlorophyll adjacent to TyrZ.

As noted several times above, an interesting model system is gi-ven by PSII of A. marina, which exhibits less spectral overlap of itsoptical bands. Photosystem II of A. marina is unique since it is theonly PSII known so far that uses Chld instead of Chla as the majorlight harvesting pigment (for recent review see e.g. [62]). Thisreplacement allows this organism to live in the shade of Chla con-taining organisms which cannot use the long-wavelength light thatChld absorbs. Chld absorbs about 35 nm to the red of Chla [63].Interestingly, there is a residual amount of Chla bound in A. marina,where the exact amount was found to depend on light intensity[64].

Evidence for the location of one Chla in the binding site of PD1

was reported by Schlodder et al. [65] who compared the P+QA� � P-

QA difference spectra measured on A. marina with those of Chlacontaining PSII in the Soret region around 435 nm and in the NIRregion. Whereas the signal in the Soret region reflects the bleach-ing in absorbance of the pigment that carries the cation, the NIRsignal reflects directly the cation absorbance. Both signatures agreevery well between A. marina and Chla containing PSII, leaving nodoubt that the site where the cation localizes, most likely the PD1

site, is occupied by Chla in A. marina.Renger and Schlodder [66] used the Hamiltonian that Raszewski

et al. [40] determined for Chla containing PSII and modified it to al-low for a replacement of Chla with Chld. Different models from theliterature concerning the identities of the RC Chls were investi-gated. A quantitative description of the P+QA

� � PQA differencespectrum in the QY region requires one Chla at the binding siteof PD1 and three Chld at the sites of PD2, ChlD1 and ChlD2, besidesthe two Pheoa at the Pheo binding sites [66]. Assuming the bindingsites of both ‘‘special pair’’ pigments to be occupied by Chla resultsin significantly less agreement with the experimental data(Fig. 10). Analyzing the equilibrium population of locally excitedstates (Eq. (5)) in A. marina, using the site energies determinedfrom the fit of the PþQ�A � PQA spectrum, allows to identify theprimary electron donor as ChlD1, even at room temperature(Fig. 11). Comparison with the respective equilibrium populationsin Chla containing PSII at room temperature (lower part of Fig. 8)demonstrates that the ‘‘spectral congestion’’ problem is much less

in A. marina. We have therefore great hope that ultrafast time-re-solved spectroscopy on A. marina will improve our understandingof the primary reactions in PSII.

3.3. Pathways of secondary electron transfer

When electron transfer between TyrZ and Pþ680 is blocked at lowtemperatures, besides charge recombination reactions, secondaryelectron transfer occurs in PSII involving Cars, Chlz and Cytb559,for recent review see, e.g., ref. [21]. From calculations of Q�ACar+ � QA Car optical difference spectra and comparison withexperimental data, it can be concluded that only CarD2 and notCarD1 is involved in secondary electron transfer in PSII [28](Fig. 12). The linear dichroism of the absorbance band of the Carcation around 1000 nm shows that the oxidized Car is orientedparallel to the membrane plane [67] which supports the assign-ment that CarD2 is oxidized by Pþ680. The position of CarD2 in theRC (upper part of Fig. 12) suggests that it connects the core pig-ments with the more peripheral secondary electron donors ChlzD2

and Cyt b559. There has been some discussion about the origin ofthe low-energy bandshift seen in the Q�A Car+ � QA Car optical dif-ference spectra. Whereas initially an interpretation in terms of asite energy shift of PheoD1 was reported [68,69], the asymmetricexciton model [40] predicted that the Stark shift of ChlD1 is respon-sible for this feature. This prediction has been verified experimen-tally by Schlodder et al. [28] who found that the position of thelow-energy bleaching changes if a residue in the vicinity of ChlD1

650 660 670 680 690 700

wavelength / nm

-0.8

-0.6

-0.4

-0.2

0

Δ O

D /

a.u.

Exp.P

D1P

D2Chl

D1Chl

D2

Chl D1

AQ

Fig. 13. T–S spectra at 5 K calculated [7] assuming the triplet state to be localizedon the respective RC Chls in comparison with experimental data [44] on T.elongatus. The inset shows the RC pigments colored in accordance with the tripletlocalization assumed in the calculations. (For interpretation of the references tocolor in this figure legend, the reader is referred to the web version of this article.)

660 670 680 690

wavelength / nm

-40

-20

0

20

40

60

Δ O

D /

a.u.

experiment

QA

-Car

D1

+- Q

ACar

D1

QA

-Car

D2

+- Q

ACar

D2

Car D1

Car D2

QA

D1Chlz

Chlz D2

cytb559

Fig. 12. Upper Part: Pigments of the RC of cyanobacteria including the twocarotenoids CarD1 and CarD2, the two peripheral ChlzD1 and ChlzD2 and the Heme ofthe Cytb559 complex. The two Cars are colored in correspondence to the cationlocalization assumed in the calculations in the lower part. Lower part: experimentallight-minus-dark difference spectra of PSII core complexes of Synechocystis sp. PCC6803 at 5 K are compared with Q�A Car+ - QA Car spectra calculated assuming twodifferent identities for Car+ [28]. (For interpretation of the references to color in thisfigure legend, the reader is referred to the web version of this article.)

