Sinjini
Bhattacharjee
,
Frank
Neese
and
Dimitrios A.
Pantazis
*
Max-Planck-Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 1, 45470 Mülheim an der Ruhr, Germany. E-mail: dimitrios.pantazis@kofo.mpg.de
First published on 17th August 2023
In oxygenic photosynthesis sunlight is harvested and funneled as excitation energy into the reaction center (RC) of Photosystem II (PSII), the site of primary charge separation that initiates the photosynthetic electron transfer chain. The chlorophyll ChlD1 pigment of the RC is the primary electron donor, forming a charge-separated radical pair with the vicinal pheophytin PheoD1 (ChlD1+PheoD1−). To avert charge recombination, the electron is further transferred to plastoquinone QA, whereas the hole relaxes to a central pair of chlorophylls (PD1PD2), subsequently driving water oxidation. Spin-triplet states can form within the RC when forward electron transfer is inhibited or back reactions are favored. This can lead to formation of singlet dioxygen, with potential deleterious effects. Here we investigate the nature and properties of triplet states within the PSII RC using a multiscale quantum-mechanics/molecular-mechanics (QM/MM) approach. The low-energy spectrum of excited singlet and triplet states, of both local and charge-transfer nature, is compared using range-separated time-dependent density functional theory (TD-DFT). We further compute electron paramagnetic resonance properties (zero-field splitting parameters and hyperfine coupling constants) of relaxed triplet states and compare them with available experimental data. Moreover, the electrostatic modulation of excited state energetics and redox properties of RC pigments by the semiquinone QA− is described. The results provide a detailed electronic-level understanding of triplet states within the PSII RC and form a refined basis for discussing primary and secondary electron transfer, charge recombination pathways, and possible photoprotection mechanisms in PSII.
Fig. 1 Reaction center pigments and other important cofactors, with schematic representation of electron flow along the active branch of Photosystem II. |
The excitation energy transfer from external light harvesting complexes and the internal antennae CP43 and CP47 initiates the electron transfer process along the D1 branch of the RC (Fig. 1). Charge-transfer excited states of mostly ChlD1δ+PheoD1δ− character are created, leading to formation of the primary charge separated radical pair ChlD1+PheoD1− (ref. 6 and 9–18) and the cationic charge is then distributed over the PD1PD2 pair (often referred to as P680+).10,19–21 This highly oxidizing radical cation (estimated Em of 1.1–1.3 V) is the strongest known oxidant in biology and drives water oxidation at the oxygen-evolving complex.3,22,23 Under normal conditions, charge recombination of the initially formed radical pairs [ChlD1+PheoD1−]4,11,16 (or possibly [PD1+PheoD1−] in some reaction centers)24 is prevented by forward electron transfer from PheoD1 to the primary plastoquinone acceptor QA within a few hundred ps. This leads to formation of the “closed RC” state with a reduced QA.5,25–29 If the plastoquinone pool remains reduced, electron transfer from QA− to the mobile acceptor QB is inhibited, thus preventing further electron transfer from PheoD1 to QA. This can facilitate charge recombination30–35 within the RC and enable formation of chlorophyll triplet states prior to relaxation to the ground state.14,30–33,36–46 Triplet states are detrimental as they can readily generate chemically active singlet oxygen (1O2) that reacts with the protein causing oxidative stress.47,48 The D1 protein embeds most crucial redox cofactors in PSII, including the oxygen-evolving complex (OEC), and thus photodamage can lead to a disruption of the entire photosynthetic machinery. Correlation has been reported between 1O2 production and the extent of photodamage of the D1 protein on exposure to excess light.49–51 All photosynthetic organisms therefore naturally adopt intrinsic strategies of photoprotection by efficiently quenching chlorophyll triplet states either by redox active cofactors (e.g. QA− in the RC)29,32,52 or carotenoids53–55 (e.g. in the bacterial RC or antenna complexes), but the exact molecular mechanisms of these phenomena are not fully understood. Therefore, it is useful to have a reliable description of the nature and localization of triplet states, as an essential basis for understanding photoprotection mechanisms in PSII.
