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The S 0 State EPR Signal from the Mn Cluster in Photosystem II Arises from an Isolated S = 1 / 2 Ground State †
Abstract
During oxygen evolution, the Mn cluster in Photosystem II cycles through five oxidation states, S0-S4. S0 and S2 are paramagnetic, and can be monitored by electron paramagnetic resonance (EPR). Recently a new EPR signal from the S0 state was discovered [Ahrling et al. (1997) Biochemistry 36, 13148-13152, Messinger et al. (1997) J. Am. Chem. Soc. 119, 11349-11350]. Here, we present a well-resolved S0 spectrum, taken at high power and low temperature. The spectrum is wider and more resolved than previously thought, with structure over more than 2500 G, and appears to have at least 20 reproducible peaks on each side of g = 2. We also present the temperature dependence of the unsaturated S0 signal amplitude. A linear relationship was found between signal intensity and reciprocal temperature (1/T) in the region 5-25 K, clearly extrapolating to 0. This obeys the Curie law, indicating that the S0 state is a ground S = 1/2 state with no thermally accessible excited state. The data are consistent with a minimum energy gap of 30 cm-1 between the ground and first excited states.
... It is well-established that the four Mn ions that constitute the OEC are magnetically coupled in all S n states and that each S n state (n = 0-3) of the Mn 4 O x Ca cluster has distinct EPR signals [6,[40][41][42][43][44][45][46][47][48][49][50]. Of particular interest is the S 2 state which has a ground spin state of total spin S T = ½. ...
... In higher plant spinach a Δ of~6 cm − 1 was measured for the non-MeOH treated OEC S 2 multiline state [48] as opposed to~25-35 cm − 1 when MeOH is present. Similar results have been observed for OEC poised in the S 1 -state. ...
... The corresponding 55 Mn-ENDOR spectrum (Fig. 3b) also changes. In samples to which MeOH is not added the [48,54] 12 [48,54], 30 [48,54] Fig. 2. The temperature dependence of the T 1 relaxation time of the OEC poised in the S 2 multiline state in PS II samples containing 4% MeOH (■) and in the absence of MeOH (no addition) (▲). The left hand side panel data plots the inverse temperature vs. the natural logarithm of the inverse of the T 1 time (Orbach process). ...
The electronic properties of the Mn4OxCa cluster in the S2 state of the oxygen-evolving complex (OEC) were studied using X- and Q-band EPR and Q-band 55Mn-ENDOR using photosystem II preparations isolated from the thermophilic cyanobacterium T. elongatus and higher plants (spinach). The data presented here show that there is very little difference between the two species. Specifically it is shown that: (i) only small changes are seen in the fitted isotropic hyperfine values, suggesting that there is no significant difference in the overall spin distribution (electronic coupling scheme) between the two species; (ii) the inferred fine-structure tensor of the only MnIII ion in the cluster is of the same magnitude and geometry for both species types, suggesting that the MnIII ion has the same coordination sphere in both sample preparations; and (iii) the data from both species are consistent with only one structural model available in the literature, namely the Siegbahn structure [Siegbahn, P. E. M. Accounts Chem. Res. 2009, 42, 1871-1880, Pantazis, D. A. et al., Phys. Chem. Chem. Phys. 2009, 11, 6788-6798]. These measurements were made in the presence of methanol because it confers favorable magnetic relaxation properties to the cluster that facilitate pulse-EPR techniques. In the absence of methanol the separation of the ground state and the first excited state of the spin system is smaller. For cyanobacteria this effect is minor but in plant PS II it leads to a break-down of the ST = ½ spin model of the S2 state. This suggests that the methanol-OEC interaction is species dependent. It is proposed that the effect of small organic solvents on the electronic structure of the cluster is to change the coupling between the outer Mn (MnA) and the other three Mn ions that form the trimeric part of the cluster (MnB, MnC, MnD), by perturbing the linking bis-μ-oxo bridge. The flexibility of this bridging unit is discussed with regard to the mechanism of O-O bond formation.
... Methanol is a key molecule in this respect, because it affects the electronic structure and modies the EPR signatures 75 of all states. [47][48][49][50][51][52][53][54][55][56][57][58][59][60][61][62][63][64][65] A major effect is that it increases the energy separation between the lowest magnetic levels of the OEC, 48,51,60,62 stabilizing the S ¼ 1/2 ground spin states of the S 0 and S 2 states and the diamagnetic ground state of S 1 . Other effects relate to the amplitude enhancement of specic spectral forms, for example of the g z 2 multiline signal of the S 2 state in plants over the g $ 4 component. ...
... The phenomenology of split EPR signals of both the S 1 Y Z c and the S 0 Y Z c states has been rationalized in terms of an increase in DE upon addition of methanol, 60 so a similar mode of interaction as for the S 1 and S 2 states (operating on the Mn3-Mn4 antiferromagnetic coupling) would be valid for the S 0 state as well. We prefer not to propose specic S 0 models at this point because of the more complex EPR phenomenology of this state 50,77,116,117 and remaining uncertainties in the protonation states of terminal waterderived ligands and oxo bridges. 35,72,[118][119][120] EPR studies of the S 3 state reveal a rich and complex phenomenology. ...
Methanol has long being used as a substrate analogue to probe access pathways and investigate water delivery at the oxygen-evolving complex (OEC) of photosystem-II. In this contribution we study the interaction of methanol with the OEC by assembling available spectroscopic data into a quantum mechanical treatment that takes into account the local channel architecture of the active site. The effect on the magnetic energy levels of the Mn4Ca cluster in the S2 state of the catalytic cycle can be explained equally well by two models that involve either methanol binding to the calcium ion of the cluster, or a second-sphere interaction in the vicinity of the "dangler" Mn4 ion. However, consideration of the latest ¹³C hyperfine interaction data shows that only one model is fully consistent with experiment. In contrast to previous hypotheses, methanol is not a direct ligand to the OEC, but is situated at the end-point of a water channel associated with the O4 bridge. Its effect on magnetic properties of plant PS-II results from disruption of hydrogen bonding between O4 and proximal channel water molecules, thus enhancing superexchange (antiferromagnetic coupling) between the Mn3 and Mn4 ions. The same interaction mode applies to the dark-stable S1 state and possibly to all other states of the complex. Comparison of protein sequences from cyanobacteria and plants reveals a channel-altering substitution (D1-Asn87 versus D1-Ala87) in the proximity of the methanol binding pocket, explaining the species-dependence of the methanol effect. The water channel established as the methanol access pathway is the same that delivers ammonia to the Mn4 ion, supporting the notion that this is the only directly solvent-accessible manganese site of the OEC. The results support the pivot mechanism for water binding at a component of the S3 state and would be consistent with partial inhibition of water delivery by methanol. Mechanistic implications for enzymatic regulation and catalytic progression are discussed.