134 T. Renger, E. Schlodder / Journal of Photochemistry and Photobiology B: Biology 104 (2011) 126–141

is mutated. Krausz and coworkers [70] measured low-temperatureabsorption and circular dichroism difference spectra upon PheoD1

reduction in a range of PSII core complexes isolated from differentorganisms, as well as in D1D2cytb559 preparations. A simplifiedasymmetric exciton model, based on the Raszewski et al. Hamiltonianwas used to analyze the data. These data support independentlythe basic assignments of the model and also helped to moreaccurately determine the energy of the exciton state dominatedby PheoD2, which in some organisms was found to be lower thanthat of the exciton state dominated by ChlD1. In spinach both stateswere found to be close to isoenergetic.

4. Triplet localization and quenching mechanism in PSII RCs

Important information about the localization of the triplet statein PSII RCs came from EPR experiments of van Mieghem et al. [27]who concluded that the triplet is localized on a pigment of whichthe chlorine plane is tilted by 30� with respect to the membraneplane. Taking into account the crystal structure of PSII, this conclu-sion leaves two possibilities for triplet localization, either on ChlD1

or ChlD2. Calculations of T–S spectra of D1D2cytb559 complexesand comparison with experimental data revealed that the tripletis localized on ChlD1 [40]. Direct evidence was provided by Schlod-der et al. [28] from T–S spectra on mutants of PSII core complexesfrom Synechocystic sp. PCC 6803. In Fig. 13, calculations [7], assum-ing triplet localization on the different RC Chls in PSII core com-plexes are compared with experimental data. Agreementbetween both is only obtained if the triplet state 3Chl is identifiedas 3ChlD1. A simulation of the temperature dependence of the T–Sspectrum revealed that at higher temperatures the thermal popu-lation of the state 3PD1 contributes to the spectrum [40,7]. From

this temperature dependence an energy difference of 10 meV(81 cm�1) was inferred between 3ChlD1 and 3PD1 in D1D2cytb559complexes [40]. Similar values of 8 meV (65 cm�1) and 13 meV(105 cm�1) were reported for this energy difference from FTIRand EPR studies on D1D2cytb559 complexes reported by Noguchiet al. [71] and Kamlowski et al. [72]. The calculations on PSII corecomplexes gave a very similar value of 11 meV (89 cm�1) [7].

Interestingly, the lifetime of the triplet state in the RC dependsstrongly on the redox state of QA. In PSII complexes, in which thefirst quinone acceptor, QA, is removed or doubly reduced (QAH2),the lifetime of 3P680 is similar to that of the Chla triplet state in sol-vents (about 1 ms under anaerobic conditions). In the presence ofsingly reduced QA the P680 triplet state was however found to de-cay two or three orders of magnitude faster [25,73]. The rapid trip-let quenching which is observed in closed PSII (singly reduced QA)may represent a protective mechanism to prevent the formation ofpotentially damaging singlet oxygen. The production of harmfulsinglet oxygen occurs in D1D2cytb559 complexes or in PSII corecomplexes after double reduction or removal of QA due to the reac-tion between the long-lived triplet state and oxygen. In this case,however, the acceptor side is already irreversibly damaged leadingto non-functional reaction centers. The quenching mechanism inthe presence of singly reduced QA is still an open question. As apossible mechanism, it has been proposed that the rapid triplet de-cay occurs via a thermally activated triplet–triplet energy transferfrom 3ChlD1 to PheoD1 which is followed by a two-step electrontransfer process consisting of (1) reduction of 3PheoD1 by QA

�

and (2) normal forward electron transfer from PheoD1� to QA

[25]. The observed efficient triplet quenching by Q�A led Noguchito conclude that triplet localization on ChlD1 might be functionallyimportant [74]. The PSII RC had to find a different way for tripletquenching than the one by Cars used in the antenna and in theRC of purple bacteria, because of the high redox potentials of theRC Chls, as discussed before.

5. Optical properties of the CP43 and CP47 core antennae

The cross section of the PSII RC to absorb light is enlarged by anumber of antenna complexes surrounding the RC. These antennacomplexes serve to deliver excitation energy to the RC, where it isused to drive the primary electron transfer reaction. All organismscontain the core antenna complexes CP43 and CP47 (Fig. 1). In cya-nobacteria and red algae these core antenna complexes are con-nected to membrane-extrinsically bound phycobilisomes,whereas in green algae and higher plants membrane-intrinsic pig-

T. Renger, E. Schlodder / Journal of Photochemistry and Photobiology B: Biology 104 (2011) 126–141 135

ment–protein complexes serve as distal antennae. Whereas thecore antenna complexes contain only Chla, the distal antennaebind, in addition, Chlb, which absorbs at higher energies, therebyincreasing the cross section of the RC also spectrally. Informationabout the optical properties of CP43 and CP47 core antennae wasobtained from high-resolution spectroscopy in the frequency do-main [80,75,78,79,81,82,51]. Excitation dependent fluorescenceline narrowing spectra [80] gave first evidence that in CP43 atlow temperatures there are two emitting states with differentinhomogeneous distributions. From the Stark and triplet-minus-singlet (T�S) spectra evidence about partially delocalized low-energy excited states in CP43 was reported [80].

