Site-directed mutations were constructed in photosystem II of Synechocystis sp. PCC6803 in which the axial ligand, D1-His198, of special pair chlorophyll PD1 was replaced with Gln and where D1-Thr179, which overlies monomeric chlorophyll ChlD1, was replaced with His. The D1-His198Gln mutation produces a 3 nm displacement to the blue of the bleaching minimum in the Soret and in the Qy region of the – absorbance difference spectrum. To a first approximation, the bleaching can be assigned to the low-energy exciton transition of the special pair chlorophylls PD1/PD2. The D1-Thr179His mutation produces a 2 nm displacement to the red of the bleaching minimum in the Qy region of the (3P–1P) absorbance difference spectrum. Analysis of the flash-induced – and (3P–1P) absorbance difference spectra of both mutants compared with wild-type at 80 K indicate that the cation of the oxidized donor P+ is predominantly localized on the chlorophyll PD1 of the special pair and that the reaction centre triplet state, produced upon charge recombination from 3[P+Pheo−], when the primary quinone electron acceptor QA is doubly reduced, is primarily localized on ChlD1.
X-ray crystallography has recently provided a great deal of structural information on the content and arrangement of the cofactors of photosystem II (PSII; Kamiya & Shen 2003; Ferreira et al. 2004; Loll et al. 2005). According to the most recent structural analysis (Loll et al. 2005), the core complex of PSII of Thermosynechococcus elongatus comprises 20 polypeptide subunits and some 77 cofactors which include 35 Chl a, two Pheo a, 11 β-carotenes, one b-type cytochrome (cytb559), one c-type cytochrome (cytc550), two plastoquinones, a non-haem iron and the Mn4Ca cluster responsible for water oxidation. Of these, six Chl a, the two Pheo a, two β-carotene, the two plastoquinones, cytb559 and the Mn4Ca cluster are located in the reaction centre. Of these, with the exception of the cytb559 and one ligand to the Mn4Ca cluster, all are coordinated by D1 and D2, two homologous polypeptides that form a complex with C2 symmetry. All of the light-driven primary and secondary electron transfer reactions of the photosystem occur within the reaction centre.
Although, Chl a and Pheo a have replaced Bchl and Bpheo, and plastoquinone has replaced ubiquinone, the cofactors of the PSII reaction centre are arranged almost identically to those of the purple bacterial reaction centres. There are two branches of cofactors (A and B) in each of these reaction centres, though only the A-branch appears to be active for light-driven electron transport under normal physiological conditions. Absorption of a photon in reaction centre preparations results in the formation within 6–11 ps of P+PheoD1− (Groot et al. 2005; Holzwarth et al. 2006) and in 200–500 ps of the charge-separated state (P+QA−; Nuijs et al. 1986; Schatz et al. 1988; Trissl & Leibl 1989; Bernarding et al. 1994). Charge separation is orthogonal to the plane of the photosynthetic membrane. Moving across the membrane from the lumenal towards the stromal side of PSII, there is a pair of excitonically coupled chlorophylls PD1 and PD2, followed on each branch by a monomeric chlorophyll ChlD1 (A-branch) and ChlD2 (B-branch), followed by PheoD1 (A-branch) and PheoD2 (B-branch), followed by QA (A-branch) and QB (B-branch). In the bacterial reaction centres, primary electron transfer is initiated by excitation energy localized at the PA/PB chlorophyll special pair (homologue to PD1/PD2) from which an electron is transferred to BA (homologue to ChlD1) and then to PheoA (homologue to PheoD1) and then to QA and ultimately to QB. The strong electronic interaction between PA and PB (500–1000 cm−1; Woodbury & Allen 1995) produces a dominant low-energy exciton band in the Qy region, which constitutes the lowest excited singlet state of the reaction centre and which consequently concentrates the excitation energy at the site of primary electron transfer. In PSII, the greater physical separation of PD1 and PD2, the smaller dipole strength of the QY transition of Chl a and slight differences in macrocycle orientation produce a substantially weaker electronic interaction of approximately 150 cm−1 between the two (Raszewski et al. (2005); see also Diner & Rappaport (2002) for a review of earlier literature), such that these chlorophylls do not represent the lowest energy sink for the excitation energy. Even though the monomeric ChlD1 has the lowest site energy (Diner et al. 2001; Diner & Rappaport 2002; Raszewski et al. 2005), the chlorophylls of the PSII reaction centre show overall much less spectral differentiation from each other in the Qy region than is observed in the bacterial reaction centres. At liquid helium temperatures, however, the excitation energy is likely to be highly localized on ChlD1, making it probable that charge separation, which still occurs at this temperature, will be initiated by ChlD1* (Diner et al. 2001; Diner & Rappaport 2002; Raszewski et al. 2005), rather than from the excited [PD1/PD2]* special pair. A consequence of the weak spectral differentiation is that the excitation energy is much more distributed over the reaction centre pigments at physiological temperatures, including ChlD1, from which charge separation could still be initiated (Tetenkin et al. 1989; Rutherford & Nitschke 1996; Dekker & Van Grondelle 2000; Prokhorenko & Holzwarth 2000; Barter et al. 2003). Recent reports from two groups (Groot et al. 2005; Holzwarth et al. 2006) place the reduction of PheoD1, even at ambient temperature, occurring prior to the oxidation of PD1/PD2, implying that the cation of the primary radical pair is localized at another site, most probably ChlD1.
