Photosynthesis Research 63: 195–208, 2000. © 2000 Kluwer Academic Publishers. Printed in the Netherlands. 195 Minireview Primary charge separation in Photosystem II Jan P. Dekker∗ and Rienk Van Grondelle Faculty of Sciences, Division of Physics and Astronomy, Vrije Universiteit, De Boelelaan 1081, 1081 HV Amsterdam, The Netherlands; ∗ Author for correspondence (e-mail: dekker@nat.vu.nl) Received 3 November 1999; accepted in revised form 10 February 2000 Key words: charge separation, disorder, exciton interaction, Photosystem II, reaction center Abstract In this Minireview, we discuss a number of issues on the primary photosynthetic reactions of the green plant Photosystem II. We discuss the origin of the 683 and 679 nm absorption bands of the PS II RC complex and suggest that these forms may reflect the single-site spectrum with dominant contributions from the zero-phonon line and a pronounced ∼80 cm−1 phonon side band, respectively. The couplings between the six central RC chlorins are probably very similar and, therefore, a ‘multimer’ model arises in which there is no ‘special pair’ and in which for each realization of the disorder the excitation may be dynamically localized on basically any combination of neighbouring chlorins. The key features of our model for the primary reactions in PS II include ultrafast (<500 fs) energy transfer processes within the multimer, ‘slow’ (∼20 ps) energy transfer processes from peripheral RC chlorophylls to the RC multimer, ultrafast charge separation (<500 fs) with a low yield starting from the singletexcited ‘accessory’ chlorophyll of the active branch, cation transfer from this ‘accessory’ chlorophyll to a ‘special pair’ chlorophyll and/or charge separation starting from this ‘special pair’ chlorophyll (∼8 ps), and slow relaxation (∼50 ps) of the radical pair by conformational changes of the protein. The charge separation in the PS II RC can probably not be described as a simple trap-limited or diffusion-limited process, while for the PS II core and larger complexes the transfer of the excitation energy to the PS II RC may be rate limiting. Abbreviations: BRC – purple bacterial reaction center, Chl – chlorophyll, LHC II – light-harvesting complex II, Phe – pheophytin, PS II – Photosystem II, P680 – primary electron donor of Photosystem II, QA – primary plastoquinone electron acceptor, RC – reaction center, YZ – redox-active Tyr-161 of D1 Introduction Photosystems are complex biological devices that are able to convert visible light into an electrochemical potential. Among all photosystems, Photosystem II (PS II) is unique in the sense that it creates a much stronger oxidant than other photosystems and that it is even capable of oxidizing water to molecular oxygen. The green plant PS II is also one of the most complex photosystems. It consists of more than 25 different protein subunits which work together to absorb substantial amounts of visible light and transfer the excitation energy to the photochemical reaction center (RC). Within the RC, the electronically excited state of a special chlorophyll (P680) induces the rapid transfer of an electron to an adjacent pheophytin (Phe) molecule. The charge separation is then stabilized by the electron transfer in about 200 ps from the reduced Phe molecule to a plastoquinone molecule called QA , by the electron transfer in nanoseconds from a redox active tyrosine (YZ ) at position 161 of the D1 protein to P680+ (Diner and Babcock 1996), and by a number of slower electron transfer reactions (millisecond timescale) which finally result in the oxidation of water to molecular oxygen, the reduction of plastoquinone to plastoquinol and the formation of a transmembrane pH gradient. Other remarkable features of green plant PS II include the very rapid turnover of one of the RC proteins (the D1 protein) in plants under illumination (Barber and Andersson 1992) and the direct involve- 196 Structure and organization Figure 1. Model of the structural arrangement of the chlorin pigments in the PS II RC. The PS II RC probably contains ‘special pair’ and ‘accessory’ chlorophylls and pheophytins in similar positions and orientations as in the RC of Rps. viridis or Rb. sphaeroides, except that the ‘special pair’ chlorophylls are wider spaced and that there are two ‘peripheral’ chlorophylls at a relatively large distance from the six central chlorins. ment of PS II in several regulatory mechanisms of the photosynthesis (Bassi et al. 1997). A detailed understanding of the primary photosynthetic reactions in PS II may, therefore, be important for the understanding of photosynthetic energy conversion in general. The primary reactions in PS II have recently been reviewed by a number of groups (see, e.g. Van Grondelle et al. 1994; Diner and Babcock 1996; Klug et al. 1998). In this Minireview, we restrict ourselves to a number of issues on the first reactions in PS II that recently have been a subject of debate and refer to the above-mentioned reviews for more detailed overviews. The discussions include our views on the suitability of the isolated PS II RC complex as a tool to study primary charge separation in PS II, the origin of the 679 and 683 nm absorption bands of the PS II RC complex, the origin of the relatively slow charge separation kinetics in the PS II RC complex at room temperature, the role and extent of excitonic coupling in the reaction center and how this is realized in the so-called multimer model (Durrant et al. 1995), the equilibrium between the excited and radical pair states and the mechanism of the primary charge separation reactions in PS II. The discussion is performed in the light of the most recent structural data (Rhee et al. 1998; Hankamer et al. 1999) and of new developments in the understanding of charge separation in photosynthetic purple bacteria (Van Brederode and Van Grondelle 1999). PS II consists of at least 25 different types of protein subunits, which are organized into two structurally and functionally different