... The total spin of the CaMn 4 -cluster is S-state dependent, suggested to be non-integral spin in the S 0 -and S 2 -states, S = 1/2, 3/2, 5/2.., and integral spin in the S 1 -and S 3 -states, S = 0, 1, 2, 3.., based on the paramagnetic EPR signals observed in the different S-states (133)(134)(135)(136). In addition to the nature of the spin-spin interaction, these differences in spin states are contributing to the different spectral shapes of the S-state dependent split signals. ...
... Hence both the S 0 multiline and the Split S 0 signal originate from the S = ½ ground state of the CaMn 4 -cluster. An increased Mn-Mn exchange coupling (J) in the presence of methanol would depopulate the higher spin states, thus increasing the population of the ground state (134). This increased ground state population could explain the more intense resonance of the ground state signal, Split S 0 , in the presence of methanol, observed in Paper II ( Figure 13, black versus red). ...
... 8,12,14 EPR and ENDOR, as well as X-ray spectroscopy have been employed to characterize the electronic structure of the Mn 4 CaO 5 cluster. 4,5,9,[15][16][17][18][19][20][21][22][23] Most groups agree that the oxidation states of the S 0 state are Mn 4 (III,III,III,IV) 24,25 and that the subsequent transitions up to S 3 involve Mn III -Mn IV oxidations 26,27 (see however refs. 28 and 29). ...
... Via the m-oxo bridges the unpaired d-electrons of the four Mn III/IV centers couple to total spins of S T = 1/2, S T = 1, S T = 1/2 (and S T = 5/2), and S T = 3 for the S 0 , S 1 , S 2 and S 3 states, respectively. The corresponding EPR signals have been observed at g = 2 (S 0 ), g = 4.9 (S 1 ), g = 2 and g Z 4.1 (S 2 ), and g = 8 and 12 (S 3 ) at liquid helium temperatures using parallel (S 1 , S 3 ) or perpendicular mode (S 0 , S 2 ) EPR. [17][18][19][20][30][31][32][33][34][35][36] Several crystal structures of PSII, which crystallizes as a dimer of 700 kDa, have been reported at progressively better resolutions of 3.8 Å to 2.9 Å, and recently 1.9 Å. 1,[37][38][39][40] This latest structure not only revealed a very detailed picture of the protein and its cofactors including the Mn 4 CaO 5 cluster, but also identified about 1300 water molecules per monomer that are associated with the stromal surface of PSII or 'bound' within the luminal extensions of the CP43 and CP47 inner lightharvesting proteins and within the three extrinsic proteins of PSII, PsbO (33 kDa), PsbU (12 kDa) and PsbV (cyt c 550 ; 15 kDa). 1 This lumenal shield protects and stabilizes the Mn 4 CaO 5 cluster, and it also forms several channels for water access, as well as for proton and oxygen release. 1,39,[41][42][43][44][45][46][47][48][49][50][51] Studying the access of water to and the escape path of protons from the catalytic site of water oxidation, as well as the binding of substrate water to the Mn 4 CaO 5 cluster are crucial aspects for deriving the mechanism of photosynthetic water oxidation. ...
The hydration of the oxygen-evolving complex (OEC) was characterized in the dark stable S1 state of photosystem II using water R1(ω) NMR dispersion (NMRD) profiles. The R1(ω) NMRD profiles were recorded over a frequency range from 0.01 MHz to 40 MHz for both intact and Mn-depleted photosystem II core complexes from Thermosynechococcus vulcanus (T. vulcanus). The intact-minus-(Mn)-depleted difference NMRD profiles show a characteristic dispersion from approximately 0.03 MHz to 1 MHz, which is interpreted on the basis of the Solomon-Bloembergen-Morgan (SBM) and the slow motion theories as being due to a paramagnetic enhanced relaxation (PRE) of water protons. Both theories are qualitatively consistent with the ST = 1, g = 4.9 paramagnetic state previously described for the S1 state of the OEC; however, an alternative explanation involving the loss of a separate class of long-lived internal waters due to the Mn-depletion procedure can presently not be ruled out. Using a point-dipole approximation the PRE-NMRD effect can be described as being caused by 1-2 water molecules that are located about 10 Å away from the spin center of the Mn4CaO5 cluster in the OEC. The application of the SBM theory to the dispersion observed for PSII in the S1 state is questionable, because the parameters extracted do not fulfil the presupposed perturbation criterion. In contrast, the slow motion theory gives a consistent picture indicating that the water molecules are in fast chemical exchange with the bulk (τw < 1 μs). The modulation of the zero-field splitting (ZFS) interaction suggests a (restricted) reorientation/structural equilibrium of the Mn4CaO5 cluster with a characteristic time constant of τZFS = 0.6-0.9 μs.
... This signal originates from the S 0 -state and is spread over 2200 G around g =2 (Table 2) with different peak intensity and separation than the S 2 -state multiline signal. The signal has been reported from spinach and cyanobacteria [45,[97][98][99]. Fig. 5 (spectrum c) shows the S 0 -state multiline signal obtained after 3 flashes in Arabidopsis. ...
... Fig. 5 (spectrum c) shows the S 0 -state multiline signal obtained after 3 flashes in Arabidopsis. The spectrum shown was obtained by subtraction of 10% of the S 2 -state multiline signal from the original EPR spectrum to eliminate contribution from the ca 10% remaining S 2 -state PSII centers after the 3 flashes [99]. The deconvoluted S 0 -state multiline signal exhibited narrower splitting between the peaks than the S 2 multiline signal (spectra a and b) and was more than 2000 G wide (Fig. 5, spectrum c, Table 2). ...