Chlorophyll triplet states, in addition to being highly reactive, serve as chemical probes to investigate primary electron transfer pathways and characterize the chemical environment of photosynthetic reaction center pigments.44 Electron paramagnetic resonance (EPR) and electron–nuclear double resonance (ENDOR) spectroscopies29,37,39,44,56–69 and other spectroscopic approaches including Fourier-transform infrared (FTIR) and optically detected magnetic resonance (ODMR)36,38,39,51,54,70–76 suggest that the “primary donor” triplet is located on an individual accessory chlorophyll (ChlD1 or ChlD2) at cryogenic temperatures.30,56–58,61,77 It has also been suggested that the triplet is partially shared with other chlorophylls at the RC at higher temperatures, but this has not been well characterized.33,62 It is important to note that many studies report varying observations depending on the type of preparation and conditions used, as in the case of D1D2Cytb559 samples60,62,78,79 or samples with chemically reduced quinone (QA−/QA2−).25,29,46,52
Various chemical and photo-physical properties of pigments such as site energies and redox potentials10,22,43,80,81 are directly or indirectly controlled by the surrounding protein matrix,82–84 as already established in the case of charge transfer states involving the RC pigments.12,24 From a methodological perspective, this establishes the need for multilayer approaches to provide an accurate quantitative description of how inter-pigment and pigment–protein interactions determine spectroscopic properties. Previous excited state calculations based on time-dependent density functional theory (TD-DFT) and quantum-mechanics/molecular-mechanics (QM/MM) simulations on pigment assemblies have shown that the lowest singlet excitations in the RC are characterized by a mixture of excitonic and [ChlD1δ+PheoD1δ−] or [PD1δ+PheoD1δ−] charge-transfer (CT) character.12,24,85,86 However, a coherent description of excited and ground triplet states is lacking. The excitation profiles of all RC pigments in their triplet states are important elements for establishing possible routes of triplet delocalization87,88 and triplet–triplet energy transfer (T-TET) onto other pigments within the PSII core complex.89
In this work, we use a membrane-bound model of an entire PSII monomer as the basis for multiscale quantum-mechanics/molecular-mechanics (QM/MM) modelling to study singlet–triplet excitations as well as relaxed triplet states within the RC pigments. The quantum chemical descriptions of both local and charge-transfer excitations in oligomeric assemblies are obtained by range-separated time-dependent density functional theory (TD-DFT). We employ our QM/MM approach to also compute EPR properties of all triplet states localized on each chromophore, and compare the results with available spectroscopic data.37,39,65 Finally, we study how charge transfer pathways and triplet formation at the RC depend on the redox state of the primary quinone (QA) acceptor and of the OEC.61,90 Overall, the present work contributes to a more complete understanding of the nature of triplet states within the RC of PSII, of their electronic and spectroscopic properties, and of the electrostatic control exerted by the PSII protein matrix.
All QM/MM calculations were performed using the multiscale module of the ORCA 5.0 suite, which incorporates the electrostatic embedding technique.96,98,99 The hydrogen link atom approach was employed to cut through C–C covalent bonds and the charge-shift (CS) scheme was used to avoid over polarization of the QM region. Along with the chlorin macrocycles, the axially coordinated ligands to Mg2+ were also treated at the QM level. For ChlD1 and ChlD2, the water molecule hydrogen bonded to the axially ligated water and ester group attached to the 132-carbon position on ring E is also included in the QM region. Similarly, the axial histidines (His198 and His197) in case of PD1 and PD2 were also treated at the QM level. The phytyl chains were included in the QM region up to C17 (truncated as a methyl group) and the rest of the chain was treated in the MM region.