parts (Hankamer et al. 1997). The first part is the core complex, a well-defined structure which is responsible for all electron transfer reactions in PS II and which is organized as a dimer in the stacked, appressed regions of the thylakoid membrane. The second part is the peripheral antenna, which in green plants and algae consists of a collection of light-harvesting complex II (LHC II) proteins and which absorbs most of the light for PS II. A variable part of the LHC II proteins is rather tightly associated with the dimeric PS II core complex to form one of the so-called PS II–LHC II super- or megacomplexes (Boekema et al. 1999a). The structure of the trimeric LHC II complex is known at 3.4 Å resolution, revealing the positions of 3 trans-membrane α-helices, 12 chlorophylls and 2 xanthophylls (Kühlbrandt et al. 1994), whereas the structure of the PS II core complex without the CP43 core antenna protein is known at 8 Å resolution, revealing the positions of 23 transmembrane α-helices, of which 6 have been assigned to the core antenna protein CP47, 10 to the reaction center proteins D1 and D2 and 7 to small proteins consisting of a single transmembrane α-helix (Rhee et al. 1998). Near the central helices of the D1 and D2 proteins, a number of masses were observed that were tentatively assigned to 4 chlorophyll (Chl) and 2 pheophytin (Phe) molecules, in line with the idea that the PS II RC binds 4 Chl a and 2 Phe a molecules in similar positions and orientations as the corresponding molecules in the RC of photosynthetic purple bacteria (see Figure 1 for a schematic representation of the chlorophylls and pheophytins of the PS II RC). Also, the proposed wider spacing between the chlorophylls of the ‘special pair’ (Braun et al. 1990; Durrant et al. 1995) became visible in the 8 Å map (Rhee et al. 1998). The D1 and D2 proteins bind most probably two additional Chl a molecules at conserved histidines at positions 118 and 117, respectively. Most of the PS II protein complexes can now be isolated and purified with at least partially retained activity. For the PS II RC complex consisting of the D1, D2, cytochrome b-559, PsbI and PsbW proteins, several purification protocolls have been reported (see, e.g. Eijckelhoff et al. 1996), but thus far, all preparations lack the quinone electron acceptors and the possibility to perform secondary electron transfer. The RC complex can also be isolated as a monomer with 197 two monomeric (CP29 and CP26) LHC II proteins around the dimeric PS II core complex (Boekema et al. 1999b). In the core complex, the positions are outlined of the D1 and D2 proteins, the CP47 and CP43 proteins and the small proteins of unknown function (Hankamer et al. 1999). The most important features are that S–LHC II can transfer excitation energy to CP43 (either directly or via CP26 as an intermediate), that S–LHC II can also transfer excitation energy to CP47 (via CP29 as a necessary intermediate), but that there is no possibility of energy transfer between the antenna proteins in the upper and lower parts of the complex without reaching the D1 and D2 reaction center proteins or the central part of the core dimer. Spectroscopic properties Absorption properties of the PS II RC Figure 2. Projection map of the negatively stained ‘standard’ PS II–LHC II supercomplex, obtained from the datasets reported in Boekema et al. (1999a,b). In the PS II core parts, the regions in red indicate the positions of the D1 and D2 proteins, while the ones in green indicate the positions of the CP47 and CP43 core antenna proteins and those in purple and yellow indicate the positions of small proteins with one or two transmembrane α-helices (adopted from Hankamer et al. 1999). The positions of trimeric (LHC II) and monomeric (CP29 and CP26) peripheral antenna proteins are also indicated in green to visualize the possible energy transfer pathways to the D1 and D2 RC proteins. The structures on the two halves of the PS II core dimer were slightly translationally shifted compared to the data in Hankamer et al. (1999) to obtain an optimal overlay with the PS II–LHC II supercomplex. one (CP47) or two (CP47 and CP43) connected antenna proteins and partially retained activity and as a dimer with even more connected antenna proteins (the PS II–LHC II supercomplexes) and fully retained activity (Schilstra et al. 1999). Figure 2 shows a projection map of the most frequently observed PS II-LHC II supercomplex and reveals the positions of one trimeric (S–LHC II) and In contrast to the situation in the purple bacterial reaction center (BRC), the Qy transitions of the chlorophylls and pheophytins of the PS II RC occur at about the same wavelength (675 nm). This spectral congestion is one of the consequences of the weak coupling between the various pigments (discussed in detail below) and considerably complicates the interpretation of spectroscopic studies on the PS II RC. Only at cryogenic temperatures some finestructure in the absorption spectrum can be observed with bands peaking near 670 and 679 nm and a shoulder at about 683 nm (Van Kan et al. 1990). There is now good evidence that at least one of the two peripheral chlorophylls absorbs maximally at about 670 nm. In the 5 Chl PS II RC (RC-5) preparations, a chlorophyll peaking at 670 nm is missing while the amplitude of the ‘slow’ (20–30 ps) energy transfer from the peripheral to core chlorophylls is about halved (Vacha et al. 1995). The second peripheral chlorophyll most likely also absorbs at 670 nm, because in the RC-5 preparations about half of the slow 670 → 680 nm energy transfer process still was observed (Vacha et al. 1995) and because the remaining 4 K absorption around 670 nm in the RC5 preparations had exactly the same bandwidth and peak wavelength as the total 4 K absorption around 670 nm in the RC-6 preparations (Eijckelhoff et al. 1997a). In the ‘red’ absorption region (679–683 nm), it is well established that P680 and the ‘active’ pheophytin show significant