Arabidopsis thaliana is widely used as a model organism in plant biology as its genome has been sequenced and transformation is known to be efficient. A large number of mutant lines and genomic resources are available for Arabidopsis. All this makes Arabidopsis a useful tool for studies of photosynthetic reactions in higher plants. In this study, photosystem II (PSII) enriched membranes were successfully isolated from thylakoids of Arabidopsis plants and for the first time the electron transfer cofactors in PSII were systematically studied using electron paramagnetic resonance (EPR) spectroscopy. EPR signals from both of the donor and acceptor sides of PSII, as well as from auxiliary electron donors were recorded. From the acceptor side of PSII, EPR signals from Q(A)- Fe²(+) and Phe- Q(A)- Fe²(+) as well as from the free Phe- radical were observed. The multiline EPR signals from the S₀- and S₂-states of CaMn₄O(x)-cluster in the water oxidation complex were characterized. Moreover, split EPR signals, the interaction signals from Y(Z) and CaMn₄O(x)-cluster in the S₀-, S₁-, S₂-, and the S₃-state were induced by illumination of the PSII membranes at 5K and characterized. In addition, EPR signals from auxiliary donors Y(D), Chl(+) and cytochrome b₅₅₉ were observed. In total, we were able to detect about 20 different EPR signals covering all electron transfer components in PSII. Use of this spectroscopic platform opens a possibility to study PSII reactions in the library of mutants available in Arabidopsis.
... There has been immense interest in elucidating the molecular and electronic structure of the OEC of PSII and, in particular, the fate of the substrate water molecules and the Mn 4 Ca-oxo cluster in each of the S-state intermediates to understand the requirements for minimizing the energetic penalty for solar water oxidation. The OEC of PSII has been studied extensively by structural, spectroscopic , biochemical, and computational methods [10, 18,[25][26][27][28][29][30][104][105][106][107][108][109][110][111]. While single-crystal X-ray diffraction has provided important insight into the molecular geometry of the OEC in the stable S 1 state [26, 27, 29, 30, 67], a high-resolution structure of the S 1 state was obtained recently after dramatic improvements in sample preparation and methodology [28]. ...
The solar water-splitting protein complex, photosystem II (PSII), catalyzes one of the most energetically demanding reactions in nature by using light energy to drive the catalytic oxidation of water to dioxygen. Light-driven electron and proton-coupled electron transfer (PCET) reactions, which are exquisitely tuned by smart protein matrix effects, are central to this water-splitting chemistry. PSII contains a series of charge-transfer cofactors, such as the special chlorophylls, pheophytin, primary and secondary plastoquinones, tetranuclear manganese-calcium-oxo cluster, and two symmetrically placed redox-active tyrosine residues, YD and YZ, that participate in the charge-transfer reactions. These cofactors are functionally very distinct and the versatility is provided by their distinct local environments in PSII. This chapter focuses on providing the reader with an outline of the primary electron transfer reactions of PSII and a description of the structure and function of the charge-transfer cofactors that participate in the primary electron transfer pathway.
... From the discussion above, it can be readily understood that the computationally derived models are characterized by a LS ground state, i.e., S GS = 1 / 2 for S 0 and S GS = 0 for S 1 , in line with the experimental EPR data for these states. [112][113][114]144,145 In the S 3 state, the cofactor instead adopts a higher-spin ground-state configuration of S GS = 3, 175,150,176 concomitant with a water binding event that renders all manganese ions sixcoordinate. S 2 displays both LS and higher-spin forms (S GS = 1 / 2 for the open-cubane form S 2 A and S GS = 5 / 2 for the closedcubane form S 2 B ) and thus represents the functional switching point in the catalytic cycle. ...
Nature’s water-splitting catalyst moves through a reaction cycle with five catalytic intermediates characterized by different spin ground states, the origin of which is connected to their geometric structures and intermetallic magnetic couplings. The early “inactive” intermediates have low-spin ground states, while the later “active” intermediates transition to high-spin states. The cofactor’s ability to switch from lower to higher-spin states via an open-to-closed cubane conversion is critical for substrate water binding.
... There has been immense interest in elucidating the molecular and electronic structure of the OEC of PSII and, in particular, the fate of the substrate water molecules and the Mn 4 Ca-oxo cluster in each of the S-state intermediates to understand the requirements for minimizing the energetic penalty for solar water oxidation. The OEC of PSII has been studied extensively by structural, spectroscopic, biochemical, and computational methods [10,18,[25][26][27][28][29][30][104][105][106][107][108][109][110][111]. While single-crystal X-ray diffraction has provided important insight into the molecular geometry of the OEC in the stable S 1 state [26,27,29,30,67], a high-resolution structure of the S 1 state was obtained recently after dramatic improvements in sample preparation and methodology [28]. ...
The solar water-splitting protein complex, photosystem II (PSII), catalyzes one of the most energetically demanding reactions in nature by using light energy to drive the catalytic oxidation of water to dioxygen. Light-driven electron and proton-coupled electron transfer (PCET) reactions, which are exquisitely tuned by smart protein matrix effects, are central to this water-splitting chemistry. PSII contains a series of charge-transfer cofactors, such as the special chlorophylls, pheophytin, primary and secondary plastoquinones, tetranuclear manganese-calcium-oxo cluster, and two symmetrically placed redox-active tyrosine residues, Y D and Y Z , that participate in the charge-transfer reactions. These cofactors are functionally very distinct and the versatility is provided by their distinct local environments in PSII.
... OEC, including the S 0 (spin S = 1/2) multiline 33,36,[41][42][43][44][45][46][47] and S 2 (S = 1/2) multiline signal, 11,[31][32][33]48,49 the two parallel-mode signals of the S 1 state of higher plants (S = 1) 50,51 and cyanobacteria (S = 2), 52 and the split signals that result from coupling of the Y z • radical to the paramagnetic forms of S 0 , S 1 , and S 3 . 35 There appear to be differences in the concentration dependence of the effects of methanol on these signals that are S-state dependent, indicating that the binding affinity of MeOH at the Mn 4 CaO 5 cluster may change as a function of the oxidation state of the cluster. ...
The binding of the substrate analogue methanol to the catalytic Mn4CaO5 cluster of the water-oxidizing enzyme photosystem II is known to alter the electronic structure properties of the oxygen-evolving complex without retarding O2-evolution under steady-state illumination conditions. We report the binding mode of 13C-labeled methanol determined using 9.4 GHz (X-band) hyperfine sublevel-correlation (HYSCORE) and 34 GHz (Q-band) electron spin-echo electron nuclear double resonance (ESE-ENDOR) spectroscopies. These results are compared to analogous experiments on a mixed-valence Mn(III)Mn(IV) complex (2-OH-3,5-Cl2-salpn)2Mn(III)Mn(IV) (salpn = N,N'-bis(3,5-dichlorosalicylidene)-1,3-diamino-2-hydroxypropane) in which methanol ligates to the Mn(III) ion (Larson, et al. 1992 J. Am. Chem. Soc. 114:6263).1 In the mixed-valence Mn(III,IV) complex, the hyperfine coupling to the 13C of the bound methanol (Aiso = 0.65 MHz, T = 1.25 MHz) is appreciably larger than that observed for 13C methanol associated with the Mn4CaO5 cluster poised in the S2 state, where only a weak dipolar hyperfine interaction (Aiso = 0.05 MHz, T = 0.27 MHz) is observed. An evaluation of the 13C hyperfine interaction using the x-ray structure coordinates of the Mn4CaO5 cluster indicates that methanol does not bind as a terminal ligand to any of the manganese ions in the OEC. We favor methanol binding in place of a water ligand to the Ca2+ in the Mn4CaO5 cluster or in place of one of the waters that form hydrogen bonds with the oxygen bridges of the cluster.