The two quasi-degenerate low energy states in CP43 were fur-ther characterized by non-photochemical hole burning (NPHB)and triplet bottleneck hole burning spectra by Jankowiak et al.[82] who termed these states A- and B-states. Interestingly, theA-state was found to mainly contribute to the triplet bottleneckspectrum, whereas the B-state was detected, instead, in the NPHBspectrum [82]. These findings were explained by assuming a longerlife time of the triplet state of A and that most of the excitation en-ergy from higher energy exciton states is trapped by the B-state[82]. Some evidence for excitonic coupling of the B-state pigmentswas reported from high energy satellite holes at 673 nm and678 nm [82]. Hughes et al. [51] investigated CP43-complexes withNPHB and non-wavelength selective photoconversion spectros-copy. They proposed a different intrinsic photoconversion (hole-burning) efficiency of the A- and the B-state due to the stronghydrogen bond of the B-state Chls reported earlier by Groot et al.[80]. From circular dichroism difference spectra, Hughes et al.[51] reported evidence that the B-state is excitonically correlatedwith higher energy states at 677 nm and 682 nm.

Fig. 14. Optical spectra of CP43 complexes. Left part: Linear absorbance (Abs) at 77 Kcalculations [7] (solid lines) are compared with experimental data [75] (symbols). Right671 nm and detection by a probe pulse centered at 670 and 682 nm (left part) or by a wlines) are compared with experimental data [76] (symbols). Right lower part: Calculated

The site energies of the pigments of the CP43 complex were in-ferred recently by Raszewski and Renger [77] from a fit of linearoptical spectra (left part of Fig. 14). Three pigments (encircled inthe left middle part of Fig. 15) are found to contribute to the lowestexciton state, as seen in the exciton pigment distribution functionin the middle left part of Fig. 15. Two of the three pigments, Chls 43and 45, belong to the same domain, whereas Chl 37 is part of a di-mer domain with Chl 34 (see Fig. 15). Hence, there are two degen-erate low energy exciton transitions around 682 nm whichrepresent the lowest excited states of the two domains in the lu-menal (containing Chls 34 and 37) and stromal layer (Chls 41,43–47). These characteristics fit to the A- and B-states identifiedin NPHB and triplet bottleneck spectra [80,82,51], discussed above.The B-state is identified as the low energy exciton state of the stro-mal domain with six pigments (shown in grey in the left part ofFig. 15), whereas the A-state is the low energy exciton state ofthe Chl 34–37 dimer on the lumenal side (shown in yellow inthe left part of Fig. 15).

Independent evidence for the trap state at Chl 37 was providedby Reppert et al. [83] from fitting of absorbance, fluorescence andholeburning data. As a second low-energy pigment state Chl 44was suggested [83], which was not identified in the calculationsby Raszewski and Renger [77], but is located in the same excitondomain as the low energy Chls 43 and 45 assigned in the latterstudy.

The low energy states of CP47 were studied in great detail by deWeerd et al. [75]. From a Gaussian band fitting of the absorptionand linear dichroism spectra, it was concluded that the low-energystate around 690 nm is due to a monomeric Chl with a distinct ori-entation of its QY-transition relative to the membrane plane (withan angle larger than 35�), and that the next higher excited state

time

/ ps

wavelength / nm650 660 670 680 690 700 710

0.1

0.51

510

50100

5001000

and 293 K, linear dichroism (LD) and circular dichroism (CD) spectra at 77 K. Theupper part: 77 K pump-probe spectra for excitation with a 60 fs pulse centered at

hite probe pulse at two different delay times (right part). The calculations [7] (solidlife time density map of excited states of CP43 complexes at 77 K.

660 670 680 690wavelength / nm

0

10

20

30

40

d m /

a.u.

Chl 37Chl 43Chl 45

CP43

660 670 680 690 700wavelength / nm

0

10

20

30

40

50

d m /

a.u.

Chl 11Chl 24Chl 29

CP47

Fig. 15. Energy sinks in CP43 (left part) and CP47 (right part) complexes, as determined from a fit of optical spectra [77]. The outer left and right parts contain thearrangements of pigments in the CP43 and CP47 complex, respectively. The pigments are numbered as in Ref. [2]. Chls with the same color belong to the same excitondomain. Arrows indicate a van der Waals contact between the conjugated p system of a carotenoid and a Chl. The three encircled Chls are those with the lowest site energies(trap states). In the case of CP47 angles of Chl transition dipoles with respect to the membrane plane that are larger than 35� are shown in parentheses. The exciton statespigment distribution functions dm(x) for the three low-energy pigments are shown in the left (CP43) and right (CP47) middle parts. (For interpretation of the references tocolor in this figure legend, the reader is referred to the web version of this article.)