contributions. There may also be absorption from a ‘trap’ chlorophyll (Groot et al. 198 1996; Den Hartog et al. 1998a) responsible for the trapping of excitations at very low temperatures (although this chlorophyll may also be part of P680 – see below), and from the ‘inactive’ pheophytin molecule, because chemical exchange of the inactive pheophytin resulted in an absorbance decrease at about 680 nm (Shkuropatov et al. 1997). There is, however, no consensus yet on the absorption properties of the inactive pheophytin and very recently Jankowiak et al. (1999) reported a very different peak wavelength at 668.3 nm. These authors recorded a difference spectrum with a bleaching at 668.3 nm and a bandshift around ∼684 nm after addition of 4 mg/ml sodium dithionite in the dark in the absence of oxygen and attributed this difference spectrum to reduction of the inactive pheophytin, because the active pheophytin and the chlorophylls should not be reduced under these conditions. Although the theoretical basis of the assignment of the 668.3 nm bleach to the inactive pheophytin seems reasonable, the experimental evidence is weak (the bleaching of the characteristic pheophytin Qx transition around 540 nm is obscured by the spectrum of the reduction of cytochrome b-559). It is also not clear if possible pH effects could have influenced the spectrum. Addition of dithionite can easily lead to an acidification of the medium, which can give rise to irreversible bleachings at 684 and 665 nm (Yruela et al. 1999). In our view, the interpretation of the results from the chemical exchange of the inactive pheophytin (Shkuropatov et al. 1997) is more straightforward and we, therefore, conclude that the inactive pheophytin most likely absorbs around 680 nm. There has also been quite some discussion and controversy in the literature on the origin of the 679 and 683 nm absorption bands. In some proposals, the 683 nm state was attributed to one of the two chlorophylls located near the periphery of the complex and was referred to as a ‘linker’ of excitation energy between the core antenna chlorophylls and P680 (Seibert 1993; Chang et al. 1994b). This interpretation is in conflict with the above-mentioned idea that both peripheral chlorophylls should absorb maximally near 670 nm. In other reports, the 683 nm state was attributed to an ‘accessory’ chlorophyll in the central core of the PS II RC (Konermann et al. 1997b), or to a special form of P680 absorbing at long wavelength (Van der Vos et al. 1992; Kwa et al. 1994). We note that the attribution of the 683 nm state to an ‘accessory’ chlorophyll or to P680 does not necessarily have to conflict, because the accessory Figure 3. Line-narrowed emission spectra of PS II RC at 5 K. With the technique of fluorescence line-narrowing (FLN), sub-nm resolution emission spectra are recorded upon narrow-bandwidth (∼cm−1 ) continuous-wave laser excitation (see, e.g. Peterman et al. 1998). The aim of these FLN experiments is to record the ‘single-site’ emission spectra of the PS II RC absorbance bands peaking at 680 and 684 nm. Generally, the single-site emission spectrum consists of a relatively narrow zero-phonon line (ZPL), caused by the purely electronic transition, a broader wing, the so-called phonon wing (PW), and a large number of less intense repeats of the ZPL-PW structure at lower energy in emission. These repeats are separated with the vibrational frequency from the purely electronic transition, provide valuable information on the vibrational modes of the emitting pigment(s) and can be used as a ‘fingerprint’ of the emitting species, because the exact vibrational frequencies depend on the molecular environment of the emitting species. Single-site emission spectra can only be recorded if (a) the excitation is ‘selective’ (i.e. occurs by a laser), (b) the temperature is below ∼40 K and (c) energy transfer does not occur. The shown spectra are the averages of six spectra selectively excited at 679.2 – 681.2 nm (upper curves) and 682.8 – 686.0 nm (lower curves), each converted to the wavenumber scale and plotted as the difference with the excitation wavenumber. The upper two spectra are 5 × magnifications to enhance details in the vibronic region. Inset: magnification of the region between 950 and 1350 cm−1 . The fact that line narrowing is observed upon excitation at ∼680 nm confirms that the 679 and 683 nm spectral forms are not connected by energy transfer. The vibrational finestructure in both spectra is very similar, which indicates that excitation of the 679 and 683 nm spectral forms leads to fluorescence from chlorophyll molecules in the same protein environments. The differences in the low-frequency region arise from the fact that the upper spectrum is excited mainly in the phonon side band, whereas the lower spectrum is excited predominantly in the zero-phonon lines. chlorophyll may in fact be a part of P680 (see below). Nevertheless, it is clear that the 679 and 683 nm absorption bands have both been observed in the states directly responsible for charge separation at 4 K (radical pair and triplet – Van Kan et al. 1990), suggesting that both absorption bands belong to P680. It has also been shown that both spectral forms are not connected by energy transfer at 4 K (Kwa et al. 1994), which would be very unlikely if the 683 nm state would be due to a peripheral chlorophyll. 