... Many propositions have been made to explain the magnetic interaction of the electron spins as well as the nuclear spins of the ions. It has been concluded that the multiline signals of the S 2 and the S 0 state originate from an S=1/2 ground state [128,161]. This should also be the case for the S −2 MLS [148]. ...
... PSII enriched membrane fragments (BBY-particles) were prepared from spinach under dim green light, as described in [53][54][55]. The isolated BBY-particles were resuspended in buffer A [50 mM Mes-NaOH (pH 6.3), 35 mM NaCl and 300 mM sucrose] at a chlorophyll concentration of 6 mg/ml and stored at )80°C until use. ...
We have investigated the electron transfer from reduced tyrosine Y D (YDred) and cytochrome b559 to the S2 and S3 states of the water oxidizing complex (WOC) in Photosystem II. The EPR signal of oxidized cyt b559, the S 2 state multiline EPR signal and the EPR signal from Y D· were measured to follow the electron transfer to the S2 and S3 states at 245 and 275 K. The majority of the S2 centers was reduced directly from YDred but at 245 K we observed oxidation of cyt b559 in about 20% of the centers. Incubation of the YDredS3 state resulted in biphasic changes of the S2 multiline signal. The signal first increased due to reduction of the S3 state. Thereafter, the signal decreased due to decay of the S2 state. In contrast, the YD· signal increased with a monophasic kinetics at both temperatures. Again, we observed oxidation of cyt b559 in about 20% of the PSII centers at 245 K. This oxidation correlated with the decay of the S2 state. The complex changes can be explained by the conversion of YDredS3 centers (present initially) to YD·S1 centers, via the intermediate YD·S2 state. The early increase of the S2 state multiline signal involves electron transfer from Y Dred to the S3 state resulting in formation of YD·S2. This state is reduced by cyt b559 resulting in a single exponential oxidation of cyt b 559. Taken together, these results indicate that the electron donor to S2 is cyt b559 while cyt b559 is unable to compete with YDred in the reduction of the S3 state in the pre-reduced samples. We also followed the decay of the S 2 and S3 states and the oxidation of cyt b559 in samples where YD was oxidized from the start. In this case cyt b559 was able to reduce both the S2 and the S3 states suggesting that different pathways exist for the electron transfer from cyt b559 to the WOC. The activation energies for the Y DredS2→YD·S1 and YDredS 3→YD·S2 transformations are 0.57 and 0.67 eV, respectively, and the reason for these large activation energies is discussed.
... The Ca 2ϩ -depleted S 2 Ј state from spinach resembles the native S 2 state from T. elongatus with regard to the spin state energies. Upon removal of the Ca 2ϩ ion, ⌬ increases to Ն35 cm Ϫ1 , which is much larger than for the native spinach S 2 state (⌬ ϭ 3-6 cm Ϫ1 ) but more similar to T. elongatus (⌬ ϭ 12-25 cm Ϫ1 ) (41,83,84). In intact spinach PS II, the energy ladder is sensitive to MeOH addition. ...
Ca²⁺ is an integral component of the Mn4O5Ca cluster of the oxygen-evolving complex in photosystem II (PS II). Its removal leads to the loss of the water oxidizing functionality. The S2′ state of the Ca²⁺-depleted cluster from spinach is examined by X- and Q-band EPR and ⁵⁵Mn electron nuclear double resonance (ENDOR) spectroscopy. Spectral simulations demonstrate that upon Ca²⁺ removal, its electronic structure remains essentially unaltered, i.e. that of a manganese tetramer. No redistribution of the manganese valence states and only minor perturbation of the exchange interactions between the manganese ions were found. Interestingly, the S2′ state in spinach PS II is very similar to the native S2 state of Thermosynechococcus elongatus in terms of spin state energies and insensitivity to methanol addition. These results assign the Ca²⁺ a functional as opposed to a structural role in water splitting catalysis, such as (i) being essential for efficient proton-coupled electron transfer between YZ and the manganese cluster and/or (ii) providing an initial binding site for substrate water. Additionally, a novel ⁵⁵Mn²⁺ signal, detected by Q-band pulse EPR and ENDOR, was observed in Ca²⁺-depleted PS II. Mn²⁺ titration, monitored by ⁵⁵Mn ENDOR, revealed a specific Mn²⁺ binding site with a submicromolar KD. Ca²⁺ titration of Mn²⁺-loaded, Ca²⁺-depleted PS II demonstrated that the site is reversibly made accessible to Mn²⁺ by Ca²⁺ depletion and reconstitution. Mn²⁺ is proposed to bind at one of the extrinsic subunits. This process is possibly relevant for the formation of the Mn4O5Ca cluster during photoassembly and/or D1 repair.
Background: EPR/⁵⁵Mn ENDOR spectroscopy of the oxygen-evolving complex (OEC) and Mn²⁺ in Ca²⁺-depleted photosystem II.
Results: Electronic model of the Ca²⁺-depleted OEC; characterization of Mn²⁺ binding.
Conclusion: Ca²⁺ is not critical for maintaining the electronic and spatial structure of the OEC. Its removal exposes a Mn²⁺ binding site supposedly in an extrinsic subunit.
Significance: Mechanistic implications for water oxidation; Mn²⁺ in photoassembly/D1 protein repair.