136 T. Renger, E. Schlodder / Journal of Photochemistry and Photobiology B: Biology 104 (2011) 126–141

around 683 nm carries the oscillator strength of approximatelythree Chls. The finding about the 690 nm emitting Chl supportedearlier investigations of van Dorson et al. [81], which provided evi-dence that the low energy fluorescing state in PSII belongs to CP47.From polarized emission measurements, de Weerd et al. [75] ob-tained additional evidence for a single emitting state at 690 nm.The depolarized 683 emission was interpreted by assuming thatat least two isoenergetic states with non-parallel transition dipolemoments contribute.

time

/ ps

wavelength / nm650 660 670 680 690 700 710

0.1650

0.51

510

50100

5001000

Fig. 16. Optical spectra of CP47 complexes. Upper left part: Linear absorbance (Abs), lineat 293 K. The calculations [7] (solid lines) are compared with experimental data [75,78,centered at 671 nm and detection by a probe pulse centered at 670, 677, 683 and 690 nmpart). The calculations [7] (solid lines) are compared with experimental data [76] (symcomplexes at 77 K.

In agreement with de Weerd et al. [75], the fit of linear opticalspectra by Raszewski and Renger [77] (left upper part of Fig. 16)suggest that the lowest excited state of CP47 is localized on amonomeric Chl, namely Chl 29, as seen in the exciton pigment dis-tribution function in the middle right part in Fig. 15. From the 16Chls in CP47 only Chls 11, 22 and 29 have an angle between theirtransition dipole moment and the membrane plane larger than 35�and therefore could be responsible for the negative LD signal ob-served at low energies in CP47.

ar dichroism (LD) and fluorescence (Flu) at 77 K, and circular dichroism (CD) spectra79] (symbols). Right part: 77 K pump-probe spectra for excitation with a 60 fs pule(right upper part) or by a white probe pulse at two different delay times (right lowerbols). Left lower part: Calculated life time density map of excited states of CP47

time

/ ps

wavelength / nm

CP43

640 650 660 670 680 690 700 710 7200.1

0.5

1

5

10

50

100

500

1000

Fig. 17. Lifetime density map of exciton states in the CP43 complex calculated at room temperature using the same parameters as in the 77 K calculation shown in the rightpart of Fig. 14.

T. Renger, E. Schlodder / Journal of Photochemistry and Photobiology B: Biology 104 (2011) 126–141 137

In agreement with anisotropy measurements [75], the calcula-tions [77] show that the second lowest optical band (at 683 nm)consists of more than one exciton transition. The two low energypigments Chls 11 and 24 which are located in different domainscontribute to different exciton transitions at this wavelength(Fig. 15).

Jankowiak and coworkers [84] measured linear absorbance,fluorescence and holeburning spectra on improved CP47 prepara-tions that in particular, exhibit a fluorescence maximum at thesame spectral position (695 nm) as intact PSII core complexes. Inearlier CP47 preparations this maximum was blueshifted by2–5 nm. Based on an analysis of their spectra, Jankowiak andcoworkers [84] concluded that Chl 29 contributes only to the sec-ond lowest exciton state, whereas the lowest state is dominated byChl 26 which is located in an exciton domain well connected to thereaction center. Unfortunately, no linear dichroism and circulardichroism spectra on the new preparations were measured yet.In particular the LD spectrum would be very important as dis-cussed above. We note that the fit of the LD spectrum of Raszewskiet al. [7] (Fig. 16) does show some discrepancies around 690 nm,the calculated LD being more negative than the measured one.An explanation of this discrepancy is needed to obtain more directevidence about which pigment dominates the low-energy excitonstate of CP47.

The new assignment of Jankowiak et al. seems to be in line withholeburning studies by Krausz and coworkers [85,86], who sug-gested that the high holeburning efficiency of PSII core complexesobserved at the energies of the trap states of CP43 and CP47 is dueto slow energy transfer to the reaction center and a conformationaltransition related to primary charge transfer. Due to this transitionthe excitation energies of the initially excited chromophores (i.e.the trap states of CP47 and CP43) are changed giving rise to thehole in the spectra. Of course, such a mechanism requires the trapstates to be situated close to the reaction center.