199 However, not only the 4 K absorption data reveal two bands in the red part of the spectrum, also the steady-state emission at 4 K shows two bands peaking near 683.5 and 680.5 nm (Peterman et al. 1998). Figure 3 shows that selective excitation of the chlorophylls absorbing around 680 and 684 nm gives virtually identical vibrational finestructures of the 4 K fluorescence, which strongly suggests that both emissions arise from the same molecule(s) or state(s) with the same molecular environments. We stress that the vibrational finestructures can be used as ‘fingerprints’ of the environment and that thusfar different finestructures were observed for different chlorophyll-protein complexes. For the CP43 complex, it was even shown that two different ‘red’ states in the same complex give rise to different vibrational finestructures (Groot et al. 1999). These observations suggest that the two spectral forms have very similar origins and are the result of an apparent ‘heterogeneity’. We note that ‘heterogeneity’ has also been observed in the absorption band of the low-exciton component of the special pair chlorophylls of the reaction center of photosynthetic purple bacteria (Hoff 1988; Friesner and Won 1989) and that the heterogeneity observed in the PS II RC does not have to be ‘real’. It can be a direct result of the particular shape of the ‘single-site’ spectrum (the lower curve in Figure 3 – see the legend of Figure 3 for an explanation of the single-site spectrum). This spectrum shows on one hand a very sharp zero-phonon line and a clear phonon band at 19 cm−1 , and on the other a characteristic and relatively intense feature around 80 cm−1 (see also Peterman et al. 1998). The nonselective spectrum (the 4 K absorption spectrum) can be regarded as a convolution of the single site spectrum with an inhomogeneous distribution function, and if the inhomogeneous width is limited, it is expected that the structures of (a) the zero-phonon line and 19 cm−1 feature and (b) the broad and relatively intense 80 cm−1 phonon side band will still be observed as separate peaks. The fact that the energetic difference between 679 and 683 nm (85 cm−1 ) matches the energetic difference between these two structures is in line with this idea. Absorption properties of P680 in intact PS II An important issue is the question to which extent the spectroscopy of the purified PS II RC complex represents that of PS II in vivo. Based on experiments on oxygen-evolving PS II membranes from higher plants and PS II preparations from cyanobacteria, it has been suggested that in intact PS II the primary electron donor has only one absorption band at 683–684 nm at cryogenic temperatures, that increased biochemical manipulation of the photosystem leads to a shift to 679–680 nm, and thus that the 679 nm state could be non-physiological (Hillmann et al. 1995). While this suggestion is in line with the idea that the 679 nm and 683 nm spectral forms arise from the same (set of) pigment(s), it is remarkable that PS II RC particles prepared by very different methods (long, short or no Triton X-100 incubation, the 5 Chl preparation) all contain about the same ratio of the 683 and 679 nm spectral forms and that increased biochemical manipulation of purified reaction centers only seems to result in a broadening of the absorption bands (Eijckelhoff et al. 1996, 1997a). We would like to point out that if intact PS II contains two (or more) spectral forms of P680, the ones peaking near 680 nm could remain undetected at 4 K. In other words, it is possible that only the red states could give rise to charge separation at 4 K. The argument is that in intact PS II many excitations will reach the long-wavelength states of the CP47 and CP43 core antenna proteins absorbing at 683–690 nm (Chang et al. 1994a; Groot et al. 1999), which at 4 K are generally too low in energy to allow uphill energy transfer to the 680 nm state of P680. Thus, in complexes with a blue RC state a large part of the excitation energy will be trapped on the low-energy states of CP47 and CP43, while in complexes with a red RC state, many excitations will be transferred to the RC and, therefore, give rise to charge separation. In both cases (blue or red RC state), a ∼684 nm state will be bleached upon cation or triplet formation, in line with the experimental results (Hillmann et al. 1995). This possibility gives a straightforward explanation for the observed blueshift of the bleaching to 680 nm upon raising the temperature in intact PS II (Hillmann et al. 1995), because at higher temperatures the uphill energy transfer to the blue RC states will become possible. We conclude that there is no evidence that the absorption properties of the chlorins in isolated PS II RC particles differ from those in intact PS II. Conversely, it should be kept in mind that the quinone electron acceptors are missing in isolated PS II RC complexes, which has a strong influence on the electron transfer reactions within this complex. 200 Cation, triplet and fluorescing states of the PS II RC At low temperatures, the triplet state of the PS II RC is localized on a single Chl with its plane tilted 30◦ relative to the membrane (Van Mieghem et al. 1991). This orientation is almost identical to that of the accessory chlorophylls in the BRC (Kwa et al. 1994). The tripletminus-singlet difference spectrum is characterized by a bleaching of the 131 C=O stretch mode at 1669 cm−1 (Noguchi et al. 1993). At higher temperatures, the situation becomes more complicated. Between 100 and 200 K, the spectrum of the triplet changes from that of the abovementioned single species with its plane tilted 30◦ relative to the membrane to that of two different species in which the species observed at low-temperature is now mixed with a second species with its plane vertical to the membrane plane (Kamlowski et al. 1996). The latter orientation is similar to that of the ‘special pair’ in the BRC. The emission at 4 K (Peterman et al. 1998) gives rise to a very similar vibrational finestructure as observed for the triplet, such as the 131 C=O stretch mode at 1669 cm−1 and a characteristic low-frequency mode at 80 cm−1 (Kwa et al. 1994). These and other observations suggest that the steady-state emission and the triplet arise from the same species. Between 70 and 150 K, the shape of the emission spectrum changes from a spectrum peaking at 684 nm to a spectrum peaking at 682 nm (Groot et al. 1994), suggesting that also here a different species starts to contribute at higher temperatures. The vibrational finestructure of the P680+ -minusP680 difference spectrum has been