Recent advances in serial femtosecond crystallography (SFX) of photosystem II (PSII), enabled by X-ray free electron lasers (XFEL), provided the first geometric models of distinct intermediates in the catalytic S-state cycle of the oxygen-evolving complex (OEC). These models are obtained by flash-advancing the OEC from the dark-stable state (S1) to more oxidized intermediates (S2 and S3), eventually cycling back to the most reduced S0. However, the interpretation of these models is controversial because geometric parameters within the Mn4CaO5 cluster of the OEC do not exactly match those expected from coordination chemistry for the spectroscopically verified manganese oxidation states of the distinct S-state intermediates. Here we focus on the first catalytic transition, S1 → S2, which represents a one-electron oxidation of the OEC. Combining geometric and electronic structure criteria, including a novel effective oxidation state approach, we analyze existing 1-flash (1F) SFX-XFEL crystallographic models that should depict the S2 state of the OEC. We show that the 1F/S2 equivalence is not obvious, because the Mn oxidation states and total unpaired electron counts encoded in these models are not fully consistent with those of a pure S2 state and with the nature of the S1 → S2 transition. Furthermore, the oxidation state definition in two-flashed (2F) structural models is practically impossible to elucidate. Our results advise caution in the extraction of electronic structure information solely from the literal interpretation of crystallographic models and call for re-evaluation of structural and mechanistic interpretations that presume exact correspondence of such models to specific catalytic intermediates of the OEC.
O2 formation in photosystem II (PSII) is a vital event on Earth, but the exact mechanism remains unclear. The presently prevailing theoretical model is "radical coupling" (RC) involving a Mn(IV)-oxyl unit in an "open-cubane" Mn4CaO6 cluster, which is supported experimentally by the S3 state of cyanobacterial PSII featuring an additional Mn-bound oxygenic ligand. However, it was recently proposed that the major structural form of the S3 state of higher plants lacks this extra ligand, and that the resulting S4 state would feature instead a penta-coordinate dangler Mn(V)=oxo, covalently linked to a "closed-cubane" Mn3CaO4 cluster. For this proposal, we explore here a large number of possible pathways of O-O bond formation and demonstrate that the "nucleophilic oxo-oxo coupling" (NOOC) between Mn(V)=oxo and μ3-oxo is the only eligible mechanism in such a system. The reaction is facilitated by a specific conformation of the cluster and concomitant water binding, which is delayed compared to the RC mechanism. An energetically feasible process is described starting from the valid S4 state through the sequential formation of peroxide and superoxide, followed by O2 release and a second water insertion. The newly found mechanism is consistent with available experimental thermodynamic and kinetic data and thus a viable alternative pathway for O2 formation in natural photosynthesis, in particular for higher plants.
Among many processes occurring in oxygenic photosynthesis, the water oxidation reaction catalyzed by the Mn4Ca cluster provides various types of insights into the field of the metal coordination chemistry. The water oxidation reaction in nature is carried out by Photosystem II (PS II), a multi subunit membrane protein complex. This light-driven reaction is made possible by a spatially separated, yet temporally connected series of cofactors along the electron transfer chain of PS II over 40 Å, through the donor—the Mn4CaO5 catalytic center, the reaction center chlorophylls, to the mobile quinone electron acceptors. Such chemical architecture provides an ideal platform to investigate how to control multi-electron/proton chemistry, using the flexibility of metal redox states, in coordination with the protein and the water network. Understanding the insights of nature's design gives inspiration of how to build artificial photosynthetic devices, where the controlled accumulation of charge and high-selectivity of products is currently challenging. The electronic and geometric structure of this catalyst have been extensively investigated, but its step-wise water oxidation mechanism is not yet completely understood. In this chapter, we summarize our current understanding of the water oxidation reaction in nature.
In photosystem II (PSII), the second-lowest oxidation state (S1) of the oxygen-evolving Mn4CaO5 cluster is the most stable, as the radical form of the redox-active D2-Tyr160 is considered to be a candidate that accepts an electron from the lowest oxidation state (S0) in the dark. Using quantum mechanical/molecular mechanical calculations, we investigated the redox potential (E m) of TyrD and its H-bond partner, D2-His189. The potential energy profile indicates that the release of a proton from the TyrD...D2-His189 pair leads to the formation of a low-barrier H-bond. The E m depends on the H+ position along the low-barrier H-bond, e.g., 680 mV when the H+ is at the D2-His189 moiety and 800 mV when the H+ is at the TyrD moiety, which can explain why TyrD mediates both the S0 to S1 oxidation and the S2 to S1 reduction.
Virtually all life on our planet is powered by the sun through the process of photosynthesis. Understanding the design and operating principles of natural photosynthesis is a central challenge for fundamental research because biology presents a unique paradigm and possible blueprint for the realization of artificial alternatives. All processes of natural photosynthesis, including light harvesting, charge separation and accumulation, water oxidation, and carbon fixation, embody “solutions” optimized through billions of years of evolution to challenges currently faced by scientists developing components for artificial photosynthetic devices. The present chapter provides an overview of the most important aspects of natural photosynthesis and discusses the ways in which they motivate biomimetic and bioinspired research into artificial photosynthesis.
Domain-based local pair natural orbital (DLPNO) coupled cluster single and double (CCSD) with triple perturbation (T) correction methods were performed to elucidate the relative stabilities of ten different intermediate structures of the CaMn4Ox cluster in the S0 state of the oxygen evolving complex (OEC) of photosystem II (PSII). Full geometry optimizations of all the S0 intermediates were performed by the UB3LYP-D3/Def2-TZVP methods, providing the assumed geometrical structures and starting natural orbitals (UNO) for DLPNO-CCSD(T)/Def2TZVP calculations. The effective exchange integrals (J) for the spin Hamiltonian models for the ten intermediates were obtained by the UB3LYP/Def2-TZVP calculations followed by the general spin projections. DLPNO-CCSD(T) calculations followed by the CBS extrapolation procedure elucidated that the (II, III, IV, IV) and (III, III, III, IV) valence states in the CaMn4O5 cluster of the OEC of the PS II were nearly degenerated in energy in the S0 state, indicating an important role of dynamical electron correlation effects for the valence and spin fluctuations in strongly correlated electron systems (SCESs) consisting of 3d transition metals.
The Mn4CaO5 cluster of Photosystem II (PSII) advances sequentially through five oxidation states (S0 to S4). Under the enzyme cycle, two water molecules are oxidized, O2 is generated and four protons are released into the lumen. Umena et al. (2011) have proposed that, with other charged amino acids, the R323 residue of the D1 protein could contribute to regulate a proton egress pathway from the Mn4CaO5 cluster and TyrZ via a proton channel identified from the 3D structure. To test this suggestion, a PsbA3/R323E site‐directed mutant has been constructed and the properties of its PSII have been compared to those of the PsbA3‐PSII by using EPR spectroscopy, polarography, thermoluminescence and time‐resolved UV–visible absorption spectroscopy. Neither the oscillations with a period four nor the kinetics and S‐state‐dependent stoichiometry of the proton release were affected. However, several differences have been found: i) the P680⁺ decay in the hundreds of ns time domain was much slower in the mutant, ii) the S2QA⁻/DCMU and S3QA⁻/DCMU radiative charge recombination occurred at higher temperatures and iii) the S0TyrZ•, S1TyrZ•, S2TyrZ• split EPR signals induced at 4.2 K by visible light from the S0TyrZ, S1TyrZ, S2TyrZ, respectively, and the (S2TyrZ•)’ induced by NIR illumination at 4.2 K of the S3TyrZ state differed. It is proposed that the R323 residue of the D1 protein interacts with TyrZ likely via the H‐bond network previously proposed to be a proton channel. Therefore, rather than participating in the egress of protons to the lumen, this channel could be involved in the relaxations of the H‐bonds around TyrZ by interacting with the bulk, thus tuning the driving force required for TyrZ oxidation.