As a first step, we computed the singlet and triplet excitation energies of individual RC pigments using TD-DFT in the QM/MM framework. The Q and B bands of the absorption spectra of porphyrin-like macrocyclic compounds are described according to the Gouterman model,118 which involves excitations within the four frontier molecular orbitals HOMO−1, HOMO, LUMO, and LUMO+1, delocalized over the chlorin ring.111 For instance, the fundamental singlet excitation of the chlorophylls is the Qy band (S1), corresponding to HOMO → LUMO and secondarily to HOMO−1 → LUMO+1 excitation. Based on the TD-DFT calculations, the lowest triplet excitations consist of two unpaired electrons, ferromagnetically coupled to each other in two singly occupied orbitals (SOMO 1, SOMO 2), also delocalized over the chlorophyll macrocycle.119,120 Our TDA-TDDFT results (see Tables S1–S5†) show that the two lowest energy triplet excited states (T1, T2) of RC chlorophylls are characterized by HOMO → LUMO (in the range of 1.22–1.30 eV) and HOMO−1 → LUMO (range of 1.73–1.78 eV) transitions. Furthermore, in all four central chlorophylls (i.e., ChlD1, PD1, PD2, ChlD2) the two lowest triplet excited states (T1 and T2) are energetically lower than the corresponding singlet excitations (S1 and S2). This observation suggests that the lowest triplet local excitations are likely to result from spin–orbit induced inter system crossing (ISC) from the corresponding first singlet excited state (S1) of each chlorophyll.84
The computation of singlet excitation energies without protein electrostatics demonstrates that both ChlD1 and ChlD2 pigments have similar site energy in the gas phase (1.88 eV and 1.90 eV, respectively, see Table 1). Moreover, the nature of excitations and participating orbitals for the chlorophyll triplet remains consistent even in the absence of the explicit PSII protein environment. On the other hand, calculations done with full inclusion of protein electrostatics red-shifts the first excited state for both pigments. This effect is more pronounced for 1ChlD1 (1.82 eV) compared to 1ChlD2 (1.88 eV). Interestingly, similar spectral shifts are obtained for the lowest triplet state (T1), where we observed protein-induced red shifts highest for 3ChlD1 (70 meV) followed by 3ChlD2 (31 meV), 3PD1 (18 meV) and 3PD2 (23 meV). The excitation energy of 3PheoD1 was found to be 17 meV higher than 3ChlD1, and about 10 meV higher than the T1 states of PD1, PD2 and ChlD2. Clearly, the signature of transverse excitonic asymmetry within the RC is preserved for the lowest localized triplet excitations. Nevertheless, it will be interesting to see how the absolute S1 and T1 excitation energies and S1–T1 gap are modulated by the protein matrix as these states should be involved in S–T intersystem crossing. The vertical excitation energies of the lowest singlet and triplet state along with the respective S–T gaps, in the presence and absence of the protein, are listed in Table 1. It is important to note that the protein matrix induces an asymmetry in tuning the S1–T1 gap for the accessory chlorophylls ChlD1 and ChlD2. In the case of ChlD1, both S1 and T1 are red-shifted by ca. 70 meV in the presence of the protein compared to the gas phase. In the case of ChlD2 the S1–T1 gap is 0.59 eV in the presence of the protein, similar to the gas phase (0.58 eV).
Method | ΔE | TD-DFT (in protein) | TD-DFT (gas-phase) | |||||
---|---|---|---|---|---|---|---|---|
RC pigment | T1–S0 (opt) | ΔT1 (opt) | S1 | T1 | S1–T1 | S1 | T1 | S1–T1 |
ChlD1 | 0.920 | 0.300 | 1.818 | 1.220 | 0.598 | 1.884 | 1.290 | 0.594 |
PD1 | 0.994 | 0.311 | 1.859 | 1.305 | 0.554 | 1.898 | 1.323 | 0.575 |
PD2 | 0.978 | 0.313 | 1.859 | 1.291 | 0.568 | 1.897 | 1.314 | 0.583 |
ChlD2 | 0.970 | 0.318 | 1.878 | 1.288 | 0.590 | 1.900 | 1.319 | 0.581 |
The singlet excited states on the central pair PD1PD2 in the presence of the protein point charges show that the lowest singlet excited states at 1.86 eV and 1.88 eV are a superposition of local excitons on PD1 and PD2, respectively (Table S6†). The lowest CT state involving the central pair (PD1δ+PD2δ−) is significantly higher (ca. 3.2 eV) than the S0. On the other hand, in the case of triplet excitations, the two lowest triplet states are isoenergetic and correspond to triplet excitons localized on PD2 (T1, 1.29 eV) and PD1 (T2, 1.30 eV) respectively (see Fig. 3a and b). Our results do not show any low-energy triplet state of the same character as the 1[PD1δ+PD2δ−] CT state mentioned above. Moreover, each triplet excitation spanning a range of 1.40–1.50 eV is attributed to individual pigments (see Table S6†), suggesting that the triplet excitons are entirely localized on either of the two chlorophyll molecules (PD1 or PD2) and therefore there is no superposition, in contrast to the singlet excitons. The absence of a low-lying triplet state with CT character is also indicative of the fact that a radical-pair charge recombination may not be favorable to form 3[PD1PD2] states in the RC. However, it cannot be excluded that delocalized triplet excited states exist at higher energies, similar to the singlet CT excitations.12,24