The excitation energy transfer within CP43 and CP47 was inves-tigated by time-resolved absorption and fluorescence spectroscopy

[76] at 77 K. In both complexes, time constants of 0.2–0.4 ps and2–3 ps were found that were attributed to exciton transfer betweenChls in the same, stromal or lumenal, layer and that between pig-ments in different layers, respectively. In CP47, in addition, a slower17–28 ps component was detected [76] for transfer to the 690 nmtrap state. The calculations by Raszewski and Renger [77] provideda structure-based analysis of these pump-probe spectra and alsoserved to check the exciton relaxation times predicted by the the-ory. The 77 K pump-probe spectra calculated for the CP43 complexare compared in the right upper part of Fig. 14 with the experimen-tal data [76] and that of the CP47 complex in the right part of Fig. 16.Both agree well with the experimental data. As seen also in the life-time density map of the exciton states in the right lower part ofFig. 14, exciton relaxation in CP43 at 77 K occurs with a main timeconstant of about 2 ps. Recent pump-probe data of Holzwarth andcoworkers [87] show that at room temperature exciton relaxationis somewhat faster with a main component of about 600 fs. The fac-tor of 3–4 difference between the time constants of exciton relaxa-tion calculated at 77 K [77] and measured at room temperature [87]led Holzwarth and coworkers to question the validity of the calcu-lations. Fig. 17 contains the previously unpublished room tempera-ture life time density map of the exciton states in CP43. As seenthere, exciton relaxation is predicted to proceed with a main timeconstant of 500 fs, in good agreement with the recent experimentaldata [87].

Finally, we note that the slow 17–28 ps components measured[76] at low temperatures for relaxation into the lowest excitonstate of CP47 complex are due to the large distance between thethree trap states in this complex, as explained by the calculations[77]. At low temperatures, the excitation energy transfer to thelowest state is calculated to become dispersive with a broad distri-bution of transfer times in the 20–60 ps time range (left lower partof Fig. 16), whereas at room temperature it is an order of magni-tude faster [77]. The main exciton relaxation time constants inCP47 are calculated to be rather temperature insensitive in the400–600 fs time range [77].

138 T. Renger, E. Schlodder / Journal of Photochemistry and Photobiology B: Biology 104 (2011) 126–141

6. Excitation energy transfer and trapping in PSII corecomplexes

The research on excitation energy transfer and trapping in PSIIhas a long history [88]. An interesting finding in studies of the timedependence of fluorescence decay of PSII is that the main decaytime roughly scales with the number N of antenna pigments con-nected to the RC (e.g. Ref. [89]). This number can be systematicallychanged by biochemical preparation of complexes with differentsize. If energy (exciton) transfer between the Chls would be fastcompared to primary electron transfer in the RC, the latter wouldstart from an excitonically equilibrated state of the whole complex.In such a state all pigments would be excited roughly with a prob-ability of 1/N, neglecting spectral differences between them. Thusthe primary donor is excited with this probability and the inverserate, that is the time constant, of charge separation is expected toscale with N, as observed in the experiments.

The above argument was particularly powerful before crystalstructure information became available and led to the excited stateradical pair equilibrium (ERPE) model, which assumes excitonequilibration between all antenna states to be fast compared toprimary charge transfer [90,91]. In these models the intrinsic rateconstant of primary charge transfer is estimated to be about (1–2 ps)�1.

For PSII core complexes (N = 35) this would result in a fluores-cence decay time of 35–70 ps in agreement with experimental data(see, e.g., Fig. 18). In the recent paper of Miloslavina et al. [14] theinterpretation was however modified. They assume that excitonequilibration occurs in about 1.5 ps, the apparent rate of primarycharge separation is about 6 ps and that the longer time constantsresult from a dynamical equilibrium between the excited and thefirst (ChlD1

+ PheoD1�) and second radical pair (PD1

+ PheoD1�). It

has been suggested that charge recombination during the lifetimeof the radical pair states can repopulate the excited singlet state ofthe primary donor, which equilibrates with the antenna pigments,and gives rise to longer-lived recombination fluorescence.

To summarize these models, the original ERPE model is simplebecause it explains the observed scaling law of the fluorescence de-cay time with N. The modified ERPE model becomes complex asthere is a need to incorporate charge recombination processes so

0 50 100 150 200

time / ps

0

0.5

1

fluo

resc

ence

/ a.

u.

experimentexact calculationfast equilibration in CP43-Chlz

D1,CP47-Chlz

D2, RC

fast equilibration in PS-II core complex

Fig. 18. Comparison between experimental fluorescence decay of PSII core com-plexes at 300 K, measured [14] after excitation with a 100 fs optical pulse that wascentered at 663 nm (open circles), in comparison to the ‘exact’ calculations (blacksolid line) and calculations that assume fast exciton equilibration in compartmentsconsisting of CP43-ChlzD1, CP47-ChlzD2 and the six core pigments of the RC (greendashed line) or fast exciton equilibration in the whole PSII core complex (bluedotted line) [77]. An intrinsic rate constant for primary electron transfer of(100 fs)�1 was obtained from the fit of the ’exact’ calculation to the experiment. Allother parameters were determined independently. (For interpretation of thereferences to color in this figure legend, the reader is referred to the web versionof this article.)

as to account for the major component of the fluorescence decayrate.