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The sunlight-powered oxidation of water by photosystem II (PSII) of algae, plants, and cyanobacteria underpins the energy conversion processes that sustain most of life on our planet. Understanding the structure and function of the “engine of life”, the oxygen-evolving complex (OEC) in the active site of PSII, has been one of the great and persistent challenges of modern science. Immense progress has been achieved in recent years through combined contributions of diverse disciplines and research approaches, yet the challenge remains. The improved understanding of the tetramanganese-calcium cluster of the OEC for the experimentally accessible catalytic states often creates a more complex picture of the system than previously imagined, while the various strands of evidence cannot always be unified into a coherent model. This review focuses on selected current problems that relate to structural–electronic features of the OEC, emphasizing conceptual aspects and highlighting topics of structure and function that remain uncertain or controversial. The Mn4CaOx cluster of the OEC cycles through five redox states (S0–S4) to store the oxidizing equivalents required for the final step of dioxygen evolution in the spontaneously decaying S4 state. Remarkably, even the dark-stable state of the OEC, the S1 state, is still incompletely understood because the available structural models do not fully explain the complexity revealed by spectroscopic investigations. In addition to the nature of the dioxygen-evolving S4 state and the precise mechanism of O–O bond formation, major current open questions include the type and role of structural heterogeneity in various intermediate states of the OEC, the sequence of events in the highly complex S2–S3 transition, the heterogeneous nature of the S3 state, the accessibility of substrate or substrate analogues, the identification of substrate oxygen atoms, and the role of the protein matrix in mediating proton removal and substrate delivery. These open questions and their implications for understanding the principles of catalytic control in the OEC must be convincingly addressed before biological water oxidation can be understood in its full complexity on both the atomic and the systemic levels.
Nature’s water splitting catalyst, an oxygen-bridged tetra-manganese calcium (Mn4O5Ca) complex, sequentially activates two substrate water molecules generating molecular O2. Its reaction cycle is composed of five intermediate (Si) states, where the index i indicates the number of oxidizing equivalents stored by the cofactor. After formation of the S4 state, the product dioxygen is released and the cofactor returns to its lowest oxidation state, S0. Membrane-inlet mass spectrometry measurements suggest that at least one substrate is bound throughout the catalytic cycle, as the rate of ¹⁸O-labeled water incorporation into the product O2 is slow, on a millisecond to second timescale depending on the S state. Here, we demonstrate that the Mn4O5Ca complex poised in the S0 state contains an exchangeable hydroxo bridge. Based on a combination of magnetic multiresonance (EPR) spectroscopies, comparison to biochemical models and theoretical calculations we assign this bridge to O5, the same bridge identified in the S2 state as an exchangeable fully deprotonated oxo bridge [Pérez Navarro et al. Proc. Natl. Acad. Sci. U.S.A. 2013 110, 15561]. This oxygen species is the most probable candidate for the slowly-exchanging substrate water in the S0 state. Additional measurements provide new information on the Mn ions that constitute the catalyst. A structural model for the S0 state is proposed that is consistent with available experimental data and explains the observed evolution of water exchange kinetics in the first three states of the catalytic cycle.
Molecular oxygen - essential for all living organisms — is produced by the photosynthetic oxidation of water in photosystem II (PSII) of higher plants, algae and cyanobacteria. The water-oxidizing complex of PSII contains a cluster of 4 Mn atoms, and turns over through five different redox states, denoted by S0-S4, during its function.
Photosystem II (PSII) catalyses the light-driven oxidation of water to molecular oxygen. The site for this process, the oxygen evolving complex (OEC), contains four Mn atoms, and cycles through the five oxidation states S0 - S4. EPR spectroscopy has been used ex-tensively to investigate the chemistry of this cycle, as well as the structure of the catalytic site. The S0 and S2 states are paramagnetic, each giving rise to a broad, fine-structured Mn EPR signal centered around g=2. The S2 multiline signal has been extensively characterized, while the S0 signal was discovered only last year [1, 2]
Plants, algae and cyanobacteria capture sunlight, extracting electrons from H2O to reduce CO2 into sugars, while releasing O2 in the oxygenic photosynthetic phenomenon. Because of the important role of water oxidation for artificial photosynthesis and many solar fuel systems, understanding the structure and function of this unique biological catalyst forms a requisite research field. Herein the structure of the water-oxidizing complex and its ligand environment are described with reference to the 1.9 Å resolution X-ray-derived crystallographic model of the water-oxidizing complex from the cyanobacterium Thermosynechococcus vulcanus. Proposed mechanisms for water oxidation by Photosystem II and nanosized manganese oxides are also reviewed and discussed in the paper.
Syntheses and magnetic functionalities of exchange-coupled magnetic systemsin a controlled fashion of molecular basis have been the focus of the current topics in chemistry and materials science; particularly extremely large spins in molecular frames and molecular high-spin clusters have attracted much attention among the diverse topics of molecule-based magnetics and high spin chemistry. Magnetic characterizations of molecule-based exchange-coupled high-spin clusters are described in terms of conventional as well as highfield/high-frequency ESR spectroscopy. Off-principal-axis extra lines as a salient feature of fine structure ESR spectroscopy in non-oriented media are emphasized in the spectral analyses. Pulse-ESR-based two-dimensional electron spin transient nutation spectroscopy applied to molecular high-spin clusters is also dealt with, briefly. Solution-phase fine-structure ESR spectroscopy is reviewed in terms of molecular magnetics. In addition to finite molecular high-spin clusters, salient features of molecule-based low-dimensional magnetic materials are dealt with. Throughout the chapter, electron spin resonance for high-spin systems is treated in a general manner in terms of theory. Hybrid eigenfield method is formulated in terms of direct products, and is described as a powerful and facile approach to the exact numerical calculation of resonance fields and transition probabilities for molecular high spin systems. Exact analytical expressions for resonance fields of high spin systems in their principal orientations are for the first time given.