The most common mechanism of triplet formation in organic chromophores involves a spin–orbit-induced intersystem crossing (ISC) but singlet fission, radical pair ISC, or spin–orbit charge-transfer ISC can result in triplet formation, particularly in systems with donor–acceptor pigment pairs.54,89,121,122 Similar studies on biomimetic assemblies have reported that low-lying CT states can promote triplet formation through a charge recombination of donor–acceptor radical pairs followed by ISC.123,124 Our TD-DFT results show that the lowest singlet excitations in the [PD1PD2ChlD1PheoD1] branch correspond to 1[PD1δ+PheoD1δ−] (1.548 eV) and 1[ChlD1δ+PheoD1δ−] (1.693 eV) CT states, respectively (Table 2). These results are further in line with recent QM/MM and TDDFT studies.18,24
Roots | E S | f osc | Transition | E T | Transition |
---|---|---|---|---|---|
1 | 1.548 | 0.00 | CT (PD1 →PheoD1) | 1.215 | LE (ChlD1) |
2 | 1.693 | 0.06 | CT (ChlD1 → PheoD1) | 1.291 | LE (PD2) |
3 | 1.801 | 0.32 | LE (ChlD1) | 1.303 | LE (PD1) |
4 | 1.807 | 0.02 | CT (PD2 → PheoD1) | 1.386 | LE (PheoD1) |
5 | 1.855 | 0.39 | LE (PD1) + LE (PD2) | 1.548 | CT (PD1 → PheoD1) |
6 | 1.882 | 0.05 | LE (PD1) + LE (PD2) | 1.681 | LE (PheoD1) |
7 | 2.023 | 0.00 | CT (PD1 → PheoD1) | 1.708 | CT (ChlD1 → PheoD1) |
8 | 2.033 | 0.17 | LE (PheoD1) | 1.731 | LE (ChlD1) |
9 | 2.251 | 0.00 | CT (ChlD1 → PheoD1) | 1.773 | LE (PD2) |
10 | 2.340 | 0.00 | CT (PD2 → PheoD1) | 1.778 | LE (PD1) |
11 | 2.385 | 0.04 | LE (ChlD1) | 1.807 | CT (PD2 → PheoD1) |
12 | 2.409 | 0.03 | LE (PD2) | 2.023 | CT (PD1 → PheoD1) |
The results presented and analyzed in terms of natural transition orbital (NTO) compositions (see Table 2) and (TDA)-TDDFT difference densities show that the lowest triplet excited state of the D1 tetramer (T1 at 1.215 eV) is fully localized on ChlD1, which also exhibits the lowest site energy (S1 at 1.801 eV) among all RC pigments. The second and third triplet states (T2 at 1.291 eV and T3 at 1.303 eV) are localized excitations on PD2 and PD1 respectively. These results are in line with those obtained for the pigment monomers and dimers. Most importantly, we identified the “spin-flipped” triplet states 3[PD1δ+PheoD1δ−] (1.548 eV) and 3[ChlD1δ+PheoD1δ−] (1.708 eV, Fig. 4) that are isoenergetic with the lowest singlet CT states (see Table 2). The corresponding TD-DFT difference densities for the low-energy CT triplet excitations 3[PD1δ+PheoD1δ−] and 3[ChlD1δ+PheoD1δ−] are depicted in Fig. 5. It is noteworthy that all the RC pigments exhibit a triplet exciton lower than the above donor–acceptor CT states.
All the low-energy triplet states are dominated by local excitations on ChlD1, PD1, PD2 and PheoD1, all lower in energy than the lowest triplet CT states. This is in contrast to singlet excitations wherein the low-energy profile is dominated by mixed local excitons and CT excitations or states with pure CT character. Furthermore, most local excitons are blue-shifted compared to the donor–acceptor CT states. Overall, our results clearly demonstrate that low-energy singlet and triplet excited state manifolds differ significantly for primary donor–acceptor pairs in the RC. A detailed schematic representation comparing the complete low-energy spectrum (singlet and triplet excitations) of the RC is provided in Fig. 6. Based on our calculations one would expect that the observable triplet state in the RC can be formed from recombination of either of these radical pairs that subsequently decays to the neutral ground-state chlorophyll triplet 3ChlD1. This mechanism is different from the formation of other triplet states (e.g. in light-harvesting antennae) where 3Chl formation is mediated by triplet–triplet energy transfer (T-TET)54,55,70 or direct intersystem crossing from a singlet excited state.54 The singlet-triplet excitation spectra of the D2 tetramer [PD1PD2ChlD2PheoD2] (see Fig. S1 and Table S7†) are also comprised of CT triplet excitations corresponding to 3[PD2δ+PheoD2δ−] (1.706 eV), 3[PD2δ+PheoD2δ−] (1.816 eV) and 3[ChlD2δ+PheoD2δ−] (2.032 eV) respectively. The lowest triplet exciton in the D2 side is localized on ChlD2 at 1.279 eV.
