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Triplet state in photosystem II reaction centers as studied by 130 GHz EPR
Chemical Physics 294 (2003) 439–449 www.elsevier.com/locate/chemphys
Triplet state in photosystem II reaction centers as studied by 130 GHz EPR S.V. Pashenko a,1, I.I. Proskuryakov b, M. Germano H.J. van Gorkom a, P. Gast a,* a
a,2
,
Huygens Laboratory, Department of Biophysics, Leiden University, P.O. Box 9504, 2300 RA Leiden, The Netherlands b Institute of Basic Biological Problems RAS, Pushchino 142290, Russia Received 3 February 2003
Abstract The triplet state in the reaction centers of photosystem II was studied by high-field/high-frequency (130 GHz) EPR in the temperature range 50–90 K. At 50 K, the zero-field splitting parameters of the EPR spectrum correspond well to those of a chlorophyll monomer, in agreement with earlier studies. In the high magnetic field of 4.6 T employed in this study, the g-anisotropy of the triplet state becomes apparent and leads to a shift of the canonical positions of the triplet EPR spectrum. Assuming that triplet g- and zero-field tensors are coaxial, the principal values of the triplet g-tensor are determined to be 2.00324, 2.00306 and 2.00231 with an error of 0.00004. Lifting this assumption results in higher ganisotropy. At higher temperatures, the shape of the spectra changes significantly. Triplet excitation hopping involving the accessory chlorophyll BA and PA or PB (equivalents of the special pair bacteriochlorophylls of the bacterial reaction centers) can partially explain those changes, but the most prominent features indicate that also the electron acceptor IA (a pheophytin molecule) must be involved. Ó 2003 Elsevier B.V. All rights reserved.
1. Introduction The primary charge separation in photosystem II (PSII) takes place in a reaction center (RC) * Corresponding author. Tel.: +31-71-527-5979; fax: +31-71527-5819. E-mail address: [email protected] (P. Gast). 1 Present address: Baker Laboratory, Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853, USA. 2 Present address: Division of Physics and Astronomy, Department of Biophysics and Physics of Complex Systems, Faculty of Sciences, Vrije Universiteit, De Boelelaan 1081, 1081 HV Amsterdam, The Netherlands.
protein that can be isolated as the so-called D1D2-cytochromeb559 complex [1]. In the isolated PSII RC the charge separation cannot be stabilized by secondary electron transfer reactions and is lost in 50–100 ns by charge recombination [2–4], at low temperature largely via the triplet state [5]. In the presence of a strong magnetic field only the T0 sublevel of the triplet state can be populated by recombination of the radical pair, generating large polarized EPR signals that provide a very sensitive probe to study the electronic structure of the molecular species involved [6]. These signals could be especially helpful in the study of the PSII RC,
0301-0104/$ - see front matter Ó 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0301-0104(03)00324-0
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where optical spectroscopy is severely handicapped by the lack of spectral distinction between the pigments. The structure of the PSII RC, as now resolved by X-ray crystallography to a precision of 3.8 A [7,8], confirms computer models based on protein sequence, homology to the RC of purple bacteria, and a wide range of spectroscopic information [9]. Like in other photosystems, the photochemically active core of the PSII RC is an approximately C2 symmetric protein structure containing a Ushaped string of 6 chlorin cofactors arranged in two branches, A and B. In PSII it is made up by the D1 and D2 subunits, each binding two chlorophyll a molecules, P and B, and one pheophytin a molecule, I. The pigments are regularly spaced at , their site energies seem to be approximately 10 A similar [10], and the excitonic interactions and energy disorder are comparable, of the order of 100 cm1 [11]. The PSII RC contains two additional Chls, which are separated from the other pigments both spatially and energetically, and will not be considered in this work. Unlike other photosystems, PA and PB do not form a strongly coupled Ôspecial pairÕ where the singlet excited state (P ), the oxidized state (Pþ ), and the triplet state (3 P) are localized and the question which pigments contribute to each of these states in PSII is not easily answered. The excited state seems to be collective even at 5 K [12], although individual pigments may outweigh all others in their contribution to particular exciton states [13]. The oxidized state, on the other hand, appears to be essentially localized on PA , and becomes distributed over more Chls only at higher temperatures [14,15]. The localization of the triplet state on a monomeric Chl at cryogenic temperatures was first demonstrated with EPR by Rutherford et al. [16]. Later, van Mieghem et al. [17] showed that the plane of this chlorophyll molecule is tilted 30° relative to the membrane plane, suggesting that the triplet state may be located on one of the accessory chlorophylls, BA or BB . Recently, Germano et al. [13] have shown that this chlorophyll is BA . The changes in the shape of the EPR spectra at higher temperatures were interpreted as delocalization of triplet excitation to other cofactors [18,19].
Evidence for such delocalization over cofactors other than BA is supported by FTIR [20] and optical spectroscopy [21]. Diner et al. [21] have shown that the triplet state is shared with PA at temperatures higher than about 80 K. High-field/high-frequency EPR has been used to study the 3 P state in the RCs of the purple bacterium Rhodobacter (Rb.) sphaeroides [22,23]. In the high applied magnetic fields used in this technique, the g-anisotropy of the triplet state becomes resolved, and the quantum yield of 3 P becomes essentially anisotropic due to increased resolution of the g-anisotropies of the Pþ and the I A radicals [22,24]. In this work, we have studied high-field EPR spectra of the triplet state in D1 D2 cytb559 complexes (PSII RCs) in the temperature range 50–90 K. The triplet g-anisotropy was determined and the temperature-induced line-shape changes were analyzed in terms of a simple model of triplet hopping from BA to other cofactors.
2. Materials and methods D1 D2 cytb559 complexes (PSII RCs) were prepared from spinach Tris-washed BBY particles [25] according to the method described in [26]. Electron spin echo (ESE) measurements were carried out on a D-band pulsed EPR spectrometer operating at 130.0 0.5 GHz. The microwave bridge and the superconducting magnet were constructed at the Donetsk Physico-Technical Institute, Ukrainian National Academy of Sciences, Ukraine. The sample was illuminated through a set of narrow slits in the wall of the cylindrical microwave cavity. Maximum microwave power at the resonator was about 40 mW. To detect the triplet state of the primary donor, the microwave pulse sequence ‘‘laser flash–DAF– P1 –s–P2 –echo’’ was employed. DAF represents the delay after the laser flash, P1 and P2 represent the two microwave pulses and s is the time between the pulses. The pulse length for P1 and P2 was kept equal. The exact duration of the microwave pulse was chosen to give maximum echo intensity at given Q-value (about 1000) of the loaded cavity and was in the range 100–140 ns. The 3 P spectra
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were obtained by recording the echo intensity while slowly sweeping the magnetic field. The laser flash at 532 nm was created by a pulsed Nd:YAG laser (Spectron Laser Systems). The width of the laser flash was less than 10 ns. The light from the laser was guided down to the EPR cavity through a multi-fiber glass light-guide. The repetition rate of the whole pulse sequence was 20 Hz. Magnetophotoselection effects [27] were neglected, because the light coming out of the light-guide was depolarized, had a large scattering angle, and was additionally scattered on the slits of the cavity. A small light-independent background signal (probably of I A ) was carefully recorded and subtracted from all experimental spectra. The g-values were determined with high precision using the g-factor standard of powder MgO doped with Mn2þ [28]. A small amount of this powder was placed on the wall of the microwave cavity. The sample and the g-standard were in the cavity simultaneously during all measurements. When detected with ESE, the narrow Mn2þ lines produce a free induction decay signal rather than an echo signal because of fast spin–spin relaxation. This leads to the absence of Mn2þ lines in the ESEdetected experimental spectra. The g-standard spectrum was therefore measured in the continuous wave mode. Switching between the ESE mode and the continuous wave mode could be done without reloading or retuning the cavity, and without changing the static magnetic field.