In order to better describe the localization of the excitation energy and of the electron transfer steps in PSII, we have been using site-directed mutagenesis to enhance the spectral differentiation of the pigments of PSII. Mutations that replace the axial ligand or that perturb a hydrogen bond to the C9=O ring V keto group of a particular chlorophyll modify the absorbance and vibrational spectra and occasionally the redox properties of that chlorophyll. The modified spectra provide spectroscopic labels on individual chlorophylls, allowing one to examine, to a first approximation, the role of that chlorophyll in energy and electron/hole localization.
2. Material and methods
(a) Mutant construction
The glucose-tolerant strain of the cyanobacterium Synechocystis sp. PCC 6803 (Williams 1988) was used for the construction of the site-directed mutants described in this paper. All strains were grown on BG-11 medium as described by Williams (1988) and Metz et al. (1989). Five millimolar glucose was included in the medium to allow the propagation of mutants inactive in PSII. Mutations at codons 179 and 198 of the psbA3 gene encoding the D1 polypeptide were introduced into the TD41 psbA triple deletion strain of Synechocystis sp. PCC 6803 according to Nixon et al. (1992).
(b) Isolation of PSII complexes
(c) Transient absorbance spectroscopy
Flash-induced absorbance difference spectra of – and (3P–1P) were recorded at low temperature as previously described (Hillmann et al. 1995) using a laboratory-built flash spectrometer. The (3P–1P) absorbance difference spectra were monitored in the presence of doubly reduced QA. QA was doubly reduced by the addition of 10 mM Na2S2O4 and preillumination at room temperature under anaerobic conditions. Under these conditions, the lifetime of 3P680 is similar to that of the chlorophyll a triplet state in solvents (τ≈1.4 ms (70%)/7 ms (30%) at 5 K and τ≈1.4 ms at T>80 K). The samples were excited with saturating flashes of approximately 15 μs in duration from a Xe flash lamp filtered by coloured glass (model CS4-96 from Corning).
3. Results and discussion
According to the 3 Å (Loll et al. 2005) and 3.5 Å (Ferreira et al. 2004) X-ray structures of PSII, D1-H198 and D2-H197 are the axial ligands, respectively, of the special pair chlorophylls PD1 and PD2. Site-directed mutations constructed at D1-His198 and D2-His197 were introduced and their effects on the (P+QA−–PQA) difference spectrum were examined for both sets of mutants in the Soret at ambient temperature using flash detection optical spectroscopy (Diner et al. 2001). Flash-induced absorbance changes in the Qy region at 80 K, attributed to the formation and the decay of P680+QA−, were measured in the presence of ferricyanide to pre-oxidize cytochrome-b559, thereby preventing the accumulation of QA−cytb559+, which is irreversibly formed with low quantum yield at low temperature (Hillmann & Schlodder 1995). The recombination of P680+QA− takes place within a few milliseconds. A satisfactory fit of the decay in wild-type (WT) PSII is obtained using two exponentials with half-lives of 1.8 ms (47%) and 6.3 ms (53%) plus a constant (1%). These difference spectra are dominated by P+–P with only a small contribution from QA−–QA. Mutations introduced at D1-198 produce displacements of the main bleaching bands to the blue of the (P+QA−–PQA) difference spectrum in both spectral regions. One of the most marked of these mutations is D1-His198Gln, which produces only a very small change in the P+/P reduction potential (Diner et al. 2001, 2004). The (P+QA−–PQA) difference spectrum of the WT measured at 80 K shows a large bleaching centred at 672.5 nm and a band shift to the blue with maxima and minima at 679 and 683 nm, respectively, and an inflection at 682 nm (figure 1a). The replacement of the D1-His198 by Gln produces a 3 nm displacement to the blue of the band bleaching with no change in the localization of the band shift. The band bleaching at 672.5 nm is assigned as a first approximation to the bleaching of the absorbance band of P680 due to its oxidation or, expressed