A frequent challenge when dealing with multinuclear transition metal clusters in biology is to determine the absolute oxidation states of the individual metal ions and to identify how they evolve during catalytic turnover. The oxygen-evolving complex of biological photosynthesis, an active site that harbors an oxo-bridged Mn4Ca cluster as the water-oxidizing species, offers a prime example of such a challenge that withstood satisfactory resolution for decades. A multitude of experimental studies have approached this question and have offered insights from different angles, but they were also accompanied by incomplete or inconclusive interpretations. Only very recently, through a combination of experiment and theory, has a definitive assignment of the individual Mn oxidation states been achieved for all observable catalytic states of the complex. Here we review the information obtained by structural and spectroscopic methods, describe the interpretation and synthesis achieved through quantum chemistry, and summarize our current understanding of the electronic structure of nature’s water splitting catalyst.
Water splitting is considered as a method to storage of renewable energies to hydrogen. The water-oxidation reaction in water splitting is an efficiency-limiting process for water splitting and, thus, there has been notable progress to find highly efficient water-oxidizing catalysts made from cost-effective and earth-abundant elements. In addition to efficiency, the stability of the water-oxidizing compounds is very important. Herein we focus on self-healing in manganese-based water-oxidizing catalysts in artificial photosynthetic systems.
Nature relies on a unique and intricate biochemical setup to achieve sunlight-driven water splitting. Combined experimental and computational efforts have produced significant insights into the structural and functional principles governing the operation of the water-oxidizing enzyme Photosystem II in general, and of the oxygen-evolving manganese-calcium cluster at its active site in particular. Here we review the most important aspects of biological water oxidation, emphasizing current knowledge on the organization of the enzyme, the geometric and electronic structure of the catalyst, and the role of calcium and chloride cofactors. The combination of recent experimental work on the identification of possible substrate sites with computational modeling have considerably limited the possible mechanistic pathways for the critical O-O bond formation step. Taken together, the key features and principles of natural photosynthesis may serve as inspiration for the design, development, and implementation of artificial systems.
Syntheses and magnetic functionalities of exchange-coupled magnetic systems in a controlled fashion of molecular basis have been the focus of the current topics in chemistry and materials science; particularly extremely large spins in molecular frames and molecular high-spin clusters have attracted much attention among the diverse topics of molecule-based magnetics and high spin chemistry. Magnetic characterizations of molecule-based exchange-coupled high-spin clusters are described in terms of conventional as well as high-field/high-frequency ESR spectroscopy. Off-principal-axis extra lines as a salient feature of fine-structure ESR spectroscopy in non-oriented media are emphasized in the spectral analyses. Pulse-ESR-based two-dimensional electron spin transient nutation spectroscopy applied to molecular high-spin clusters is also dealt with, briefly. Solution-phase fine-structure ESR spectroscopy is reviewed in terms of molecular magnetics. In addition to finite molecular high-spin clusters, salient features of molecule-based low-dimensional magnetic materials are dealt with. Throughout the chapter, electron spin resonance for high-spin systems is treated in a general manner in terms of theory. Hybrid eigenfield method is formulated in terms of direct products, and is described as a powerful and facile approach to the exact numerical calculation of resonance fields and transition probabilities for molecular high spin systems. Exact analytical expressions for resonance fields of high spin systems in their principal orientations are for the first time given.
Photosystem II is the enzyme that catalyzes the thermodynamically demanding splitting of water, yielding dioxygen, protons, and electrons, a sunlight-driven reaction that forms the foundation of oxygenic photosynthesis. The latest results from high-resolution crystallographic models have led to a refined view of the overall structure of the enzyme and the arrangement of the cofactors involved in light harvesting, charge separation, and electron transfer. In addition, combined efforts from protein crystallography, spectroscopy, and computational chemistry have greatly improved the understanding of the tetramanganese–calcium cluster of the oxygen-evolving complex, the site of water oxidation. The most important features of the geometric and electronic structures of the oxygen-evolving complex for the earlier reaction cycle intermediates are now sufficiently well understood such that connections between several structural features and spectroscopic observables can be made with confidence. Advanced spectroscopic techniques have also identified possible sites where the substrate water binds. Although the details of the actual mechanism of biological water oxidation remain elusive, experimental and theoretical studies impose an increasing number of constraints that significantly limit the number of mechanistic possibilities for O–O bond formation.Keywords:photosynthesis;photosystem II;water oxidation;water splitting;oxygen-evolving complex;manganese
The solar water-splitting protein complex, photosystem II (PSII), catalyzes one of the most energetically demanding reactions in Nature by using light energy to drive a catalyst capable of oxidizing water. The water oxidation reaction takes place at the tetra-nuclear manganese calcium-oxo (Mn4Ca-oxo) cluster at the heart of the oxygen-evolving complex (OEC) of PSII. Previous studies have determined the magnetic interactions between the paramagnetic Mn4Ca-oxo cluster and its environment in the S2 state of the OEC. The assignments for the electron-nuclear magnetic interactions that were observed in these studies were facilitated by the use of synthetic dimanganese di-μ-oxo complexes. However, there is an immense need to understand the effects of the protein environment on the coordination geometry of the Mn4Ca-oxo cluster in the OEC of PSII. In the present study, we use a proteinaceous model system to examine the protein ligands that are coordinated to the dimanganese catalytic center of manganese catalase from Lactobacillus plantarum. We utilize two-dimensional hyperfine sublevel correlation (2D HYSCORE) spectroscopy to detect the weak magnetic interactions of the paramagnetic dinuclear manganese catalytic center of superoxidized manganese catalase with the nitrogen and proton atoms of the surrounding protein environment. We obtain a complete set of hyperfine interaction parameters for the protons of a water molecule that is directly coordinated to the dinuclear manganese center. We also obtain a complete set of hyperfine and quadrupolar interaction parameters for two histidine ligands as well as a coordinated azide ligand, in azide-treated superoxidized manganese catalase. On the basis of the values of the hyperfine interaction parameters of the dimanganese model, manganese catalase, and those of the S2 state of the OEC of PSII, for the first time, we discuss the impact of a proteinaceous environment on the coordination geometry of multinuclear manganese clusters.