3. Results Fig. 1 shows the light-induced triplet spectra in the PSII RCs at D-band at 50 K. The parameters D and E of the zero-field splitting (zfs) tensor are D ¼ 30:9 0:2 mT, E ¼ 4:6 0:1 mT, assuming assignment of the canonical positions as indicated in Fig. 1. These D and E values agree well with those obtained by others [16–19,29]; they indicate that at low temperature, the triplet state in PSII RC resides on a Chl monomer. Recently, it was shown that this Chl is the accessory chlorophyll BA [13]. The asymmetry in the canonical field positions of the 50 K spectrum is due to the g-tensor anisotropy of the triplet. In the high magnetic field
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approximation (B D, E), the electron spins are quantized along the field direction into Tþ , T0 and T states (corresponding to the projection of S along B of +1, 0 and )1, respectively). The energies of those states are [6] 1 Eþ ¼ geff be B þ DZZ ; E0 ¼ DZZ ; 2 1 E ¼ geff be B þ DZZ ; 2
ð1Þ
where DZZ depends on the orientation of the magnetic field 1 2 DZZ ¼ D bZ þ E b2X b2Y ; ð2Þ 3 and geff is defined as qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi geff ¼ gx2 b2x þ gy2 b2y þ gz2 b2z ;
ð3Þ
in which bx , by and bz are the coordinates of the unit vector b directed along the magnetic field in the zero-field frame. For each orientation of the magnetic field, there are two transitions in the vicinity of the resonant magnetic field corresponding to Eq. (1) – those between T0 and Tþ and between T0 and T . Assuming that the g- and zfs tensors are coaxial, the principal g-values of the triplet state may be obtained as follows: gxðy;zÞ ¼ gMn
BMn ; BXþ ðYþ ;Zþ Þ þ BX ðY ;Z Þ =2
ð4Þ
where gMn is the g-factor of the Mn2þ g-standard, BMn is the magnetic field corresponding to gMn , BX þ is the magnetic field corresponding to the Xþ canonical position, etc. Substituting the experimental magnetic field values into Eq. (4) results in the following principal g-values for the triplet state in D1 D2 cytb559 : gx ¼ 2:00324, gy ¼ 2:00306 and gz ¼ 2:00231, with an error of 0.00004. Since the g- and zfs tensors reflect quite different properties of the spin density distribution in the triplet state, they may be non-coaxial. We found it impossible to determine from the canonical positions of the experimental spectrum both the principal g-values and the orientation of the principal g-tensor axes relative to the zfs axes. However, for (almost) any assumed relative orientation of the gand zfs tensors, it is possible to find D, E and
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Fig. 1. Dependence of the experimental light-induced ESE-detected D-band EPR spectra in PSII RCs on delay between the microwave pulses, s (a, b), and delay after the laser flash, DAF, (c, d). Temperature ¼ 50 K. In (a) and (b), DAF ¼ 5 ls; in (c) and (d), s ¼ 100 ns. The spectra in (b, d) are the same as in (a, c), but they are normalized to have the same intensity as the spectrum recorded at DAF ¼ 5 ls and s ¼ 100 ns at magnetic field positions shown by the arrows. While (a) and (c) show the overall decay of the spectra, (b) and (d) demonstrate the anisotropy of the decay. The labels X , Y and Z mark the canonical positions.
principal g-values such that the obtained canonical positions will correspond quite well to the experimental data. (For example, assuming that gx and gy principal axes are rotated 40° in the zero-field XY plane gives gx ¼ 2:00367, gy ¼ 2:00263 and E ¼ 4:53 mT.) Orientations where the angles between the principal axes of the tensors are close to 45° may result in unrealistically large or small gvalues. The assumption of non-coaxiality of the gand zfs tensors always results in smaller D and E values and larger g-anisotropy. Thus, the g-values obtained above assuming coaxial g- and zfs tensors define the ‘‘minimal’’ g-anisotropy required to describe the canonical positions of the experimental spectrum. The small difference between gx and gy might indicate that the g-tensor is rotated
relative to the zfs tensor in the XY plane, but since gx > gy the angle of this rotation is less than 45°. The experimental triplet EPR spectra have been obtained by recording the electron spin echo intensity for a fixed delay-time s between the microwave pulses, while sweeping the magnetic field. With increasing s, the intensity of the echo decreases (Fig. 1(a)). The spin–spin echo decay time TM is 1100 ns at the Yþ canonical position. Fig. 1(b) shows the same spectra after normalization at the indicated field position and reveals that the spectral shape is slightly dependent on s. Therefore TM must also depend slightly on the field position, but this anisotropy does not significantly affect the spectra detected at s ¼ 100 ns.
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The triplet state decays to the ground state by the intersystem crossing (ISC) mechanism, which is selective with respect to the zero-field triplet states TX , TY and TZ [6]. The decay times for these states may be substantially different, leading to significant changes in the shape of the spectrum with increasing DAF. From the data in Fig. 1(c), the decay times of 1100, 750 and 1900 ls (100 ls) for the TX , TY and TZ states, respectively, are obtained. We estimate that at 50 K, the spin–lattice relaxation time T1 between the high-field triplet states Tþ , T0 and T is longer than 6 ms. Normalized triplet spectra measured at temperatures from 50 to 90 K are shown in Fig. 2. The overall intensity decreased significantly with increasing temperature, and no signal could be detected above 90 K. The observed changes in spectral shape are in general similar to those observed by Bosch et al. [18] and Kamlowski et al. [19] at X-band, but our spectra show more resolved features. With increasing temperature, several distinct features, marked by letters A–E, emerge. Feature C, as well as the decrease at the highest and lowest-field parts of the spectra (fea-
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tures A and E), may be indicative of the admixture of a triplet state with smaller zfs parameters (with feature C arising from its Yþ canonical peak). The nature of the features B and D is less obvious; as we will show below, they may be due to the admixture of a pheophytin triplet state. The triplet EPR spectrum decay rates with increasing DAF become faster at higher temperatures. The decay times at the field corresponding to the Yþ canonical position of the 50 K spectrum are 400 30, 190 20 and 125 20 ls at 70, 80 and 90 K, respectively. With increasing temperature, the decay becomes more isotropic.
4. Discussion We have presented here the EPR spectra of the triplet state in D1 D2 cytb559 PSII RCs measured for the first time at high microwave frequency of 130 GHz (Fig. 1) and for a range of temperatures (Fig. 2). The spectra were measured by recording the echo amplitude in an ESE experiment with a 5 ls delay time between laser excitation and detection and with 100 ns between the two microwave pulses. As is demonstrated in Figs. 1(a)–(d), this rapid detection scheme ensures that the spectral shapes are not distorted by fast relaxation, either by anisotropic TM or T1 or ISC. In the following discussion we will first address the possible origin of the amplitude asymmetry of the 50 K spectrum. In the second part we will consider the unusual temperature dependence of the triplet spectra. 4.1. Triplet state at 50 K
Fig. 2. The experimental triplet spectra at temperatures of 50, 70, 80 and 90 K. DAF ¼ 1 ls, s ¼ 100 ns. The spectra are normalized at the central positive peak of the 50 K spectrum (the 70, 80 and 90 K spectra are multiplied by a factor of 2.6, 8.9 and 16, respectively). The arrows labeled A–E mark the features emerging with increasing temperature that are discussed in the text.
In Fig. 3 the experimental triplet spectrum at 50 K from Fig. 1 is compared to a calculated triplet spectrum assuming that only the triplet T0 sublevel is populated, and that the triplet quantum yield is isotropic. Virtually the same lineshapes were obtained for any relative orientation of the g- and zfs tensors, if the triplet g-values were chosen so that the experimental canonical positions were reproduced well. The intensities of the experimental spectrum are more asymmetric than the simulation. This effect cannot arise from the decay with s or DAF, because the experimental spectrum in
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Fig. 3. Experimental triplet spectrum at 50 K (solid line; same as in Fig. 1), and the spectrum calculated assuming pure T0 -population and isotropic triplet quantum yield. The simulated spectrum is normalized to the maximum of the experimental one. The labels X , Y and Z mark the canonical positions of the triplet spectra. The shift of the X and Y positions relative to Z is due to the triplet g-anisotropy, resolved at D-band. Simulation parameters: D ¼ 30:9 mT, E ¼ 4:6 mT, the principal values of the triplet g-tensor gx ¼ 2:00324, gy ¼ 2:00306, gz ¼ 2:00231, and triplet zfs and g-tensors are assumed to be coaxial.