recorded at 150 K (Noguchi et al. 1998) and shows features of charge delocalization. The 131 C=O stretch mode is under these circumstances characterized by bleachings at 1679 and 1704 cm−1 . These energies differ from those observed for the triplet difference spectrum at 85 K, although it is not clear to which extent the much higher temperature gives rise to these differences. Nevertheless, the different finestructures have been used as an argument in favour of triplet migration to a Chl different from the species responsible for charge separation (Noguchi et al. 1998). In our view, there is no clear evidence for such a migration. Apart from the temperature problem mentioned above, it can not be excluded that the observed frequencies of vibrational modes vary as a function of experimental conditions. The presence of chemicals to produce the radical pair state could influence the charge distribution around the secondary donors, which could influence the frequencies of the 131 C=O stretch modes. The absence of triplet migration would in every case explain the highly polarized triplet-minus-singlet absorbance-difference spectrum obtained upon 4 K excitation at λ > 680 nm (Kwa et al. 1994). Excitonic coupling in PS II When two or more pigments are sufficiently close in space, they will interact and, consequently, the transition energies, orientations and relative intensities of the original transition dipoles will be modified (see, e.g. Van Grondelle et al. 1994; Van Amerongen et al. 2000). This excitonic interaction is optimal when the frequencies of the involved transitions are the same and weakens progressively upon increasing the difference between these frequencies. Experimental evidence for a relatively weak excitonic interaction (∼140 cm−1 ) in the PS II RC has been presented by Kwa et al. (1994), who detected differently-oriented low- and high-exciton components at 680–683 and 667 nm, respectively, and by Chang et al. (1994b), who reported a high-exciton component at 667 nm on the basis of hole-burning experiments. This coupling most probably occurs between the chlorins of the primary donor P680, and is much weaker than the coupling between the primary donor pigments in the BRC of Rb. sphaeroides (∼550 cm−1 ) or Rps. viridis (∼950 cm−1 ). There has been a debate on the extent of excitonic interactions between the other chlorins in the central core part of the PS II RC. Some authors argued that these interactions can be neglected and that P680 should be viewed as a weakly-coupled dimer of chlorophylls (see, e.g. Bosch et al. 1995; Konermann and Holzwarth 1996), whereas others argued that the interactions between all six chlorins should be taken into account (Tetenkin et al. 1989; Durrant et al. 1995) and that the central part of the PS II RC should be viewed as a weakly-coupled multimer of four Chl a and two Phe a molecules. The interaction energy or coupling strength V12 between two interacting identical pigments can be calculated using the point-dipole approximation: V 12 = cκ D |R312 | in which V12 is given in cm−1 , c is a constant (5.04 in theory and about 5.3 in practice – Kwa 1993), κ is a dimensionless geometrical factor that can be 201 Table 1. Relative angles between calculated exciton bands of a trimer consisting of two Chl a and one Phe a molecule coordinated as the PM , BL , and HL molecules of the Rps. viridis RC, respectively (see Figure 1). Also listed are the dipole strengths in Debye2 . The simple point-dipole approximation was used (see text), and the monomer wavelengths were taken to be 674 nm for all three pigments. The dipole strengths of Chl a and Phe a were assumed to be 23 and 15 Debye2 , while c was assumed to be 5.3 Exciton band λ (nm) Dipole strength (Debye2 ) Angle with band 1 (degrees) Angle with band 2 (degrees) Angle with band 3 (degrees) 1 2 3 680.5 673.2 668.4 49.3 (82%) 8.0 (13%) 3.1 (5%) – 68.3 80.9 68.3 – 49.2 80.9 49.2 – calculated from the crystal structure, D is the dipole strength in (Debye)2 and R̄12 is the vector connecting the dipoles (in nm). The point-dipole approximation has been used successfully to map the excitonic interactions in structurally well-resolved FMO complexes from green sulphur bacteria (Louwe et al. 1997). Kwa (1993) used the point-dipole approximation to calculate the extent of excitonic coupling in a trimer consisting of one Phe a and two Chl a molecules positioned exactly as the HL , BL and PM molecules of the Rps. viridis RC (see also Table 1) and found three exciton bands with similar energies, intensities and orientations to those observed experimentally in PS II RC complexes. Calculations using a complete RC structure have suggested that the interaction energy between the two ‘special pair’ chlorophylls is a very critical parameter (Durrant et al. 1995). In a situation as in the bacterial reaction center, this interaction is so strong that a special pair dimer is formed with very minor contributions from the other RC pigments and with one major red-shifted low-exciton band. The other extreme is a situation in which this interaction is negligible, such as in the situation as in Table 1. In this situation, there are two groups of three chlorins which give rise to two sets of exciton bands, each delocalized over separate ‘arms’ of the reaction center, and thus to two nearly degenerate low-exciton bands. It is not clear which situation occurs in the PS II RC. Some observations suggest the presence of two nearly degenerate exciton bands (see below), suggesting a situation of a very small interaction energy between the two ‘special pair’ chlorophylls, but an intermediary situation of equal interaction energy between all chlorins (and thus of a ‘multimer’ of maximally six interacting chlorins) is also very well possible. The calculations by Kwa (1993) refer to a situation of degenerate site energies. This is, however, not very realistic, for two different reasons. First, the average site energies of pigments at different sites in a pigment–protein