Previous site-directed mutagenesis experiments on D1-H198G, combined with low-temperature optical difference spectroscopy, conducted by Diner et al.,9 reported shifts in the difference spectra of PD1+/PD1 and YZ˙/YZ, as well as displacements in the midpoint potential of PD1+/PD1. However, the mutation had no effect on the difference spectra or EPR properties corresponding to 3P680. Schlodder et al.125 performed similar studies on D1-T179H mutants, which involve the ligand H-bonded to the axially bound water of ChlD1, and observed shifts in the Qy band and EPR signals upon triplet formation. The T–S absorption spectra of photosynthetic pigments in D1D2Cytb559 complexes were also investigated by Renger et al.,15,42 and more recent phosphorescence measurements73,74 supported the notion that the triplet state is localized on an RC chlorophyll different from the one accommodating the stable positive charge. FTIR measurements indicated that the triplet is localized on a chlorophyll distinct from the primary cation-stabilizing chlorophyll, based on the vibrational peak of the 131-keto CO keto arising from differences in H-bonding interactions.20 These experimental observations, combined with the latest experimental and theoretical descriptions of the primary events at the RC of PSII that identify ChlD1 as the primary donor, consistently support the idea that the accessory chlorophyll ChlD1 is the site of the most stable triplet state.
Here, we determined the TD-DFT vertical excitation energies for 3[PD1PD2] and 3ChlD2 to be 1.29 eV and 1.28 eV, respectively (see Table 1). Consequently, the lowest energy triplet excitation was found to be localized on ChlD1, consistent with the above findings. Additionally, we observed that the QM/MM geometry relaxation had a similar effect of approximately 0.3 eV on the triplet state for each chlorophyll in the reaction center (Table 1). The EPR/ENDOR and FTIR spectra obtained from temperature-dependent studies estimated energy differences between 3ChlD1 and 3PD1 of 8–13 meV from isolated RCs and 11 meV from core complexes.9,62,74,126 Our computational results align with these experimental observations, indicating that the triplet state on ChlD1 is also the lowest in energy among all pigments at the reaction center.15,42 However, given the close spacing of energy levels, it is expected that at higher temperatures, an equilibrium would exist among the triplet states of PD1, PD2, ChlD1, and ChlD2, resulting in the delocalization of the observable triplet state over more than one chlorophyll molecule. These conclusions are consistent with recent FTIR studies conducted by Noguchi and co-workers.87 Therefore, our findings support both the localization of the triplet on the specific chlorophyll center (ChlD1) at low temperatures and the decrease in triplet intensities due to delocalization at ambient temperatures.
The accurate determination of zero field splitting (ZFS) parameters D and E is important to characterize the spatial extent and specific location of the triplet-state spin densities. From a methodological perspective, the accuracy of the spin–spin contribution of the D-tensors (Dss) for organic radicals is significantly affected by spin contamination, and ROKS approaches show better performance than UKS approaches for predicting the correct sign and the ZFS tensor orientation in organic triplets.117 Based on our calculations (see Table S9†) we observe good agreement despite a small systematic underestimation of the magnitude of the ZFS for the RC triplets, as also reported in the past for isolated Chl a triplets.117 Our calculations nevertheless confirm that the lowest triplet state is localized on a monomeric chlorophyll at the RC, as can be concluded from the corresponding ZFS parameters and comparison with those of isolated Chl a. This appears to rule out the possibility that the observed triplet is delocalized at low temperatures. From the first series of EPR studies on chlorophyll triplets in photosynthetic RCs, Rutherford et al.56,61 and Van Mieghem et al.58 proposed that the observable triplet is localized on a pigment whose ring plane is tilted at an angle of 30° with respect to the membrane plane. Following on the 1.9 Å crystal structure of PSII,8 this was assumed to be either of the accessory chlorophylls, ChlD1 or ChlD2. Based on our QM/MM model and EPR calculations, we estimated an angle of about 37° between the chlorophyll plane and the approximate membrane plane, the z-axis of the ZFS tensor and the molecular z-axis (perpendicular to the porphyrin ring plane) being approximately collinear. However, one still cannot assign the triplet state of the RC to a specific pigment only based on the ZFS parameters.