Fig. 3 is recorded at s ¼ 100 ns and DAF ¼ 5 ls, which are much shorter than the corresponding decay times. Besides, the anisotropy of the decay with s and DAF are ‘‘symmetric’’, see Figs. 1(b) and (d). Thus this asymmetry is not caused by asymmetry in the relaxation of the triplet state. Bosch et al. [18] concluded from the unusually fast relaxation of the triplet state in D1 D2 cytb559 that they found for T > 20 K that slow triplet-hopping between cofactors could start at this temperature. It therefore may be argued that the asymmetry found in Fig. 3 results from such hopping. However, Kamlowski et al. [19] have shown that at 40 K and at X-band the triplet spectrum in D1 D2 cytb559 is still symmetric in amplitude. Thus the asymmetry seen in Fig. 3 cannot be explained by triplet relaxation and is not found at X-band. At the high magnetic field of 4.6 T employed by D-band, significant changes in the intensity across the triplet spectrum of the primary donor of Rb. sphaeroides have been observed [22]. It was explained as the manifestation of the anisotropy of triplet quantum yield, arising from the g-anisotropy of the primary radical pair constituents, Pþ
and I A [24]. The primary RP recombination in PSII is in general similar to that in bacteria [30]. Then, in principle, the PSII triplet quantum yield might be also anisotropic at D-band. We have conducted extensive simulations to check if the anisotropy of the triplet quantum yield could lead to the observed asymmetry. The spectra were calculated as described in [22]. During the simulation, all the important parameters (the orientations of the triplet, Pþ and I A g-tensors relative to the zfs tensor, the rates of the primary RP decay, and the exchange and dipolar interactions in the RP) were varied. For the triplet g-tensor being coaxial to the zfs tensor, all the calculated spectra were symmetric. Significantly asymmetric triplet spectra were obtained only when the angle between one of the g-tensor axes and one of the zfs axes was close to 45°. None of the parameter combinations that were tried produced a good fit to the experimental spectrum. The triplet state EPR spectrum could also become asymmetric if the Tþ and T sublevels are differently populated. One of the mechanisms leading to population of Tþ and T is the triplet state formation through ISC from the P state. However, the population rates of Tþ and T are equal in the high-field approximation, thus, this mechanism results in equal populations of Tþ and T , and cannot explain the observed asymmetry. Finally, the triplet Tþ and T sublevels might become populated due to S–Tþ or S–T transitions in the primary RP. Above, when considering the case of purely T0 -populated triplet state, we have assumed that the exchange interaction in the primary radical pair is much smaller than the Zeeman splitting. (Then, the Tþ and T sublevels of the RP are well separated from T0 and S, and only S–T0 evolution in the primary RP may take place [31].) However, it is possible that the primary þ RP is a mixture of the Pþ A IA and BA IA states (see Fig. 4 in [10]). The exchange interaction in the latter may be orders of magnitude larger than in the former. The exchange interaction is proportional to expðr=dÞ, where r is the distance be [32]. tween the unpaired electrons and d ¼ 0:59 A The edge-to-edge distance between BA and IA in [7]. Assuming that the exPSII is about 4.5 A change interaction between Pþ A and IA is 1 mT in
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Rb. sphaeroides [24,33], where the P–I edge-to-edge , we can roughly estimate that it distance is 9.5 A will be 4.8 T between Bþ A and IA . This value is very close to the Zeeman splitting at a D-band field of 4.6 T. Such large exchange interaction could lead to the crossing of S with Tþ or T (depending on the sign of the interaction) and to the population of Tþ or T sublevels in the primary RP.
expðDG=kB T Þ, where kB is the Boltzmann constant and T is temperature. It is then convenient to express the rates as 1 DG=2 ¼ khop exp and s1 kB T 1 DG=2 ¼ khop exp þ : ð6Þ s2 kB T
4.2. Triplet state at >50 K
Then reaction (5) is described by two independent parameters, DG=T and the average, temperatureindependent rate of hopping khop . The EPR spectra in the presence of hopping were calculated as follows: for each of 3200 orientations of the RC in the magnetic field, the two field positions, where the T0 –Tþ transition of 3 C1 and 3 C2 occur, were determined. The ‘‘elementary’’ lineshape resulting from the interconversion between two lines at those positions was calculated according to [34]. The same was done for the T0 –T transitions. The triplet spectra were obtained by summing up the obtained elementary lineshapes. It was assumed that both 3 C1 and 3 C2 are exclusively T0 -populated, and that the triplet quantum yield is isotropic. The calculations were done for various values of the energy gap DG=T in the range from 0 to 3 and for the average hopping rate khop in the range from 0.1 to 103 mT (khop ðTÞ ¼ khop ðs1 Þ h=2pge be ). An average hopping rate of 0.1 mT corresponds well to the limit of slow hopping, and that of 103 mT – to the limit of fast hopping (the hopping rate being, respectively, much smaller and much larger than the values for the zfs parameters). In the slow hopping limit, the resulting spectrum is simply a sum of the 3 C1 and 3 C2 spectra weighted by their Boltzmann probabilities f1 and f2 . In the limit of fast hopping, the resulting spectrum is described by a zfs tensor
As mentioned above, Bosch et al. [18] have found that the decay of the triplet state EPR signal in PSII is governed by the triplet state lifetime at temperatures lower than 30 K, and by relaxation between the triplet sublevels at higher temperatures. The latter was found to be unusually fast as compared to the 3 Chl state in solution or 3 P in bacterial RCs. This enhancement was attributed to slow triplet hopping between different cofactors. Delocalization of the triplet excitation was also suggested in [20,21]. Although temperature-activated hopping of the triplet state in PSII is now an accepted phenomenon, its exact mechanism remains unknown and to our knowledge no simulations of the PSII triplet EPR spectra have been published that included hopping of the triplet state. In the following, we calculate the EPR spectra in the presence of triplet excitation hopping using a two-site model. Our aim is to check if such a simple model is capable to account for the temperature-induced changes in the experimental spectra (the emergence of the features A–E in Fig. 2), and to find out which cofactors could be involved in this process. To explain the changes in the experimental spectra at temperatures above 50 K (Fig. 2), we have simulated the EPR spectra resulting from triplet excitation hopping between two photosynthetic cofactors, C1 and C2 . If s1 and s2 are the hopping times, then the hopping scheme can be written as 3
1=s1
C1 C2 C1 3 C2 1=s2
ð5Þ
The probability of finding the triplet excitation on Ci is then fi ¼ si =ðs1 þ s2 Þ ði ¼ 1; 2Þ. If the free energy difference between 3 C1 and 3 C2 is DG, the ratio f2 =f1 is governed by the Boltzmann factor
^ 1 þ f2 D ^2; ^ fast ¼ f1 D D
ð7Þ
^ 1 and D ^ 2 are the zfs tensors of C1 and C2 . where D ^ 1 and D ^ 2 can have different orientations, Because D ^ fast does not coincide with either of them, and the D spectrum in the limit of fast hopping is different from that obtained for slow hopping. Temperature-activated hopping of a triplet state in PSII was detected in FTIR [20], EPR [18,19] and optical absorption [21] spectra. 3
3
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Fig. 4. Simulations of EPR spectra in the presence of triplet excitation hopping between BA and PA . Spectra for different in-plane orientations of zero-field tensors are shown: (a, b) principal axes of both zfs tensors are rotated 45° around z molecular axes; (c, d) zfs axes of 3 BA rotated for 80°, and that of 3 PA – for )60°. (a, c) Spectra for hopping rate khop ¼ 103 mT (fast hopping limit) and for different DG=T ; arrows show the spectra for which their maxima have the same field position as the feature C in Fig. 1. (b, d) Dependence of the spectra on the hopping rate for DG=T ¼ 1 (b) and DG=T ¼ 2 (d). The molecular axes were defined as follows: z-axis is perpendicular to the plane of the corresponding molecule, and x-axis is directed along the line connecting NII and NIV nitrogens of the chlorin ring (this convention is used widely for Chls – e.g. [6,35]. The directions of the molecular axes were calculated using the structure given in [7]. Since ring V of the chlorin cofactors is not shown in the structure, the assignment of labels NI –NIV was done so that the directions of the molecular axes of the cofactors would correspond as good as possible to those of purple bacteria (e.g. [5,36]).
Noguchi et al. [15] have suggested that the triplet resides on one of the accessory Chls at low temperatures, and is shared with PA and/or PB at higher temperatures. Diner et al. [21] concluded that the triplet is shared between BA and PA . Since BA , PA and PB are all Chl molecules, it is reasonable to assume that their triplet states have similar D and E parameters and g-tensors. Then the spectrum in the slow hopping limit is indistinguishable from the spectrum of the pure 3 BA state (spectra for khop ¼ 0:1 mT in Figs. 4(b) and (d)), and it cannot explain the temperature-activated changes.