complex may not be the same (Gudowska-Nowak et al. 1990). Second, there is a spread of site energies of pigments at the same site in the complete ensemble of molecules (the inhomogeneous width or energetic disorder). An energetic disorder of the order of about 100 cm−1 was taken into account in the calculations presented by Durrant et al. (1995). This number may be higher if exchange narrowing of the absorption band has occurred. However, there are, as yet, no indications for large differences in the average site energies of the constituting chlorins. Reduction of the active pheophytin, chemical exchange of the inactive pheophytin molecule, or the formation of a triplet, either at very low temperatures on a chlorophyll oriented as the ‘accessory’ bacteriochlorophyll in the BRC, or at higher temperatures on a chlorophyll oriented as a ‘special pair’ bacteriochlorophyll in the BRC, results in all cases in an absorbance decrease around 680 nm (see above), which would not be expected if one of these components would have a very different average site energy. We conclude from these considerations that excitonic interactions between the six central chlorins of the PS II RC should not be neglected. An additional argument for this can be found in the most recent structural data (Rhee et al. 1998), which seem to suggest that the mutual distances between the six central chlorins, except those of the special pair, do not differ very much in the PS II RC and BRC and that the distance parameters in the exciton calculations mentioned above must have been estimated about cor- 202 rectly. Thus, a situation arises in the PS II RC in which the coupling between the chlorins has about the same value as the intrinsic disorder. Consequently, a multimer model arises in which there is no ‘special pair’ and in which for each realization of the disorder, the excitation may be dynamically localized on basically any combination of neighbouring chlorins. It is not difficult to imagine that at sufficiently low temperatures such a system shows a very complex behaviour, and that large variations of transition energies and dipole strengths can be expected from RC to RC (see, e.g. Figures 2 and 3 in Durrant et al. 1995). We note that the multimeric organization of the central chlorins of the PS II RC (and of any other organization of nearby chlorophylls) implies that the redox potentials of all constituting chlorophylls must be very high, as discussed recently by Mulkidjanian (1999). In this view, both the electrostatic influence of Arg-181 of the D2 subunit and a retarded protonic relaxation near the central chlorins contribute to the raise of the redox potential of the oxidized chlorophyll to up to about 1.15 V. Kinetics of energy transfer and trapping in the PS II RC The kinetics of energy transfer and trapping in the PS II RC have been studied by several groups using both ultrafast absorbance-difference and fluorescence techniques. After intense debates in the literature, it seems now generally accepted that primary charge separation at room temperature is a strongly multiphasic process with main components of about 8, 20 and 50 ps (see, e.g. Müller et al. 1996; Greenfield et al. 1997; Donovan et al. 1997; Klug et al. 1998). Thus, the formation of the primary radical pair takes more time in the isolated PS II RC than in the BRC, where most components are shorter than 10 ps. Excitation energy transfer within the six central core pigments has been reported to occur in maximally 500 fs (Durrant et al. 1992; Merry et al. 1996), whereas the energy transfer from the peripheral chlorophylls to the central core occurs in about 20–30 ps (Roelofs et al. 1993; Schelvis et al. 1994; Rech et al. 1994). Several origins have been proposed for the relatively slow charge separation kinetics in the PS II RC: 1) Delocalization of the excitation energy (Van Grondelle et al. 1994). In case of trap-limited kinetics (i.e. when the equilibration of the excitation energy is much faster than charge separation), the rate of charge separation is proportional to the probability to find the excitation on the primary electron donor. This probability is 1/N, where N is the number of pigments among which the excitation energy can be divided. In the PS II RC N equals 6–8, depending on the extent to which the two peripheral chlorophylls contribute to the equilibration of the excitation energy. In the BRC N equals 1, because only the lowest excitonic state of the special pair contributes to the 870 nm absorption band and the 800 and 770 nm states of the accessory chlorophylls and the pheophytins are too high in energy to become populated. 2) Slow energy transfer (Groot et al. 1997). It has been suggested on the basis of low-temperature experiments that the PS II RC contains pigments or states degenerate with P680 (the so-called ‘trap’-states, which trap excitation energy at cryogenic temperatures – Groot et al. 1996), that the energy transfer between the P680 and trap states is rate-limiting at intermediate temperatures, and that energy transfer may also be limiting at room temperature (Groot et al. 1997). This latter suggestion, however, seems at variance with the conclusion of Merry et al. (1996) and Leegwater et al. (1997) that the energy transfer between such states is about 500 fs at room temperature, which is too fast to be rate-limiting. 