Probably the weakest point of the ERPE model is that it does nottake into account the crystal structure information. The structureshows that the distance between pigments within the core antennasubunits CP43 and CP47 are much smaller than the inter-subunitpigment–pigment distances between CP43/47 and the six core pig-ments in the RC. Rapid exciton equilibration, required by the ERPEmodel, is not consistent with the relatively large distances betweenpigments.

First calculations of excitation energy transfer, performed byVasiliev et al. [18] reported much slower exciton equilibrationtimes and located the bottleneck for the decay of excited statesas the transfer between CP43/CP47 and the RC. Despite the manyshortcomings of this study (orientation of transition dipole mo-ments of pigments were not known, no optical spectra were calcu-lated, local transition energies of the pigments were not known,localized excited states were assumed) this study reported the firststructure-based calculations that questioned the assumptions ofthe ERPE model.

A detailed theoretical analysis using the site energies deter-mined as described above was performed by Raszewski and Rengerto model the excited state decay in whole PSII core complexes [77].Generalized Förster theory was applied to model excitation energytransfer between different domains of strongly coupled pigmentsand Redfield theory for exciton relaxation within the domains.No adjustable parameter was used for the excitation energy trans-fer rate constants. The experimental fluorescence decay (Fig. 18)could only be described by two assumptions: (i) recombinationfluorescence does not contribute significantly and (ii) primarycharge transfer in the RC is ultrafast, occurring with a time con-stant of 300 fs, that corresponds to an intrinsic rate constant of(100 fs)�1, assuming about 30% equilibrium population of ChlD1

(see Fig. 8 bottom). In order to find the bottleneck for the decayof excited states the ‘exact’ calculations were compared with sim-pler models, where fast exciton relaxation was assumed to occur incertain domains [77]. The simplest model that could still reproducethe ‘exact’ result is a 3-compartment model containing the six corepigments of the RC in one compartment and CP43-ChlzD1 andCP47-ChlzD2 in the other two. In this model, excitation energytransfer between the CP43 and the CP47 antenna and the RC occurswith 41 and 50 ps at 300 K, respectively, i.e., light-harvesting isfound to be limited by the transfer to the trap (Fig. 19).

Whereas the ERPE model provides a simple explanation of thescaling law of fluorescence decay, a structure-based calculationprovides a simple explanation of the slow fluorescence decay time

Fig. 19. Minimal compartment model [77] that describes the experimentalfluorescence decay in Fig. 18. The red numbers show disorder averaged inverserate constants for excitation energy transfer between the three compartments. Therate constants between the compartments were obtained from the individualmicroscopic rate constants by assuming fast exciton equilibration between theexcited states within the compartments, i.e. without free parameters. The 0.3 pstime constant in the RC characterizes the primary charge transfer between theequilibrated excited state of the RC and the first radical pair state, assumingprimary electron transfer starting at ChlD1 with an intrinsic rate constant of(100 fs)�1. Pigments colored in the same way belong to the same domain of stronglycoupled pigments in which delocalization of excited states occurs.

T. Renger, E. Schlodder / Journal of Photochemistry and Photobiology B: Biology 104 (2011) 126–141 139

constants measured. So, the question arises: Can a structure-basedcalculation also explain the linear scaling of the fluorescence decaytime constant with the size of the antenna? Unfortunately, at pres-ent there exist no crystal structure data on larger complexes thanPSII core complexes that would allow to perform excitation energytransfer calculations as described above. However, there is the fol-lowing indirect evidence for fast excitation energy transfer be-tween the distal antenna and the core complex.

In experiments on PSII membrane fragments of spinach by vanAmerongen and coworkers [92], the fluorescence decay for two dif-ferent excitation conditions was compared, one in the Chlb spectralregion, that, therefore, excites predominantly the distal antennaeand one in the Chla spectral region with no spatial selection ofexcitation. The minor difference of about 4 ps between the decaytimes suggests fast excitation energy transfer between the periph-eral and the core antenna complexes. Since this excitation energytransfer is fast compared to the transfer to the trap, i.e., betweenCP43/47 and the RC, a quasi-equilibrium of excited states is estab-lished in the antenna, before the excitation energy is transferred tothe RC. This equilibrium gives rise to a linear scaling of the decayconstant with antenna size, since the populations of CP43 andCP47 states that transfer excitation energy to the RC scale accord-ingly. Of course, the question arises: Why is the transfer betweenmore peripheral complexes so much faster than between the coreantennae and the RC. The most straightforward explanation lies inthe high redox potential of the RC pigments, needed for the watersplitting reaction. Nature had to isolate these pigments from therest in order to avoid an oxidation of antenna pigments.

It is, however, interesting to note that, despite the lower oxida-tion potential, also purple bacteria exhibit a transfer-to-the-traplimited light-harvesting kinetics [93].