If hopping is not slow, the shape of the resulting spectrum depends on the relative orientations of the monomers zfs tensors. The latter are determined by the molecular axes of the pigment cofactors. To define them, we have used the X-ray structure of Zouni et al. [7] (see caption of Fig. 4). resolution is sufficient to determine the Its 3.8 A orientations of the Chl and Pheo planes, but apparently not their in-plane rotation (only the pyrrole rings are shown, the ring V position of Chl and Pheo cofactors, necessary to identify the NI – NIV nitrogens, are not resolved in this structure). The molecular axes were defined as follows: z-axis
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is perpendicular to the plane of the corresponding molecule, and x-axis is directed along the line connecting NII and NIV nitrogens of the chlorin ring (this convention is used widely for Chls – e.g. [6,35]). Since ring V of the chlorin cofactors is not shown in the structure, the assignment of labels NI –NIV was done so that the directions of the inplane molecular axes of the cofactors would correspond as good as possible to those of purple bacteria (e.g. [5,36]). Figs. 4(a) and (b) show the spectra calculated for zfs tensors of 3 BA and 3 PA rotated 45° in-plane relative to thus chosen molecular axes of the cofactors. (For a monomeric 3 Chl, the angles between the X and Y zfs axes and the Qy optical transition are about 45° [29,37]. Qy is often taken to be parallel to the NI –NIII direction, i.e., to the molecular y-axis.) Fig. 4(a) shows the dependence of the shape of the spectrum on DG=T for the fast hopping limit. The field position of the maximum of the spectrum calculated for DG=T ¼ 1 (shown with an arrow in Fig. 4(a)) is about the same as the position of feature C in Fig. 2 – this means that temperatureactivated admixture of this spectrum could in principle explain the appearance of feature C. This spectrum, however, cannot explain any of the other experimental features, and this situation is not improved for slower hopping rates (Fig. 4(b)). The spectra for other values of DG=T and khop (those shown in Fig. 4(a), and calculated but not shown) deviate even more from the experimental spectrum. The molecular axes of the cofactors have been chosen rather arbitrarily, so we are in a position to test various in-plane rotations of the zfs axes. In such calculations, the described molecular axes are used simply as a reference coordinate system, so that they do not specify the direction of the actual Qy transition dipole. Figs. 4(c) and (d) show spectra calculated with zero-field axes of 3 BA rotated in-plane for 80°, and those of 3 PA – for )60° relative to this system. The admixture of the spectrum calculated for DG=T ¼ 2 in the fast hopping limit (shown with an arrow in Fig. 4(c)) could account for the experimental features A, C and E in Fig. 2. From the dependence of the spectra for DG ¼ 200 K on hopping rate (Fig. 4(d)) it is seen that features A and E (Fig. 2) can be explained if heterogeneity in khop is assumed.
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The same general conclusions may be obtained assuming 3 BA –3 PB hopping, because the planes of the PA and PB are close to parallel [7], and their zfs tensors differ only by in-plane rotation. Thus we conclude that hopping of triplet excitation from 3 BA to 3 PA or 3 PB could explain the features A, C, and E observed in the experiment (Fig. 2). However, none of the calculated spectra can account for the prominent features B and D. 4.2.1. 3 Pheo state The 250 K X-band EPR spectrum of PSII measured by Kamlowski et al. [19] has noticeable ‘‘wings’’. The field separation between the outmost positions of those wings is significantly larger than 2 30:9 mT (twice the parameter D of the 3 Chl a state), and thus those wings cannot be due to 3 Chl. In [29,38,39], formation of a triplet state of pheophytin molecule was observed in PSII RCs. This 3 Pheo state has jDj ¼ 36:1 mT and jEj ¼ 2:3 mT [39], and may account very well for the wings seen at 250 K in [19]. Thus, it is possible that the admixture of 3 Pheo is also present in our experimental spectra. The simulation of the 3 Pheo EPR spectrum is shown in Fig. 5(a). One can see immediately that the central peaks of the 3 Pheo EPR spectrum may account for the features B and D in experimental spectra. The other two spectra shown in Fig. 5(a) correspond to the 3 BA state (or to slow 3 BA –3 PA hopping), and to fast 3 BA –3 PA hopping (the latter can account for the features A, C and E, as discussed above). A combination of those three spectra has all the A–E features observed in the experiment (Fig. 5(b)). The remaining deviations are partially due to the asymmetry of the experimental spectra, which is discussed above. Simple addition of the 3 Pheo spectrum leads to the appearance of the ‘‘wings’’ in the higher and lower parts of the spectrum, which are not observed in our experiment (Fig. 5(b)). Those wings could disappear if 3 Pheo state also participates in the triplet hopping. Since at low temperature triplet excitation is localized on BA , there might be temperature-activated hopping of triplet excitation between BA and IA . From the calculations for triplet hopping between BA and IA (made similar to the 3 BA –3 PA hopping described above; spectra
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simulated very well assuming exclusive RPM-population; our simulations in Fig. 3 deviate somewhat from the experimental spectrum at 50 K, but, as discussed above, this deviation cannot be due to ISC). Partial population of the triplet state by ISC, however, cannot be easily ruled out at higher temperatures, where the spectral lineshape changes significantly. The direct evidence of ISC population comes from the E–A polarization of the outer wings of the 250 K EPR spectrum in [19]. Such polarization of the Zþ and Z canonical positions of a triplet state can not arise if the triplet state is produced through RPM, but it is easily explained by ISC [6,40] Partial ISC population of the triplet state could also account for the faster disappearance of the Z-peaks in the EPR spectra in [18].
5. Conclusions
Fig. 5. Demonstration that an admixture of 3 Pheo state spectrum could account for the features B and D in the experimental spectra. (a) EPR spectrum of 3 Pheo with D ¼ 36:1 mT and E ¼ 2:3 mT [29] (the g-tensor was assumed to be coaxial to zfs tensor and to have principal values gx ¼ 2:0038, gy ¼ 2:0033 and gz ¼ 2:0023); also shown are the spectrum of 3 BA and the spectrum in the presence of fast hopping between 3 BA and 3 PA (calculated for DG ¼ 200 K; this spectrum is the same as the one marked with the arrow in Fig. 4(c)). (b) Combination of the spectra from a (with weights 0.33, 0.37 and 0.30, respectively) as compared to the experimental spectra.
not shown), we conclude that this two-site model is also unable to explain all the features in the experimental spectra, and triplet hopping between three (BA , IA and PA or PB ) or more cofactors should be assumed. Van der Vos and Hoff [39] concluded that in PSII, the population of the triplet state may proceed not only through radical pair mechanism (RPM), but also through ISC from P . The analysis of the shape of the triplet EPR spectra rules out the major contribution of ISC mechanism at low temperatures (the 40 K X-band EPR spectrum in [19] is
The first high-field/high-frequency (D-band) EPR spectra of a triplet state in D1 D2 cytb559 complexes (PSII RCs) are reported for the temperature range 50–90 K. At 50 K, the zfs parameters of the EPR spectrum correspond well to those of a Chl monomer. The principal values of the triplet g-tensor are determined to be gx ¼ 2:00324, gy ¼ 2:00306 and gz ¼ 2:00231 with an error of 0.00004, assuming that triplet g-tensor and zfs tensors are coaxial. The g-anisotropy is increased when the assumption of coaxiality is lifted. The asymmetry seen in the experimental spectra can be qualitatively explained by the usual T0 population of the triplet state due to S–T0 interconversion in the primary radical pair. For that, the necessary conditions are that the triplet g- and zfs tensors are significantly non-collinear, and the triplet quantum yield is anisotropic. Another proposed mechanism to explain the asymmetry is the occurrence of S–Tþ or S–T transitions in the primary radical pair. This could probably happen if the primary radical pair exists as equilibrium þ between Pþ A IA and BA IA states. At temperatures higher than 50 K, the shape of the experimental spectra changes significantly. The model of triplet excitation hopping between BA and PA or PB can explain some of those changes, but not
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the most prominent features emerging in the experimental spectra with increasing temperature. We propose that in PSII, triplet excitation is hopping between at least three cofactors, BA ; PA and IA . Acknowledgements I.I.P. and P.G. acknowledge support from NWO Grant 047-009-008 of the Russian-Dutch Scientific Cooperation and from INTAS Grant 2000-404; M.G. acknowledges support from the Portuguese National Foundation for Scientific and Technical Research (JNICT), Grant PRAXISXXI/BD/2870/94. References [1] K. Satoh, in: D.R. Ort, C.F. Yocum (Eds.), Oxygenic Photosynthesis: The Light Reactions, Kluwer Academic Publishers, Dordrecht, 1996, p. 196. [2] R.V. Danielius, K. Satoh, P.J.M. van Kan, J.J. Plijter, A.M. Nuijs, H.J. van Gorkom, FEBS Lett. 213 (1987) 241. [3] Y. Takahashi, O. Hansson, P. Mathis, K. Satoh, Biochim. Biophys. Acta 893 (1987) 49. [4] P.J.M. van Kan, S.C.M. Otte, F.A.M. Kleinherenbrink, M.C. Nieveen, T.J. Aartsma, H.J. van Gorkom, Biochim. Biophys. Acta 1020 (1990) 146. [5] M.L. Groot, E.J.G. Peterman, P.J.M. van Kan, I.H.M. van Stokkum, J.P. Dekker, R. van Grondelle, Biophys. J. 67 (1994) 318. [6] D.E. Budil, M.C. Thurnauer, Biochim. Biophys. Acta 1057 (1991) 1. [7] A. Zouni, H.T. Witt, J. Kern, P. Fromme, N. Krauss, W. Saenger, P. Orth, Nature 409 (2001) 739. [8] N. Kamiya, J.R. Shen, Proc. Natl. Acad. Sci. USA 100 (2003) 98. [9] B. Svensson, C. Etchebest, P. Tuffery, P. van Kan, J. Smith, S. Styring, Biochemistry 35 (1996) 14486. [10] J.P. Dekker, R. van Grondelle, Photosynt. Res. 63 (2000) 195. [11] J.R. Durrant, D.R. Klug, S.L.S. Kwa, R. van Grondelle, G. Porter, J.P. Dekker, Proc. Natl. Acad. Sci. USA 92 (1995) 4798. [12] E.J.G. Peterman, H. van Amerongen, R. van Grondelle, J.P. Dekker, Proc. Natl. Acad. Sci. USA 95 (1998) 6128. [13] M. Germano, A.Y. Shkuropatov, H. Permentier, R. de Wijn, A.J. Hoff, V.A. Shuvalov, H.J. van Gorkom, Biochemistry 40 (2001) 11472. [14] S.E.J. Rigby, J.H.A. Nugent, P.J. OÕMalley, Biochemistry 33 (1994) 10043.