A more sensitive tool that offer insights into the electronic nature of the triplet states is the electron-nuclear hyperfine coupling (HFC) for protons and heavier nuclei strongly interacting with it. We computed the 1H HFC constants for each of the chlorophyll triplet states explicitly accounting for the protein electrostatics. From our calculations, we can assign the EPR coupling constants to each proton corresponding to the chlorophyll triplet state (Table 3). It has been argued based on experiments that 3P680 is localized on ChlD1 or ChlD2, based on the low number of contacts of the three methyl groups (2, 7 and 12). We also conclude that the peak corresponding to the highest positive HFC should be assigned to the freely rotating methyl group at position 12, followed by that of 2, and this is consistent for all the RC pigments. Our assignment of the hyperfine coupling constants is also consistent with DFT computed Mulliken spin populations of the neighboring carbon atoms of the chlorin macrocycle (see Fig. S2†). Overall, C12 has the highest spin population (0.293 in ChlD1) in the chlorin ring, which consequently leads to a large proton hyperfine coupling in the C12 methyl protons. The spin population at C2 and C7 are comparatively lower. The assignment of the HFC constants at position 2 is also interesting, because the signal corresponding to these protons is not clearly assigned in ENDOR studies of isolated RC (D1D2Cytb559) samples.39 Interestingly, the largest contribution for each chlorophyll is seen to arise for the methyl protons oriented towards the perpendicular z-axis of the molecule. The negative values of the HFCs are assigned to the methine (CH) protons on the plane of the chlorin macrocycle (5, 10 and 20) because their isotropic couplings arise from spin polarization effects. Among these methine (CH) protons the carbon with highest spin density leads to more a negative value of 1H HFC due to a higher spin polarization and this trend is consistent among all the four RC pigments. In the recent work by Niklas and coworkers,37 the hyperfine coupling constants for the protons at C17 and C18 were not clearly determined for 3P680. From our calculations, we observe that for all the chlorophylls the proton at position 18 has a higher isotropic 1H HFC than position 17. Also, the corresponding spin population analysis of the macrocyclic carbon atoms indicate a higher spin density at C19 than C16. This trend is also consistent among all the RC chlorophylls (ChlD1, ChlD2, PD1 and PD2), and therefore our QM/MM calculations indicate the experimentally observed HFC of 2.99 in 3P680 likely arises from position 18.
Triplet state | 10 (CH) | 20 (CH) | 5 (CH) | 7 (CH3) | 12 (CH3) | 2 (CH3) | 18 (CH) | 17 (CH) | 3′ (CH) | 3′′ (CH2) | |
---|---|---|---|---|---|---|---|---|---|---|---|
ENDOR37,39,127 | 3P680 | −10.03 | −7.88 | −4.79 | 0.62 | 10.35 | 4.80 | 2.99 | n.d. | 0.91 | −1.30 |
3Chl a (WSCP) | −10.20 | −7.70 | −5.70 | 1.10 | 10.70 | 4.70 | 2.60 | n.d. | |||
3Chl a (MTHF) | −11.44 | −7.20 | −6.20 | n.d. | 7.40 | n.d. | |||||
DFT | 3Chl a (gas-phase) | −5.12 | −5.21 | −3.32 | 0.97 | 10.77 | 5.61 | 4.81 | 3.96 | 0.69 | −1.64 |
3Chl a (MTHF) | −7.20 | −7.32 | −4.96 | 0.39 | 10.61 | 5.69 | 3.14 | 2.46 | 0.16 | −1.65 | |
3ChlD1 (gas-phase) | −6.63 | −6.77 | −5.61 | 0.61 | 10.95 | 5.35 | 2.90 | 1.78 | 0.39 | −2.04 | |
QM/MM | 3ChlD1 | −6.98 | −6.18 | −5.64 | 1.25 | 12.27 | 5.68 | 2.59 | 1.28 | 0.52 | −2.80 |
3ChlD2 | −7.41 | −6.42 | −5.63 | 0.71 | 12.41 | 5.31 | 3.06 | 1.58 | 0.64 | −2.19 | |
3PD1 | −6.02 | −5.17 | −5.59 | 1.05 | 10.86 | 4.78 | 2.47 | 1.19 | 0.58 | −2.42 | |
3PD2 | −1.82 | −4.13 | −1.08 | 1.07 | 11.67 | 5.93 | 4.18 | 4.69 | 3.59 | −1.76 |
We have also identified contributions from the vinyl group (3′, 3′′), the peaks of which were not clearly assigned in previous spectroscopic studies. The negative HFC at 3′′ is likely due to spin polarization from C3′′, and the magnitude is consistent with the corresponding spin populations. However, the orientation of the vinyl group of PD2 is particularly noteworthy here. It is known that in PD2 the vinyl CH2 is slightly out of plane from the chlorin macrocycle, and our results indicate that this significantly affects the spin density distribution of the vinyl carbons. This clearly explains why the 1H HFC of the vinyl protons in PD2 differ significantly from the other RC chlorophylls.