7. Challenging questions and hypotheses

7.1. How fast is the primary charge transfer and what is themechanism?

The values suggested for the time constants of primary chargeseparation range from 300 fs from modeling of fluorescence decaydata of PSII core complexes [77] to 3 ps inferred from time-re-solved pump-probe experiments on D1D2cytb559 complexes[15,16] and PSII core complexes [15].

Raszewski and Renger [77] noted, that using instead of the300 fs a 600 fs time constant (corresponding to an intrinsic timeconstant of 200 fs), inferred experimentally from femtosecond IRstudies [13], in the calculation of the fluorescence decay (Fig. 18),still results in a reasonably agreement with the experimental data,however a 3 ps time constant cannot describe the data (see sup-porting online material of Ref. [77]). Of course, such short timeconstants, described above, raise the question about the underly-ing mechanism and whether electron transfer between twopigments that do not show considerable wavefunction overlap asChlD1 and PheoD1 can really be so fast.

Considering the almost perfect overlay of ChlD1–PheoD1 with itscounter part BA–HA in purple bacteria suggests a similar electrontransfer coupling matrix element between the two pigments (leftpart of Fig. 4). Since in purple bacteria sub 400 fs electron transferoccurs starting at BA [52,53] it is very reasonable to assume thatthe fast, but unused, side pathway for primary electron transferin purple bacteria becomes the main electron transfer pathwayin PSII. It remains an open question whether such a fast electrontransfer reaction can be described by the standard non-adiabaticelectron transfer theory (e.g. [94]) that uses a second order pertur-bation theory in the electron transfer coupling. Comparative exper-iments on PSII of A. marina, discussed before, could be very helpful

also in this respect. They allow to investigate the driving forcedependence of this reaction, since primary electron transfer occursbetween a Chld and Pheoa instead of Chla and Pheoa in Chla con-taining PSII RC.

Related to the question of the kinetics of primary electron trans-fer is the question about the identity of the primary electron donor.Whereas in the RC of purple bacteria, the excitation energy sink iscreated by the coupling of excited states to intra special pair chargetransfer states [95,35] in the ‘‘special pair’’, in PSII RCs the lowestexcited state is strongly localized at ChlD1 [40,7]. Indeed, there isa significant difference in the two special pair structures (Fig. 4).Whereas, there is significant overlap of the p-electron wavefunc-tion of one ring in the case of purple bacteria, the ‘‘special pair’’of the PSII RC is in a staggered rather than an eclipsed configura-tion. It seems a slight tilt of the two ‘‘special pair’’ Chls with respectto each other has disrupted the wavefunction overlap in PSII. Thissmall tilt had large consequences for the optical and redox proper-ties of the ‘‘special pair’’. It lifted the transition energy of the ‘‘spe-cial pair’’ to an extent that it does not represent the excitationenergy sink anymore and it helped to increase the redox potentialas needed for the water splitting reaction [96,7].

The multiple ET pathway hypothesis of van Grondelle andcoworkers that is based on an interpretation of time resolved opti-cal pump-probe data [16] and a theoretical analysis of the Starkspectrum [48] seems to be in line with the finding of Krausz andcoworkers [50,51] of a low lying excited state of the PSII RC capableof initiating primary electron transfer. However, besides the differ-ent broadening mechanisms, discussed above, Novoderezhkin et al.[48] predicted that the absorbance of the CT state in D1D2cytb559complexes extends to wavelengths longer than 690 nm, in dis-agreement with experimental data of Krausz and coworkers [42],which show that the long-wavelength absorbance of ‘‘native’’ PSIIcore complexes is lost when D1D2cytb559 complexes are formed.The nature of the persistent spectral hole-burning in D1D2cytb559complexes was explained [97] by using a simplified Raszewskiet al. [40,7] asymmetric exciton model. According to this explana-tion, it is the fraction of exciton realizations arising from inhomo-geneous broadening in which PheoD2 dominates the lowest excitonstate, that gives rise to efficient persistent spectral hole burning.

The inhomogeneous broadening assumption for the CT state ofNovoderezhkin et al. contradicts the large permanent dipole mo-ment of 30 D assumed for this state in the calculations [48]. How-ever, since modified Redfield theory cannot describe dynamiclocalization effects [47] of the excitonic wavefunction it cannotbe applied for strong exciton-vibrational coupling, i.e., homoge-neous broadening. It is not clear whether the large inhomogeneousbroadening assumed by Novoderezhkin et al. for the CT state canreally compensate for the above short-coming of modified Redfieldtheory. Therefore, the conclusions concerning the nature of the CTstate and its role in the primary charge transfer in photosystem IIshould be reexamined including dynamic localization effects in thedescription. The hierarchical equation of motion approach devel-oped by Tanimura and Ishizaki [98] seems to be suitable for thistask.

We note that, in agreement with the finding of Krausz andcoworkers at low temperature, water splitting activity of func-tional plant leaves [99] and PSII enriched membranes and PSII corecomplexes from spinach [57] at physiological temperatures, fol-lowing far-red light excitation, was reported.