3) An initially about zero free energy difference of charge separation combined with slow charge-induced relaxations in the protein surroundings (Konermann et al. 1997a). These authors concluded that charge separation to the initial radical pair state is actually very fast in the PS II RC, that the yield of this first radical pair state is rather low because the equilibrium between the excited states and the radical pair is towards the excited states, and that subsequent energetic relaxations in the protein surroundings, induced by the creation of the two charges, shift this equilibrium towards the radical pair, ultimately resulting in a high yield of radical pair states and, apparently, relatively slow charge separation kinetics. An about zero free-energy difference of the primary charge separation reaction of PS II was also concluded from quantum chemical calculations using a density-functional theory (Blomberg et al. 1998). A consequence of the latter explanation is that the intrinsic rate of charge separation must be very fast, perhaps even in the order of a few hundred femtoseconds, and perhaps even faster than the rate of excitation equilibration in the central core part of the PS II RC. In the BRC, similar ultrafast rates have already been recorded for charge separation from the excited ‘accessory’ bacteriochlorophyll on the active 203 branch (Van Brederode et al. 1997, 1999). In intact bacteria, the importance of this very fast charge separation route is limited because most excitation energy will end up in the bacteriochlorophylls of the LH1 antenna, which are too low in energy to excite the accessory bacteriochlorophyll. In PS II, however, the energetic differences between the various chlorophylls are negligible at room temperature, and a significant part of the excitation energy can become localized on the ‘accessory’ chlorophylls. The delocalization of the excitation energy (the first possibility mentioned above) probably plays a significant role as well. It should, however, be realized that within the PS II RC the description in terms of trap-limited kinetics may not be appropriate in view of the possible faster rate of charge separation than excited state equilibration. If in the first charge separated state the electron hole is on the ‘accessory’ chlorophyll of the active branch, then electron transfer from one of the ‘special pair’ chlorophylls to this hole may occur, and if the free energy of the latter charge separated state is lower than the former, as is usually the case in bacterial reaction centers (Van Brederode and Van Grondelle 1999), then this electron transfer will shift the equilibrium between the excited state and the radical pair towards the radical pair, thus providing a stabilization of the charge separation reaction. A good candidate for this process is the ∼8 ps phase, which has been observed in both transient absorption (Müller et al. 1996; Greenfield et al. 1997) and ultrafast fluorescence (F. van Mourik et al., unpublished observations). Slow energy transfer reactions (mainly involving the ‘peripheral’ chlorophylls) and protein relaxations may then be primarily responsible for the ∼20 and 50 ps processes, respectively. In contrast to the situation at room temperature, only a few studies have been performed on the kinetics of charge separation at cryogenic temperatures. However, even from the limited available evidence it is clear that also the temperature dependence of charge separation differs considerably in RCs of PS II and purple bacteria. In the latter RC, charge separation occurs in about 3 ps at room temperature and in about 1 ps at 8 K (Fleming et al. 1988). In the PS II RC, however, the kinetics have been shown to slow down upon cooling. Data between 240 K and 20 K could best be described by phases of 0.4 ps and 18 ps at 240 K that progressively retard to 2.6 ps and 120 ps at 20 K (Groot et al. 1997), while at 7 K phases of about 5 and 120 ps were observed (Greenfield et al. 1999). The main implication of these results is that charge separation is activationless in the BRC and at least to some extent activated in the PS II RC. The situation of an initially about zero free energy difference of charge separation can have large effects on the efficiency of charge separation at very low temperatures. In such a situation, the charge separation reaction will have a small negative 1G in some RC complexes and a small positive 1G in others (depending on the energetic disorder of the system). In other words, charge separation will proceed nearly activationless and fast in some complexes, but activated and (very) slow in others, because the thermal energy will not be sufficient to activate the reaction at very low temperatures. The latter situation may lead to a longlived primary electron donor unable to perform charge separation at very low temperatures, thus explaining a number of very similar properties of P680 and the species responsible for the fluorescence at very low temperatures (Peterman et al. 1998). It is, therefore, very well possible that the chlorins responsible for the ‘trap’ of excitation energy at very low temperatures (Groot et al. 1994, 1996) and for P680 are the same and that the difference is only determined by the precise value of the free energy difference of charge separation. The excited-state radical pair equilibrium in PS II It is now beyond doubt that a dynamic equilibrium between the primary radical pair and chlorophyll excited states plays a very important role in the charge separation reaction in PS II RC complexes. A nice example was reported by Merry et al. (1998), who analyzed a set of isolated PS II RC complexes from site-directed mutants of the cyanobacterium Synechocystis PCC 6803, and found that a modulation of the free energy of the radical pair by changes in the direct environment of the P680+ or Phe− states correlates linearly with the equilibrium constant of the charge separation reaction and the quantum yield of charge separation. The free energy change of the charge separation reaction was estimated to be –27 meV for PS II RC complexes from green plants at a time of 60 ps after the initiation of the charge separation reaction (Merry et al. 1998). This number is probably less negative at earlier times and more negative at later times due to dynamic relaxations of the radical pair. We note that there may also be considerable variation in the free energy values at fixed times due to structural heterogeneities (Groot et al. 1994). 