more properly, to the disappearance of the low-energy exciton band of the special pair as in the bacterial reaction centres or in PSI. We would like to make it clear that the position of the bleaching band at 672.5 nm does not reflect the transition energy (site energy) of PD1 alone but corresponds to the transition from the groundstate to an exciton state which is dominated by a contribution from PD1 and PD2. That the spectral shift of the bleached band has indeed contributions from the PD1 chlorophyll was shown by measurement of the (YZ−YZ) difference spectrum (Diner et al. 2001) which, owing to retention within the reaction centre of the phenolic proton which dissociates upon oxidation, produces an electrochromic shift of the nearby PD1. The (P+–P) difference spectrum measured in the Soret also produces a 3 nm displacement to the blue (Diner et al. 2001). The equivalent site-directed mutation introduced at the PD2 axial ligand D2-197 (His to Gln) produces practically no change in the (P+–P) difference spectrum in the Soret region. These experiments are interpreted as signalling the localization of the P+ cation primarily on PD1 (Diner et al. 2001). The band shift to the blue in the (P+–P) difference spectrum is attributed to an electrochromic shift of the nearby ChlD1, induced by the positive charge on PD1 and a negative charge on QA. The band shift of ChlD1 is unaffected by the D1-His198Gln site-directed mutation.
When electron transfer to the first PSII quinone acceptor, QA, is blocked, the primary radical pair, P680+Pheo−, decays with high yield at low temperature, after singlet–triplet mixing in the radical pair, by recombination to the lowest excited triplet state of P680, 3P680 (Schlodder et al. 1998). The P680 triplet state has been monitored in the presence of singly reduced QA by transient absorbance spectroscopy (van Mieghem et al. 1995; Schlodder et al. 1998) and time-resolved electron paramagnetic resonance (EPR) (Feikema et al. 2005). Under these conditions, the triplet decay is two or three orders of magnitude faster than when QA is doubly reduced. Double reduction of QA can be achieved by illumination of PSII core complexes in the presence of dithonite (van Mieghem et al. 1995). Under these conditions, 3P680 can be detected by its characteristic spin-polarized EPR spectrum (Rutherford et al. 1981). High triplet yields have also been observed at low temperature in Dl/D2/cytb559 reaction centre preparations that lack QA (Durrant et al. 1990; Kwa et al. 1994). Orientation studies by van Mieghem et al. (1991) indicated that the triplet state was localized on a chlorophyll, the macrocycle ring plane of which was oriented at 30° with respect to the membrane plane. This orientation is consistent with the localization of the triplet state on either ChlD1 or ChlD2 but could not distinguish between the two. Fourier transform infrared spectroscopy (FTIR) measurements of the triplet also indicated that the triplet state was localized at T<80 K on a chlorophyll other than that on which the P+ cation was localized (Breton et al. 1997; Noguchi et al. 1998, 2001; Sarcina et al. 1998). This conclusion was based on differences in the position of the stretch vibrational mode of the C9O keto carbonyl arising from differences in hydrogen bond strength. Noguchi et al. (2001) attributed the triplet localization to ChlD1 at T<80 K but could not in principle distinguish ChlD1 from ChlD2. The triplet is expected to be localized on the chlorophyll with the lowest triplet energy, and not necessarily on the one on which it was initially formed. A comparison of the (3P–P) absorbance difference spectra in the WT and D1-His198Gln strains of Synechocystis 6803 at 80 K (Figure 1b) and at 5 K (not shown) indicated practically no effect of the mutation, consistent with the localization of the triplet state on a chlorophyll other than PD1 and probably ChlD1 considering that the triplet bleaching at 80 K, centred at approximately 682 nm, matches the inflection of the band shift observed in the (P+QA−–PQA) difference spectrum (figure 1a; Diner et al. 2001).