7.2. Structure-based calculation of site energies and short-range effectsin the ‘‘special pair’’

An important parameter class of the exciton Hamiltonian is gi-ven by the site energies of the pigments. So far these energies wereinferred indirectly from a fit of optical spectra of the subunits.

140 T. Renger, E. Schlodder / Journal of Photochemistry and Photobiology B: Biology 104 (2011) 126–141

However, to draw conclusions about structure–function relation-ships, giving rise to these values, a direct structure-based calcula-tion is needed. First successful calculations of site energies ofpigment-protein complexes were reported for the Fenna–Mat-thews–Olson protein, a light-harvesting complex of green sulfurbacteria [100,101], photosystem I core complexes [102] and thelight-harvesting complex LHC2 of photosystem II [103]. Applyingthis methodology, it will be interesting to find out, for example,which protein-cofactor-interactions are responsible for the asym-metry of site energy values in the RC of PSII. Another importantquestion concerns the location of the so-called antenna trap statesin CP47 and CP43, i.e., the sites with low-excitation energies[81,80,104,82,75,105,77,83]. Of course, a structure based calcula-tion of site energies should ultimately also take into account theconformational transitions of the protein that are responsible forstatic and dynamic disorder. For example, it is an open question,why the site energy of ChlD1 differs in D1D2cytb559 complexesand PSII core complexes and why its inhomogeneous width issmaller in PSII core complexes [40,7], whereas the site energiesand inhomogeneous distribution functions of the remaining pig-ments are the same in the two preparations.

Once a higher resolution crystal structure will be available, astructure based calculation of short-range effects on the excitoniccoupling of the ‘‘special pair’’ and of the coupling of excited statesto intradimer charge transfer states will become possible. A meth-od exists to extract these effects from monomer and dimer quan-tum chemical calculations [35]. The finding of Krausz andcoworkers[50,51], that charge separation can be initiated at lowtemperature with long-wavelength excitation of a strongly homo-geneously broadened excited state raises the question about a low-lying charge transfer state in the RC of PSII. For an accurate calcu-lation of its energy, most likely solvent and protein environmentaleffects have to be included in the calculations.

8. Functional implications

A major challenge for PSII is to cope with the high redox poten-tial of its four central RC Chls that is needed for the water splittingreaction. The structure based analysis of optical spectra discussedabove allows to identify some building principles that make thephotosystem relatively stable.

One is the large spatial separation between RC and antenna pig-ments in order to avoid an oxidation of the antenna pigments. Thislarge separation gives rise to a transfer-to-the trap limited light-harvesting kinetics, where the primary electron transfer is twoorders of magnitude faster than the excitation energy transferbetween the core antenna complexes CP43 and CP47 and the RC.The ratio of time constants for forward and back excitation energytransfer between the core antennae and the RC resembles approx-imately the inverse ratios of bound pigments, i.e. roughly 1/2 forCP43/RC and 1/3 for CP47/RC (Fig. 19). This result shows that theRC represents a very shallow trap for excitation energy and there-fore the number of pigments, i.e., the entropic factor, determinesthe inverse rate constants between the compartments in Fig. 19.The two orders of magnitude faster primary charge transfer timeallows an efficient trapping of excitation energy despite an unfa-vorable ratio of forward and back excitation energy transfer rateconstants between the core antennae and the RC. Moreover, byslowing down the primary charge transfer time, the RC couldswitch the PSII core complex into a photoprotective mode wherea large part of the excitation energy can escape the RC back intothe antennae [77].

Another building principle is the limitation of unavoidablephotophysical damage to the D1-protein. This limitation is ob-tained by using one branch for primary and the second branch

for secondary electron transfer. In this way only the D1-proteinneeds to be replaced in a controlled way within the repair cycleof PSII. One of the deciding factors that guide primary electrontransfer along the D1-branch probably is the low site energy ofChlD1, which allows this pigment to function as primary electrondonor in PSII. Secondary electron transfer, which involves CarD2

leads to a controlled reduction of Pþ680 by Cytb559, that may becompleted by a subsequent charge recombination with the re-duced QB [106]. A second photoprotective function of CarD2 dis-cussed [21,107] is the quenching of singlet oxygen, that isgenerated by reaction of triplet oxygen with triplet states of Chl.

A third building principle is the use of a unique mechanism forthe quenching of triplet states of Chl, which cannot be quenched bycarotenoids, as in the antennae. The new mechanism is thequenching by the reduced plastoquinone QA

�. The Chl with thelowest triplet energy is therefore the one closest to QA, i.e. ChlD1

(Figs. 2 and 13).

Acknowledgements

It is a pleasure to acknowledge support from the German Re-search Foundation (DFG) through Collaborative Research Center(SFB) 498, TP A5 (E.S.) and TP A7 (T.R.) and through Emmy Noetherproject RE 1610 (T.R.) We thank Prof. Rienk van Grondelle for dis-cussions on primary charge transfer in PSII and for sharing with usunpublished work.

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