Our QM/MM methodology therefore not only reproduces the experimental EPR/ENDOR results obtained from intact PSII core samples but also accounts for local perturbations that might affect EPR signals from isolated RC samples. Overall, the triplet spin distribution of individual chlorophylls (Fig. 7b) remain unchanged for isolated RC samples.87 The EPR parameters however, are not sufficiently sensitive to the protein environment to enable confident differentiation between the chlorophylls of the RC and it is not possible to assign the spectroscopically observable triplet state to a single RC chlorophyll based on EPR parameters alone. Nevertheless, the lowest triplet excitations and the energetically most stable triplet state are found on ChlD1 and, hence, the combined results of all our calculations show a clear preference to assign this state to a triplet state localized on the accessory chlorophyll ChlD1.
Fig. 7 (a) Structure of Chl a with carbon atom numbering and spectroscopically important hydrogen positions indicated. (b) Computed spin density distribution of triplet (S = 1) Chl a. |
In view of the above, as a next step we performed TD-DFT calculations on the “closed” RC, where the OEC is modelled in the S2 state of the Kok–Joliot cycle and QA is reduced, i.e., the S2QA− state. Our excited state calculations on the [PD1PD2ChlD1PheoD1] assembly (Table S8†) reveal interesting results. The low-energy spectrum (see Fig. 8) in the presence of the semiquinone QA− is dominated by local excitations both for singlets and triplets, in stark contrast to the case when QA is neutral and available to accept electrons. The relative stability of site energies (ChlD1, PD1, PD2 and PheoD1) also explains the longer lifetime of chlorophyll excited states and high fluorescence yields observed in closed RCs.26,45 Moreover, the 1[ChlD1δ+PheoD1δ−] CT state is 2.23 eV higher than the ground state and thus considerably blue-shifted compared to the open RC (1.69 eV). This is in line with previous experimental hypotheses regarding reduced charge separation due to the electrostatic repulsion of QA−.26,32,79 Interestingly, we also find that the two low-energy CT states 1[ChlD1δ+PheoD1δ−] (2.231 eV) and 1[PD1δ+PheoD1δ−] (2.276 eV) are almost isoenergetic for the closed RC (Table S8†). This is clearly an effect of the differential influence of oxidized OEC and QA− on the primary donor–acceptor pairs, with PheoD1− and PD1+ being more destabilized than ChlD1+ due to their spatial proximity to QA− and/or the oxidized OEC respectively (Fig. S4;† PheoD1 is the closest pigment to QA with an edge-to-edge distance of 8.8 Å, and a center-to-center distance of 13.2 Å, while PD1 is closest to OEC with a distance of about 17.2 Å).
Studies on charge recombination reactions have shown that formation of RC triplet states can be influenced not only by the presence of the semiquinone (QA−) but also by the complete absence of QA (e.g., isolated D1D2Cytb559 samples) or the double reduction of QA.45,46 In some experiments conducted at cryogenic temperatures the spin-polarized triplet state was only detected when QA was doubly reduced (QA2− or QAH2) and not when it was singly reduced, which led to controversies about whether or not primary charge separation can occur in the presence of QA−. Studies that monitored the light-induced triplet signals with different redox states of QA using EPR spectroscopy, reported higher triplet yields but shorter life times (t1/2 < 20 μs) with QA− (closed RC).29,32,33 On the other hand, Feikema et al. based on time-resolved EPR measurements on PSII core samples reported that the yield of the triplet state with a singly reduced QA− did not differ significantly from those with QAH2.29 In the case of QAH2 however, the chlorophyll triplet was reported to have a much extended lifetime (t1/2 ∼ 1–2 ms) and this has been attributed to the absence of QA− to quench chlorophyll triplet states in PSII. Moreover, flash-induced PSII activity measurements showed the extent of D1-photodamage due to 1O2 to be most pronounced in the S2 and S3 states of the OEC, and this also has been correlated to other competing back reactions.47,50 Hence, the pathway of triplet formation and the dependence of the singlet–triplet excitations on the redox state of the QA and OEC remain unclear, yet they are crucial to understand both the control of primary processes by the transmembrane electrostatic gradient and the photoprotection mechanisms of PSII.