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Triplet state in photosystem II reaction centers as studied by 130 GHz EPR S.V. Pashenko a,1, I.I. Proskuryakov b, M. Germano H.J. van Gorkom a, P. Gast a,* a
a,2
,
Huygens Laboratory, Department of Biophysics, Leiden University, P.O. Box 9504, 2300 RA Leiden, The Netherlands b Institute of Basic Biological Problems RAS, Pushchino 142290, Russia Received 3 February 2003
Abstract The triplet state in the reaction centers of photosystem II was studied by high-field/high-frequency (130 GHz) EPR in the temperature range 50–90 K. At 50 K, the zero-field splitting parameters of the EPR spectrum correspond well to those of a chlorophyll monomer, in agreement with earlier studies. In the high magnetic field of 4.6 T employed in this study, the g-anisotropy of the triplet state becomes apparent and leads to a shift of the canonical positions of the triplet EPR spectrum. Assuming that triplet g- and zero-field tensors are coaxial, the principal values of the triplet g-tensor are determined to be 2.00324, 2.00306 and 2.00231 with an error of 0.00004. Lifting this assumption results in higher ganisotropy. At higher temperatures, the shape of the spectra changes significantly. Triplet excitation hopping involving the accessory chlorophyll BA and PA or PB (equivalents of the special pair bacteriochlorophylls of the bacterial reaction centers) can partially explain those changes, but the most prominent features indicate that also the electron acceptor IA (a pheophytin molecule) must be involved. Ó 2003 Elsevier B.V. All rights reserved.
1. Introduction The primary charge separation in photosystem II (PSII) takes place in a reaction center (RC) * Corresponding author. Tel.: +31-71-527-5979; fax: +31-71527-5819. E-mail address: [email protected] (P. Gast). 1 Present address: Baker Laboratory, Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853, USA. 2 Present address: Division of Physics and Astronomy, Department of Biophysics and Physics of Complex Systems, Faculty of Sciences, Vrije Universiteit, De Boelelaan 1081, 1081 HV Amsterdam, The Netherlands.
protein that can be isolated as the so-called D1D2-cytochromeb559 complex [1]. In the isolated PSII RC the charge separation cannot be stabilized by secondary electron transfer reactions and is lost in 50–100 ns by charge recombination [2–4], at low temperature largely via the triplet state [5]. In the presence of a strong magnetic field only the T0 sublevel of the triplet state can be populated by recombination of the radical pair, generating large polarized EPR signals that provide a very sensitive probe to study the electronic structure of the molecular species involved [6]. These signals could be especially helpful in the study of the PSII RC,
0301-0104/$ - see front matter Ó 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0301-0104(03)00324-0
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where optical spectroscopy is severely handicapped by the lack of spectral distinction between the pigments. The structure of the PSII RC, as now resolved by X-ray crystallography to a precision of 3.8 A [7,8], confirms computer models based on protein sequence, homology to the RC of purple bacteria, and a wide range of spectroscopic information [9]. Like in other photosystems, the photochemically active core of the PSII RC is an approximately C2 symmetric protein structure containing a Ushaped string of 6 chlorin cofactors arranged in two branches, A and B. In PSII it is made up by the D1 and D2 subunits, each binding two chlorophyll a molecules, P and B, and one pheophytin a molecule, I. The pigments are regularly spaced at , their site energies seem to be approximately 10 A similar [10], and the excitonic interactions and energy disorder are comparable, of the order of 100 cm1 [11]. The PSII RC contains two additional Chls, which are separated from the other pigments both spatially and energetically, and will not be considered in this work. Unlike other photosystems, PA and PB do not form a strongly coupled Ôspecial pairÕ where the singlet excited state (P ), the oxidized state (Pþ ), and the triplet state (3 P) are localized and the question which pigments contribute to each of these states in PSII is not easily answered. The excited state seems to be collective even at 5 K [12], although individual pigments may outweigh all others in their contribution to particular exciton states [13]. The oxidized state, on the other hand, appears to be essentially localized on PA , and becomes distributed over more Chls only at higher temperatures [14,15]. The localization of the triplet state on a monomeric Chl at cryogenic temperatures was first demonstrated with EPR by Rutherford et al. [16]. Later, van Mieghem et al. [17] showed that the plane of this chlorophyll molecule is tilted 30° relative to the membrane plane, suggesting that the triplet state may be located on one of the accessory chlorophylls, BA or BB . Recently, Germano et al. [13] have shown that this chlorophyll is BA . The changes in the shape of the EPR spectra at higher temperatures were interpreted as delocalization of triplet excitation to other cofactors [18,19].
Evidence for such delocalization over cofactors other than BA is supported by FTIR [20] and optical spectroscopy [21]. Diner et al. [21] have shown that the triplet state is shared with PA at temperatures higher than about 80 K. High-field/high-frequency EPR has been used to study the 3 P state in the RCs of the purple bacterium Rhodobacter (Rb.) sphaeroides [22,23]. In the high applied magnetic fields used in this technique, the g-anisotropy of the triplet state becomes resolved, and the quantum yield of 3 P becomes essentially anisotropic due to increased resolution of the g-anisotropies of the Pþ and the I A radicals [22,24]. In this work, we have studied high-field EPR spectra of the triplet state in D1 D2 cytb559 complexes (PSII RCs) in the temperature range 50–90 K. The triplet g-anisotropy was determined and the temperature-induced line-shape changes were analyzed in terms of a simple model of triplet hopping from BA to other cofactors.
2. Materials and methods D1 D2 cytb559 complexes (PSII RCs) were prepared from spinach Tris-washed BBY particles [25] according to the method described in [26]. Electron spin echo (ESE) measurements were carried out on a D-band pulsed EPR spectrometer operating at 130.0 0.5 GHz. The microwave bridge and the superconducting magnet were constructed at the Donetsk Physico-Technical Institute, Ukrainian National Academy of Sciences, Ukraine. The sample was illuminated through a set of narrow slits in the wall of the cylindrical microwave cavity. Maximum microwave power at the resonator was about 40 mW. To detect the triplet state of the primary donor, the microwave pulse sequence ‘‘laser flash–DAF– P1 –s–P2 –echo’’ was employed. DAF represents the delay after the laser flash, P1 and P2 represent the two microwave pulses and s is the time between the pulses. The pulse length for P1 and P2 was kept equal. The exact duration of the microwave pulse was chosen to give maximum echo intensity at given Q-value (about 1000) of the loaded cavity and was in the range 100–140 ns. The 3 P spectra
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were obtained by recording the echo intensity while slowly sweeping the magnetic field. The laser flash at 532 nm was created by a pulsed Nd:YAG laser (Spectron Laser Systems). The width of the laser flash was less than 10 ns. The light from the laser was guided down to the EPR cavity through a multi-fiber glass light-guide. The repetition rate of the whole pulse sequence was 20 Hz. Magnetophotoselection effects [27] were neglected, because the light coming out of the light-guide was depolarized, had a large scattering angle, and was additionally scattered on the slits of the cavity. A small light-independent background signal (probably of I A ) was carefully recorded and subtracted from all experimental spectra. The g-values were determined with high precision using the g-factor standard of powder MgO doped with Mn2þ [28]. A small amount of this powder was placed on the wall of the microwave cavity. The sample and the g-standard were in the cavity simultaneously during all measurements. When detected with ESE, the narrow Mn2þ lines produce a free induction decay signal rather than an echo signal because of fast spin–spin relaxation. This leads to the absence of Mn2þ lines in the ESEdetected experimental spectra. The g-standard spectrum was therefore measured in the continuous wave mode. Switching between the ESE mode and the continuous wave mode could be done without reloading or retuning the cavity, and without changing the static magnetic field.