204 The influence of the equilibrium between the chlorophyll excited states and the radical pair in larger systems (such as PS II core complexes or membranes) is more difficult to establish. The exciton/radical pair equilibrium model, originally proposed by Van Grondelle (1985) and Van Gorkom (1985), has been used successfully to explain the time-resolved fluorescence kinetics in PS II core particles (Schatz et al. 1988), in particular because it provides a good explanation for the biphasic decay in ‘open’ centers (with oxidized quinone acceptor QA ). In this view, the first phase with about 50–100 ps kinetics reflects the trapping of the excitation energy in the primary radical pair P680+ Phe− , while the second phase with about 300–500 ps kinetics reflects the electron transfer from to Phe− toQA ,which indeed has been shown to proceed with these kinetics (see, e.g. Nuijs et al. 1986). There are a number of indications that the situation is much more complex than originally proposed by Schatz et al. (1988): (1) a 250–300 ps phase has also been detected in PS II core particles with doubly reduced QA (Van Mieghem et al. 1992), which indicates that this phase can not be attributed solely to charge stabilization; (2) a non-radiative decay channel has been proposed in the PS II RC with kinetics of, again, about 300 ps (Merry et al. 1998), and it can not be excluded that this process is also important for the 300–500 ps kinetics in PS II core particles; (3) the assumption of Schatz et al. (1988) of a fast (< 10 ps) equilibration in the PS II core antenna may not be correct. The most important hint for this is the 8 Å structure of the CP47–RC complex (Rhee et al. 1998), which seems to point to a relatively large distance between the groups of chlorophylls in the CP47 and RC parts of the complex, although it cannot be excluded yet that both groups of chlorophylls are connected by one or more linker chlorophylls not observed in the 8 Å structure. The helices I and II of the D1 and D2 proteins, however, seem to not be located between CP47 and the central part of the RC, which suggests that if the ‘peripheral’ chlorophylls of the PS II RC are really located near these helices, as is expected from their slow (∼ 20 ps) energy transfer to the central chlorins, then they probably do not function as ‘linkers’ of excitation energy transfer from core antenna to RC. Moreover, there is now conclusive evidence that the CP47 and CP43 core antenna proteins are located at opposite sites from the PS II RC (see, e.g. Eijckelhoff et al. 1997b; Harrer et al. 1998; Rhee et al. 1998) and that, therefore, direct excitation energy transfer between these antenna systems is impossible. All these new structural details point to a relatively large distance between core antenna and RC, a situation which is also encountered in PS I and purple bacterial photosystems (Van Grondelle et al. 1994), which favours a situation of transfer-to-thetrap-limited kinetics (Beekman et al. 1994; Valkunas et al. 1995), and which suggests that the 50–100 ps trapping phase may to at least some extent be determined by a slow transfer of excitation energy from the CP43 or CP47 core antenna to the PS II RC. We would furthermore like to mention that experiments on PS II membranes or complete thylakoids are very hard to interpret in view of the very heterogeneous organization of the peripheral antenna LHC II in these systems (see, e.g. Boekema et al. 1999 and Dekker et al. 1999). The observed trapping times of 80, 200 and 390 ps in ‘open’ PS II membranes (Vass et al. 1993) may relate to PS II centers with increasing amounts of LHC II, while minor phases with lifetimes of 2–3 ns may reflect the fluorescence properties of LHC II trimers or multimers that are not directly connected to the PS II core. Fluorescence lifetime measurements on carefully prepared PS II–LHC II supercomplexes and LHC II multimers will shed more light on this issue. Conclusions In Figures 4 and 5 we show schematic models that may account for the most elementary energy transfer and charge separation mechanisms at physiological temperatures discussed above for the PS II RC and the PS II core, respectively. The most essential features of the model of the processes in the PS II RC (Figure 4) are: (A) energy transfer on two levels: ultrafast population transfer of delocalized exciton states within the six central chlorins of the RC (with time constants of ∼100 and ∼500 fs), and slow (∼20 ps) energy transfer from the peripheral chlorophylls to the RC core, (B) ultrafast charge separation with a low yield, primarily starting from the singlet-excited ‘accessory’ Chl on the active branch, (C) cation transfer from the ‘accessory’ Chl to a ‘special pair’ Chl and/or charge separation starting from the ‘special pair’ Chl on the active branch (∼8 ps), and (D) slow relaxation (∼50 ps) of the radical pair by conformational changes of the protein. At very low temperatures, the triplet delocalization does not occur, indicating that process (C) does not occur to a significant extent, but now part of the energy transfer and/or initial charge separation re- 205 Figure 5. Schematic representation of the energy transfer and primary charge separation reactions in a monomeric PS II core complex. For details see text. Figure 4. Schematic representation of the energy transfer and primary charge separation reactions in the PS II RC. For details see text. actions may slow down very considerably. According to our model, both processes proceed between almost isoenergetic states, which in view of the disorder means that they can either be slightly downhill (and thus proceed fast) or slightly uphill (and thus become retarded very significantly at very low temperatures). The most essential point of the model in Figure 5 is a possibly slow energy transfer from the core antenna to the PS II RC. We note that all lifetimes depicted in both models are very rough estimates of the real values, and that at room temperature some energy transfer routes may proceed in the same time-range as charge separation. 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