As seen from the excitation energy profiles (Fig. 8), the energetics of the singlet and triplet charge transfer excitations can be directly influenced by the redox state of surrounding cofactors, particularly QA. A more comprehensive overview of the singlet and triplet excitation energies, charge transfer pathways, charge recombination and triplet forming routes, is provided in Fig. 9. Based on our results, it can be suggested that formation of triplet states at the RC should be preceded by charge recombination of the primary radical pair [ChlD1+PheoD1−] or [PD1+PheoD1−] formed from the corresponding CT states. Subsequently, a very important aspect when discussing molecular mechanisms of photoprotection involves the acceptor side of PSII. PheoD1 is the site of the primary anion radical PheoD1−, following charge separation.4,11,12,15,79 In normal physiological conditions the electron is rapidly transferred to QA (PheoD1−QA → PheoD1QA−). The thermodynamic driving force for this step is governed by the relative midpoint potentials of PheoD1/PheoD1− and QA/QA− and is controlled by local pigment–protein interactions.32 However, the reduction of QA to QA− can lead to the following alternate possibilities: (a) direct charge recombination with P680+ to 1P680* and finally the ground state, (b) backward electron transfer onto PheoD1 to form 1[P680+PheoD1−] or (c) formation of the charge recombination triplet 3[P680+PheoD1−] which finally localizes on ChlD1i.e., the triplet route. Calculation of the PheoD1 electron affinity suggests that PheoD1− formation is disfavored by ca. 0.5–1 eV in the presence of a reduced QA−. The electrostatic repulsion of QA− destabilizes the primary radical pair [P680+PheoD1−], but also inhibits forward electron transfer. This might cause spin inversion from 1[P680+PheoD1−] to 3[P680+PheoD1−], the excess excitation energy dissipated through the non-radiative triplet route (Fig. 9). Experiments suggest that the observable triplet in the closed RC has an extremely short lifetime (t1/2 < 20 μs), and it has been proposed that this is because QA− quenches RC triplet states through 3PheoD1.32 However, this mechanism of triplet quenching involving the semiquinone (QA−) and 3PheoD1 is not well understood. Based on our computed excitation profile of the closed RC (Fig. 9), we find numerous thermodynamically accessible triplet states that are localized on the individual pigments (ChlD1, PD1, PD2 and PheoD1). All these local excitations are in fact lower in energy than the CT 3[ChlD1δ+PheoD1δ−] and 3[PD1δ+PheoD1δ+] excitations, which is in contrast to the triplet energy profile of open RC [S1QA] (see Fig. 8). Specifically, all D1 pigments in the closed RC possess at least two triplet excitations (T1 to T8) energetically lower than the first CT state. Thus, non-radiative energy dissipation involving multiple RC pigments might be a possibility in the closed RC, in line with arguments regarding triplet delocalization pathways discussed in recent FTIR studies.87 When QA is doubly reduced as QAH2, the PheoD1− anion is expected to be more stable in the absence of a negative charge in its vicinity.25,45,46,130 This can stabilize 1[P680+PheoD1−] and a subsequent spin inversion to 3[P680+PheoD1−] may again lead to more centers favoring the triplet route as opposed to a direct charge recombination to the singlet state.
It is known that formation of triplet states is detrimental to photosynthetic organisms as long-lived triplets in the RC can accelerate the formation of reactive oxygen species and subsequent photodamage.43,49,51 In this respect, we provided a quantitative explanation of how the PSII protein matrix and redox active cofactors may work in tandem to tune the energetics of primary charge separation and triplet formation in photosynthetic reaction centers. Our results have implications for photoprotection mechanisms in both the open and the closed states of active PSII. The next line of photoprotection in the RC may involve the delocalization of triplet states away from ChlD1 onto other pigments at ambient temperatures to avoid the selective damage of the D1 protein. However, if this still leads to photoinactivation, the D1 protein is selectively degraded and regenerated, thereby allowing photosynthetic organisms to preserve functionality even under extreme conditions.48,131,132
Footnote |
† Electronic supplementary information (ESI) available: Tables S1–S9 and Fig. S1–S4. See DOI: https://doi.org/10.1039/d3sc02985a |
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