3. Results Fig. 1 shows the light-induced triplet spectra in the PSII RCs at D-band at 50 K. The parameters D and E of the zero-field splitting (zfs) tensor are D ¼ 30:9 0:2 mT, E ¼ 4:6 0:1 mT, assuming assignment of the canonical positions as indicated in Fig. 1. These D and E values agree well with those obtained by others [16–19,29]; they indicate that at low temperature, the triplet state in PSII RC resides on a Chl monomer. Recently, it was shown that this Chl is the accessory chlorophyll BA [13]. The asymmetry in the canonical field positions of the 50 K spectrum is due to the g-tensor anisotropy of the triplet. In the high magnetic field
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approximation (B D, E), the electron spins are quantized along the field direction into Tþ , T0 and T states (corresponding to the projection of S along B of +1, 0 and )1, respectively). The energies of those states are [6] 1 Eþ ¼ geff be B þ DZZ ; E0 ¼ DZZ ; 2 1 E ¼ geff be B þ DZZ ; 2
ð1Þ
where DZZ depends on the orientation of the magnetic field 1 2 DZZ ¼ D bZ þ E b2X b2Y ; ð2Þ 3 and geff is defined as qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi geff ¼ gx2 b2x þ gy2 b2y þ gz2 b2z ;
ð3Þ
in which bx , by and bz are the coordinates of the unit vector b directed along the magnetic field in the zero-field frame. For each orientation of the magnetic field, there are two transitions in the vicinity of the resonant magnetic field corresponding to Eq. (1) – those between T0 and Tþ and between T0 and T . Assuming that the g- and zfs tensors are coaxial, the principal g-values of the triplet state may be obtained as follows: gxðy;zÞ ¼ gMn
BMn ; BXþ ðYþ ;Zþ Þ þ BX ðY ;Z Þ =2
ð4Þ
where gMn is the g-factor of the Mn2þ g-standard, BMn is the magnetic field corresponding to gMn , BX þ is the magnetic field corresponding to the Xþ canonical position, etc. Substituting the experimental magnetic field values into Eq. (4) results in the following principal g-values for the triplet state in D1 D2 cytb559 : gx ¼ 2:00324, gy ¼ 2:00306 and gz ¼ 2:00231, with an error of 0.00004. Since the g- and zfs tensors reflect quite different properties of the spin density distribution in the triplet state, they may be non-coaxial. We found it impossible to determine from the canonical positions of the experimental spectrum both the principal g-values and the orientation of the principal g-tensor axes relative to the zfs axes. However, for (almost) any assumed relative orientation of the gand zfs tensors, it is possible to find D, E and
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Fig. 1. Dependence of the experimental light-induced ESE-detected D-band EPR spectra in PSII RCs on delay between the microwave pulses, s (a, b), and delay after the laser flash, DAF, (c, d). Temperature ¼ 50 K. In (a) and (b), DAF ¼ 5 ls; in (c) and (d), s ¼ 100 ns. The spectra in (b, d) are the same as in (a, c), but they are normalized to have the same intensity as the spectrum recorded at DAF ¼ 5 ls and s ¼ 100 ns at magnetic field positions shown by the arrows. While (a) and (c) show the overall decay of the spectra, (b) and (d) demonstrate the anisotropy of the decay. The labels X , Y and Z mark the canonical positions.
principal g-values such that the obtained canonical positions will correspond quite well to the experimental data. (For example, assuming that gx and gy principal axes are rotated 40° in the zero-field XY plane gives gx ¼ 2:00367, gy ¼ 2:00263 and E ¼ 4:53 mT.) Orientations where the angles between the principal axes of the tensors are close to 45° may result in unrealistically large or small gvalues. The assumption of non-coaxiality of the gand zfs tensors always results in smaller D and E values and larger g-anisotropy. Thus, the g-values obtained above assuming coaxial g- and zfs tensors define the ‘‘minimal’’ g-anisotropy required to describe the canonical positions of the experimental spectrum. The small difference between gx and gy might indicate that the g-tensor is rotated
relative to the zfs tensor in the XY plane, but since gx > gy the angle of this rotation is less than 45°. The experimental triplet EPR spectra have been obtained by recording the electron spin echo intensity for a fixed delay-time s between the microwave pulses, while sweeping the magnetic field. With increasing s, the intensity of the echo decreases (Fig. 1(a)). The spin–spin echo decay time TM is 1100 ns at the Yþ canonical position. Fig. 1(b) shows the same spectra after normalization at the indicated field position and reveals that the spectral shape is slightly dependent on s. Therefore TM must also depend slightly on the field position, but this anisotropy does not significantly affect the spectra detected at s ¼ 100 ns.
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The triplet state decays to the ground state by the intersystem crossing (ISC) mechanism, which is selective with respect to the zero-field triplet states TX , TY and TZ [6]. The decay times for these states may be substantially different, leading to significant changes in the shape of the spectrum with increasing DAF. From the data in Fig. 1(c), the decay times of 1100, 750 and 1900 ls (100 ls) for the TX , TY and TZ states, respectively, are obtained. We estimate that at 50 K, the spin–lattice relaxation time T1 between the high-field triplet states Tþ , T0 and T is longer than 6 ms. Normalized triplet spectra measured at temperatures from 50 to 90 K are shown in Fig. 2. The overall intensity decreased significantly with increasing temperature, and no signal could be detected above 90 K. The observed changes in spectral shape are in general similar to those observed by Bosch et al. [18] and Kamlowski et al. [19] at X-band, but our spectra show more resolved features. With increasing temperature, several distinct features, marked by letters A–E, emerge. Feature C, as well as the decrease at the highest and lowest-field parts of the spectra (fea-
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tures A and E), may be indicative of the admixture of a triplet state with smaller zfs parameters (with feature C arising from its Yþ canonical peak). The nature of the features B and D is less obvious; as we will show below, they may be due to the admixture of a pheophytin triplet state. The triplet EPR spectrum decay rates with increasing DAF become faster at higher temperatures. The decay times at the field corresponding to the Yþ canonical position of the 50 K spectrum are 400 30, 190 20 and 125 20 ls at 70, 80 and 90 K, respectively. With increasing temperature, the decay becomes more isotropic.
4. Discussion We have presented here the EPR spectra of the triplet state in D1 D2 cytb559 PSII RCs measured for the first time at high microwave frequency of 130 GHz (Fig. 1) and for a range of temperatures (Fig. 2). The spectra were measured by recording the echo amplitude in an ESE experiment with a 5 ls delay time between laser excitation and detection and with 100 ns between the two microwave pulses. As is demonstrated in Figs. 1(a)–(d), this rapid detection scheme ensures that the spectral shapes are not distorted by fast relaxation, either by anisotropic TM or T1 or ISC. In the following discussion we will first address the possible origin of the amplitude asymmetry of the 50 K spectrum. In the second part we will consider the unusual temperature dependence of the triplet spectra. 4.1. Triplet state at 50 K
Fig. 2. The experimental triplet spectra at temperatures of 50, 70, 80 and 90 K. DAF ¼ 1 ls, s ¼ 100 ns. The spectra are normalized at the central positive peak of the 50 K spectrum (the 70, 80 and 90 K spectra are multiplied by a factor of 2.6, 8.9 and 16, respectively). The arrows labeled A–E mark the features emerging with increasing temperature that are discussed in the text.
In Fig. 3 the experimental triplet spectrum at 50 K from Fig. 1 is compared to a calculated triplet spectrum assuming that only the triplet T0 sublevel is populated, and that the triplet quantum yield is isotropic. Virtually the same lineshapes were obtained for any relative orientation of the g- and zfs tensors, if the triplet g-values were chosen so that the experimental canonical positions were reproduced well. The intensities of the experimental spectrum are more asymmetric than the simulation. This effect cannot arise from the decay with s or DAF, because the experimental spectrum in
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Fig. 3. Experimental triplet spectrum at 50 K (solid line; same as in Fig. 1), and the spectrum calculated assuming pure T0 -population and isotropic triplet quantum yield. The simulated spectrum is normalized to the maximum of the experimental one. The labels X , Y and Z mark the canonical positions of the triplet spectra. The shift of the X and Y positions relative to Z is due to the triplet g-anisotropy, resolved at D-band. Simulation parameters: D ¼ 30:9 mT, E ¼ 4:6 mT, the principal values of the triplet g-tensor gx ¼ 2:00324, gy ¼ 2:00306, gz ¼ 2:00231, and triplet zfs and g-tensors are assumed to be coaxial.