In order to pin down the assignment of the band shifted pigment in the – difference spectrum and the band bleaching in (3P–1P) spectrum to ChlD1, site-directed mutations were constructed at D1-Thr179. According to the X-ray crystal structures, this threonine overlies the Mg2+ of the ChlD1 (figure 2; Ferreira et al. 2004; Loll et al. 2005). However, the distance between the -OH group of this residue and the Mg2+ is too great (4.8 Å; Loll et al. 2005) for this residue to be a direct ligand to ChlD1. Instead, it is probable that a water molecule, hydrogen bonded to D1-Thr179, is the axial ligand. Figure 1c shows that when D1-Thr179 is replaced by His, there is a marked displacement of approximately 2 nm to the red relative to WT of the band shift observed in the – difference spectrum. In addition, the amplitudes of the positive and negative peaks become more equal. There is, however, no significant change in the location of the bleached component of the difference spectrum. These observations are the inverse of what was observed in the case of the D1-His198Gln mutation (figure 1a). Measurement of the (3P–1P) difference spectrum in the D1-Thr179His mutant cores at 80 K, generated in the presence of QAH2, shows a displacement to the red of approximately 2 nm relative to WT (figure 1d), whereas the D1-His198Gln mutation (Figure 1b) produced no change. The minimum of the (3P–1P) difference spectrum measured at 80 K in the mutant is located at 684.5 nm. The location of the band shift inflection point in the – difference spectra and the minima of the (3P–1P) difference spectrum all measured at 80 K are at about the same wavelength and track each other in the wild-type and in the two mutants. It must therefore be the same pigment molecule that produces the electrochromic shift in response to the charge on P+ (primarily on PD1) and on which the reaction centre triplet state is localized at ≤80 K. As the mutation at D1-Thr179 produces the same displacement of both spectra, it must be the monomeric ChlD1 that detects the charge on PD1+ and on which the triplet is localized.
The loss of histidine as the axial ligand in the D1-His198Gln and the replacement of Thr with His in the case of the D1-Thr179His produce 2–3 nm displacements of PD1 to the blue and of ChlD1 to the red, respectively. These observations are consistent with the electronic polarizability of the π-system of the imidazole moiety of histidine which stabilizes the excited state of nearby chromophores by dispersive interactions, shifting the absorbance spectrum of the pigment to the red whenever it is present. We cannot be sure that in the case of the D1-Thr179His mutant His has replaced a water molecule as a ligand to ChlD1. In fact, substitution of Thr with His using the program Swisspdbviewer and the T. elongatus crystal structure (Ferreira et al. 2004; Loll et al. 2005) suggests that His may still be too distant to be a direct ligand. However, even if a water molecule remains the immediate ligand, it is probably hydrogen bonded to the imidazole group of histidine. The polarizable His residue is still close enough to ChlD1 to exert a stabilizing effect on the excited state.
The (3P–1P) difference spectra were recorded on the millisecond time scale. The clear localization of the triplet energy on ChlD1 indicates that at 1 ms there is no triplet energy in the CP43 and CP47 antenna complexes. Presumably, any triplet generated in CP43 and CP47 disappears on a much more rapid time scale (≪1 ms), quenched by antenna carotenoids.
Having introduced spectroscopic tags that permit one to distinguish between the reaction centre core pigments that are probably involved in primary and secondary charge separation, we are now attempting to track the pathways for electron transfer involved in primary and secondary radical pair formation.
G. Brudvig. Yale University, Yale, USA. When measuring the equilibrium-binding constant for Mn2+ during the first step of photooxidation, it is important to determine the site occupancy before the flash, otherwise there will be a second-order Mn2+ oxidation reaction from oxidation of Mn2+ that diffuses into the OEC site during the lifetime of the charge separation. This could be a factor for different values for Km determined by different types of experiments.
B. Diner. The value that you are asking for is a Kd, a dissociation constant. We have been careful to specify that what we are measuring is a Km, which combines the binding and oxidation of Mn2+ following a light flash in a second-order mechanism, and not a Kd. There is a small amount of prebound Mn2+ (typically 20–30%), the oxidation of which appears to be first order. It is, however, very difficult to measure the Kd, both because the fraction of centres with prebound Mn2+ is small in wild-type (smaller still or non-existent in some D1-Asp170 mutants, see for example Diner & Nixon (1992)) and because the Kd is so low (≪1 μM) where adventitious Mn2+ is difficult to exclude. As long as one is clear about what one is measuring (Km versus Kd) and the concentration of the core complexes is submicromolar, it is possible to demonstrate a clear-cut dependence of the Km on site-directed mutations constructed at the oxygen-evolving complex (OEC). The binding and oxidation, represented by the Km, is consistent with what occurs physiologically in the first step of assembly of the OEC.
The authors would like to thank Marianne Çetin at the TU Berlin for excellent technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 498, TP A6 (to E.S.) and TP A7 (to Th.R.)), by the National Research Initiative of the USDA Cooperative State Research, Education and Extension Service, grant number 2003-35318-13589 (to B.A.D.) and by the BBSRC (Biotechnology and Biological Sciences Research Council) (to P.J.N.).
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