Fig. 3 is recorded at s ¼ 100 ns and DAF ¼ 5 ls, which are much shorter than the corresponding decay times. Besides, the anisotropy of the decay with s and DAF are ‘‘symmetric’’, see Figs. 1(b) and (d). Thus this asymmetry is not caused by asymmetry in the relaxation of the triplet state. Bosch et al. [18] concluded from the unusually fast relaxation of the triplet state in D1 D2 cytb559 that they found for T > 20 K that slow triplet-hopping between cofactors could start at this temperature. It therefore may be argued that the asymmetry found in Fig. 3 results from such hopping. However, Kamlowski et al. [19] have shown that at 40 K and at X-band the triplet spectrum in D1 D2 cytb559 is still symmetric in amplitude. Thus the asymmetry seen in Fig. 3 cannot be explained by triplet relaxation and is not found at X-band. At the high magnetic field of 4.6 T employed by D-band, significant changes in the intensity across the triplet spectrum of the primary donor of Rb. sphaeroides have been observed [22]. It was explained as the manifestation of the anisotropy of triplet quantum yield, arising from the g-anisotropy of the primary radical pair constituents, Pþ
and I A [24]. The primary RP recombination in PSII is in general similar to that in bacteria [30]. Then, in principle, the PSII triplet quantum yield might be also anisotropic at D-band. We have conducted extensive simulations to check if the anisotropy of the triplet quantum yield could lead to the observed asymmetry. The spectra were calculated as described in [22]. During the simulation, all the important parameters (the orientations of the triplet, Pþ and I A g-tensors relative to the zfs tensor, the rates of the primary RP decay, and the exchange and dipolar interactions in the RP) were varied. For the triplet g-tensor being coaxial to the zfs tensor, all the calculated spectra were symmetric. Significantly asymmetric triplet spectra were obtained only when the angle between one of the g-tensor axes and one of the zfs axes was close to 45°. None of the parameter combinations that were tried produced a good fit to the experimental spectrum. The triplet state EPR spectrum could also become asymmetric if the Tþ and T sublevels are differently populated. One of the mechanisms leading to population of Tþ and T is the triplet state formation through ISC from the P state. However, the population rates of Tþ and T are equal in the high-field approximation, thus, this mechanism results in equal populations of Tþ and T , and cannot explain the observed asymmetry. Finally, the triplet Tþ and T sublevels might become populated due to S–Tþ or S–T transitions in the primary RP. Above, when considering the case of purely T0 -populated triplet state, we have assumed that the exchange interaction in the primary radical pair is much smaller than the Zeeman splitting. (Then, the Tþ and T sublevels of the RP are well separated from T0 and S, and only S–T0 evolution in the primary RP may take place [31].) However, it is possible that the primary þ RP is a mixture of the Pþ A IA and BA IA states (see Fig. 4 in [10]). The exchange interaction in the latter may be orders of magnitude larger than in the former. The exchange interaction is proportional to expðr=dÞ, where r is the distance be [32]. tween the unpaired electrons and d ¼ 0:59 A The edge-to-edge distance between BA and IA in [7]. Assuming that the exPSII is about 4.5 A change interaction between Pþ A and IA is 1 mT in
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Rb. sphaeroides [24,33], where the P–I edge-to-edge , we can roughly estimate that it distance is 9.5 A will be 4.8 T between Bþ A and IA . This value is very close to the Zeeman splitting at a D-band field of 4.6 T. Such large exchange interaction could lead to the crossing of S with Tþ or T (depending on the sign of the interaction) and to the population of Tþ or T sublevels in the primary RP.
expðDG=kB T Þ, where kB is the Boltzmann constant and T is temperature. It is then convenient to express the rates as 1 DG=2 ¼ khop exp and s1 kB T 1 DG=2 ¼ khop exp þ : ð6Þ s2 kB T
4.2. Triplet state at >50 K
Then reaction (5) is described by two independent parameters, DG=T and the average, temperatureindependent rate of hopping khop . The EPR spectra in the presence of hopping were calculated as follows: for each of 3200 orientations of the RC in the magnetic field, the two field positions, where the T0 –Tþ transition of 3 C1 and 3 C2 occur, were determined. The ‘‘elementary’’ lineshape resulting from the interconversion between two lines at those positions was calculated according to [34]. The same was done for the T0 –T transitions. The triplet spectra were obtained by summing up the obtained elementary lineshapes. It was assumed that both 3 C1 and 3 C2 are exclusively T0 -populated, and that the triplet quantum yield is isotropic. The calculations were done for various values of the energy gap DG=T in the range from 0 to 3 and for the average hopping rate khop in the range from 0.1 to 103 mT (khop ðTÞ ¼ khop ðs1 Þ h=2pge be ). An average hopping rate of 0.1 mT corresponds well to the limit of slow hopping, and that of 103 mT – to the limit of fast hopping (the hopping rate being, respectively, much smaller and much larger than the values for the zfs parameters). In the slow hopping limit, the resulting spectrum is simply a sum of the 3 C1 and 3 C2 spectra weighted by their Boltzmann probabilities f1 and f2 . In the limit of fast hopping, the resulting spectrum is described by a zfs tensor
As mentioned above, Bosch et al. [18] have found that the decay of the triplet state EPR signal in PSII is governed by the triplet state lifetime at temperatures lower than 30 K, and by relaxation between the triplet sublevels at higher temperatures. The latter was found to be unusually fast as compared to the 3 Chl state in solution or 3 P in bacterial RCs. This enhancement was attributed to slow triplet hopping between different cofactors. Delocalization of the triplet excitation was also suggested in [20,21]. Although temperature-activated hopping of the triplet state in PSII is now an accepted phenomenon, its exact mechanism remains unknown and to our knowledge no simulations of the PSII triplet EPR spectra have been published that included hopping of the triplet state. In the following, we calculate the EPR spectra in the presence of triplet excitation hopping using a two-site model. Our aim is to check if such a simple model is capable to account for the temperature-induced changes in the experimental spectra (the emergence of the features A–E in Fig. 2), and to find out which cofactors could be involved in this process. To explain the changes in the experimental spectra at temperatures above 50 K (Fig. 2), we have simulated the EPR spectra resulting from triplet excitation hopping between two photosynthetic cofactors, C1 and C2 . If s1 and s2 are the hopping times, then the hopping scheme can be written as 3
1=s1
C1 C2 C1 3 C2 1=s2
ð5Þ
The probability of finding the triplet excitation on Ci is then fi ¼ si =ðs1 þ s2 Þ ði ¼ 1; 2Þ. If the free energy difference between 3 C1 and 3 C2 is DG, the ratio f2 =f1 is governed by the Boltzmann factor
^ 1 þ f2 D ^2; ^ fast ¼ f1 D D
ð7Þ
^ 1 and D ^ 2 are the zfs tensors of C1 and C2 . where D ^ 1 and D ^ 2 can have different orientations, Because D ^ fast does not coincide with either of them, and the D spectrum in the limit of fast hopping is different from that obtained for slow hopping. Temperature-activated hopping of a triplet state in PSII was detected in FTIR [20], EPR [18,19] and optical absorption [21] spectra. 3
3
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Fig. 4. Simulations of EPR spectra in the presence of triplet excitation hopping between BA and PA . Spectra for different in-plane orientations of zero-field tensors are shown: (a, b) principal axes of both zfs tensors are rotated 45° around z molecular axes; (c, d) zfs axes of 3 BA rotated for 80°, and that of 3 PA – for )60°. (a, c) Spectra for hopping rate khop ¼ 103 mT (fast hopping limit) and for different DG=T ; arrows show the spectra for which their maxima have the same field position as the feature C in Fig. 1. (b, d) Dependence of the spectra on the hopping rate for DG=T ¼ 1 (b) and DG=T ¼ 2 (d). The molecular axes were defined as follows: z-axis is perpendicular to the plane of the corresponding molecule, and x-axis is directed along the line connecting NII and NIV nitrogens of the chlorin ring (this convention is used widely for Chls – e.g. [6,35]. The directions of the molecular axes were calculated using the structure given in [7]. Since ring V of the chlorin cofactors is not shown in the structure, the assignment of labels NI –NIV was done so that the directions of the molecular axes of the cofactors would correspond as good as possible to those of purple bacteria (e.g. [5,36]).
Noguchi et al. [15] have suggested that the triplet resides on one of the accessory Chls at low temperatures, and is shared with PA and/or PB at higher temperatures. Diner et al. [21] concluded that the triplet is shared between BA and PA . Since BA , PA and PB are all Chl molecules, it is reasonable to assume that their triplet states have similar D and E parameters and g-tensors. Then the spectrum in the slow hopping limit is indistinguishable from the spectrum of the pure 3 BA state (spectra for khop ¼ 0:1 mT in Figs. 4(b) and (d)), and it cannot explain the temperature-activated changes.
If hopping is not slow, the shape of the resulting spectrum depends on the relative orientations of the monomers zfs tensors. The latter are determined by the molecular axes of the pigment cofactors. To define them, we have used the X-ray structure of Zouni et al. [7] (see caption of Fig. 4). resolution is sufficient to determine the Its 3.8 A orientations of the Chl and Pheo planes, but apparently not their in-plane rotation (only the pyrrole rings are shown, the ring V position of Chl and Pheo cofactors, necessary to identify the NI – NIV nitrogens, are not resolved in this structure). The molecular axes were defined as follows: z-axis
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is perpendicular to the plane of the corresponding molecule, and x-axis is directed along the line connecting NII and NIV nitrogens of the chlorin ring (this convention is used widely for Chls – e.g. [6,35]). Since ring V of the chlorin cofactors is not shown in the structure, the assignment of labels NI –NIV was done so that the directions of the inplane molecular axes of the cofactors would correspond as good as possible to those of purple bacteria (e.g. [5,36]). Figs. 4(a) and (b) show the spectra calculated for zfs tensors of 3 BA and 3 PA rotated 45° in-plane relative to thus chosen molecular axes of the cofactors. (For a monomeric 3 Chl, the angles between the X and Y zfs axes and the Qy optical transition are about 45° [29,37]. Qy is often taken to be parallel to the NI –NIII direction, i.e., to the molecular y-axis.) Fig. 4(a) shows the dependence of the shape of the spectrum on DG=T for the fast hopping limit. The field position of the maximum of the spectrum calculated for DG=T ¼ 1 (shown with an arrow in Fig. 4(a)) is about the same as the position of feature C in Fig. 2 – this means that temperatureactivated admixture of this spectrum could in principle explain the appearance of feature C. This spectrum, however, cannot explain any of the other experimental features, and this situation is not improved for slower hopping rates (Fig. 4(b)). The spectra for other values of DG=T and khop (those shown in Fig. 4(a), and calculated but not shown) deviate even more from the experimental spectrum. The molecular axes of the cofactors have been chosen rather arbitrarily, so we are in a position to test various in-plane rotations of the zfs axes. In such calculations, the described molecular axes are used simply as a reference coordinate system, so that they do not specify the direction of the actual Qy transition dipole. Figs. 4(c) and (d) show spectra calculated with zero-field axes of 3 BA rotated in-plane for 80°, and those of 3 PA – for )60° relative to this system. The admixture of the spectrum calculated for DG=T ¼ 2 in the fast hopping limit (shown with an arrow in Fig. 4(c)) could account for the experimental features A, C and E in Fig. 2. From the dependence of the spectra for DG ¼ 200 K on hopping rate (Fig. 4(d)) it is seen that features A and E (Fig. 2) can be explained if heterogeneity in khop is assumed.
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The same general conclusions may be obtained assuming 3 BA –3 PB hopping, because the planes of the PA and PB are close to parallel [7], and their zfs tensors differ only by in-plane rotation. Thus we conclude that hopping of triplet excitation from 3 BA to 3 PA or 3 PB could explain the features A, C, and E observed in the experiment (Fig. 2). However, none of the calculated spectra can account for the prominent features B and D. 4.2.1. 3 Pheo state The 250 K X-band EPR spectrum of PSII measured by Kamlowski et al. [19] has noticeable ‘‘wings’’. The field separation between the outmost positions of those wings is significantly larger than 2 30:9 mT (twice the parameter D of the 3 Chl a state), and thus those wings cannot be due to 3 Chl. In [29,38,39], formation of a triplet state of pheophytin molecule was observed in PSII RCs. This 3 Pheo state has jDj ¼ 36:1 mT and jEj ¼ 2:3 mT [39], and may account very well for the wings seen at 250 K in [19]. Thus, it is possible that the admixture of 3 Pheo is also present in our experimental spectra. The simulation of the 3 Pheo EPR spectrum is shown in Fig. 5(a). One can see immediately that the central peaks of the 3 Pheo EPR spectrum may account for the features B and D in experimental spectra. The other two spectra shown in Fig. 5(a) correspond to the 3 BA state (or to slow 3 BA –3 PA hopping), and to fast 3 BA –3 PA hopping (the latter can account for the features A, C and E, as discussed above). A combination of those three spectra has all the A–E features observed in the experiment (Fig. 5(b)). The remaining deviations are partially due to the asymmetry of the experimental spectra, which is discussed above. Simple addition of the 3 Pheo spectrum leads to the appearance of the ‘‘wings’’ in the higher and lower parts of the spectrum, which are not observed in our experiment (Fig. 5(b)). Those wings could disappear if 3 Pheo state also participates in the triplet hopping. Since at low temperature triplet excitation is localized on BA , there might be temperature-activated hopping of triplet excitation between BA and IA . From the calculations for triplet hopping between BA and IA (made similar to the 3 BA –3 PA hopping described above; spectra
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simulated very well assuming exclusive RPM-population; our simulations in Fig. 3 deviate somewhat from the experimental spectrum at 50 K, but, as discussed above, this deviation cannot be due to ISC). Partial population of the triplet state by ISC, however, cannot be easily ruled out at higher temperatures, where the spectral lineshape changes significantly. The direct evidence of ISC population comes from the E–A polarization of the outer wings of the 250 K EPR spectrum in [19]. Such polarization of the Zþ and Z canonical positions of a triplet state can not arise if the triplet state is produced through RPM, but it is easily explained by ISC [6,40] Partial ISC population of the triplet state could also account for the faster disappearance of the Z-peaks in the EPR spectra in [18].
5. Conclusions
Fig. 5. Demonstration that an admixture of 3 Pheo state spectrum could account for the features B and D in the experimental spectra. (a) EPR spectrum of 3 Pheo with D ¼ 36:1 mT and E ¼ 2:3 mT [29] (the g-tensor was assumed to be coaxial to zfs tensor and to have principal values gx ¼ 2:0038, gy ¼ 2:0033 and gz ¼ 2:0023); also shown are the spectrum of 3 BA and the spectrum in the presence of fast hopping between 3 BA and 3 PA (calculated for DG ¼ 200 K; this spectrum is the same as the one marked with the arrow in Fig. 4(c)). (b) Combination of the spectra from a (with weights 0.33, 0.37 and 0.30, respectively) as compared to the experimental spectra.
not shown), we conclude that this two-site model is also unable to explain all the features in the experimental spectra, and triplet hopping between three (BA , IA and PA or PB ) or more cofactors should be assumed. Van der Vos and Hoff [39] concluded that in PSII, the population of the triplet state may proceed not only through radical pair mechanism (RPM), but also through ISC from P . The analysis of the shape of the triplet EPR spectra rules out the major contribution of ISC mechanism at low temperatures (the 40 K X-band EPR spectrum in [19] is
The first high-field/high-frequency (D-band) EPR spectra of a triplet state in D1 D2 cytb559 complexes (PSII RCs) are reported for the temperature range 50–90 K. At 50 K, the zfs parameters of the EPR spectrum correspond well to those of a Chl monomer. The principal values of the triplet g-tensor are determined to be gx ¼ 2:00324, gy ¼ 2:00306 and gz ¼ 2:00231 with an error of 0.00004, assuming that triplet g-tensor and zfs tensors are coaxial. The g-anisotropy is increased when the assumption of coaxiality is lifted. The asymmetry seen in the experimental spectra can be qualitatively explained by the usual T0 population of the triplet state due to S–T0 interconversion in the primary radical pair. For that, the necessary conditions are that the triplet g- and zfs tensors are significantly non-collinear, and the triplet quantum yield is anisotropic. Another proposed mechanism to explain the asymmetry is the occurrence of S–Tþ or S–T transitions in the primary radical pair. This could probably happen if the primary radical pair exists as equilibrium þ between Pþ A IA and BA IA states. At temperatures higher than 50 K, the shape of the experimental spectra changes significantly. The model of triplet excitation hopping between BA and PA or PB can explain some of those changes, but not
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