A central question in biological water splitting concerns the oxidation states of the manganese ions that comprise the oxygen-evolving complex of photosystem II. Understanding the nature and order of oxidation events that occur during the catalytic cycle of five S
i
states (i = 0-4) is of fundamental importance both for the natural system and for artificial water oxidation catalysts. Despite the widespread adoption of the so-called "high-valent scheme"-where, for example, the Mn oxidation states in the S2 state are assigned as III, IV, IV, IV-the competing "low-valent scheme" that differs by a total of two metal unpaired electrons (i.e. III, III, III, IV in the S2 state) is favored by several recent studies for the biological catalyst. The question of the correct oxidation state assignment is addressed here by a detailed computational comparison of the two schemes using a common structural platform and theoretical approach. Models based on crystallographic constraints were constructed for all conceivable oxidation state assignments in the four (semi)stable S states of the oxygen evolving complex, sampling various protonation levels and patterns to ensure comprehensive coverage. The models are evaluated with respect to their geometric, energetic, electronic, and spectroscopic properties against available experimental EXAFS, XFEL-XRD, EPR, ENDOR and Mn K pre-edge XANES data. New 2.5 K 55Mn ENDOR data of the S2 state are also reported. Our results conclusively show that the entire S state phenomenology can only be accommodated within the high-valent scheme by adopting a single motif and protonation pattern that progresses smoothly from S0 (III, III, III, IV) to S3 (IV, IV, IV, IV), satisfying all experimental constraints and reproducing all observables. By contrast, it was impossible to construct a consistent cycle based on the low-valent scheme for all S states. Instead, the low-valent models developed here may provide new insight into the over-reduced S states and the states involved in the assembly of the catalytically active water oxidizing cluster.
The current understanding of the structure of the Mn4CaO 5 cluster, as well as the water oxidation reaction based on the insights are reviewed. There are seven ligands directly ligated to the Mn 4CaO5 cluster, six carboxyl ligands (aspartate and glutamate) and one imidazole ligand (histidine). These ligands are from side chains from two domains of the D1 subunit, the interhelical CD luminal loop and the C-terminal region and one domain of CP43, the large helical EF luminal loop. Most of the ligands are arranged in a bidentate fashion bridging two metals. Simultaneous data collection of XRD and X-ray spectroscopy probes the overall protein structure from XRD and the electronic structure of the Mn 4CaO5 cluster in the oxygen-evolving complex from the spectroscopic data using the same samples under the same conditions. The use of the X-ray free electron laser (XFEL) pulses is critical for this approach.
Two reaction paths from S2 to S3 and S4 states of OEC have been studied by the hybrid density functional method. The path 1 where the added H2O molecule is irrelevant to the substrate of oxidation and the path 2 where the added H2O molecule is relevant to the substrate have been investigated. The S3 states of the paths 1 and 2 are isoenergetic, while the S4 state of the path 1 is remarkably stable (16.1 kcal/mol) rather than the path 2. [Mn4O(W2)] (S3) and [Mn4OOH(W2,W6)] (S4) structures are recommended for the S2 → S3 → S4 transitions.
This article provides an overview of the application of electron paramagnetic resonance (EPR) and related hyperfine spectroscopies for the investigation of the electronic and geometric structure of the tetramanganese cluster that constitutes the oxygen-evolving complex (OEC) of photosystem II. Starting from the spin physics of Mn ions, a quantum-mechanical description of exchange-coupled oligonuclear Mn systems is given. Then, the focus shifts to the characterization of the OEC with emphasis on the two half-integer spin states of its catalytic cycle, namely the S0 and S2. The development of electronic models based on EPR and related spectroscopies, such as electron nuclear double resonance (ENDOR), is described in detail. The role of broken-symmetry density functional theory (BS-DFT) is also outlined. The characterization of Mn ligands and bound substrate waters via electron nuclear magnetic interactions is illustrated. Implications for the mechanism of water oxidation catalysis are discussed.
Based on the structure of 1.9 Å resolved OEC, which was published as PDBid = 3ARC, geometries and electronic structures of the CaMn4O5 core and surrounding water molecules at the S0 state are examined by the hybrid B3LYP and broken-symmetry methods. Two doublet states 1 and 2 with energy difference of 15.2 kcal/mol were obtained. 1 is lower than 2. The oxidation states of four manganese atoms of 1 are Mn4[III, III, IV, III], while those of 2 are Mn4[III, IV, IV, II]. It is concluded that the oxidation states of Mn4(II, III, IV, IV) is ruled out at the S0 state.
The water-splitting protein, photosystem II, catalyzes the light-driven oxidation of water to dioxygen. The solar water oxidation reaction takes place at the catalytic center, referred to as the oxygen-evolving complex, of photosystem II. During the catalytic cycle, the oxygen-evolving complex cycles through five distinct intermediate states, S0–S4. In this study, we trap the oxygen-evolving complex in the S2 intermediate state by low temperature illumination of photosystem II isolated from three different species, Thermosynechococcus vulcanus, the PsbB variant of Synechocystis PCC 6803 and spinach. We apply two-dimensional hyperfine sublevel correlation spectroscopy to detect weak magnetic interactions between the paramagnetic tetra-nuclear manganese cluster of the S2 state of the OEC and the surrounding protons. We identify five groups of protons that are interacting with the tetra-nuclear manganese cluster. From the values of hyperfine interactions and using the recently reported 1.9 Å resolution X-ray structure of the OEC in the S1 state [Umena et al., Nature, 2011, 473, 55], we discuss the assignments of the five groups of protons and draw important conclusions on the structure of the oxygen-evolving complex in the S2 state. In addition, we conclude that the structure of OEC is nearly identical in photosystem II from Thermosynechococcus vulcanus, the PsbB variant of Synechocystis PCC 6803 and spinach.
The oxygen evolving complex (OEC) of photosystem II (PSII) gives rise to manganese-derived electron paramagnetic resonance (EPR) signals in the S-0 and S-2 oxidation states. These signals exhibit different microwave power saturation behavior between 4 and 10 K. Below 8 K, the S-0 state EPR signal is a faster relaxer than the S-2 multiline signal, but above 8 K, the So signal is the slower relaxer of the two. The different temperature dependencies of the relaxation of the S-0 and S-2 ground-state Mn signals are due to differences in the spin-lattice relaxation process, The dominating spin-lattice relaxation mechanism is concluded to be a Raman mechanism in the So slate, with a T-4.1 temperature dependence of the relaxation rate. It is proposed that the relaxation of the St State arises from a Raman mechanism as well, with a T-6.8 temperature dependence of the relaxation rate, although the data also fit an Orbach process. If both signals relax through a Raman mechanism, the different exponents are proposed to reflect structural differences in the proteins surrounding the Mn cluster between the S-0 and S-2 states. The saturation of SIIslow from the Y-D(OX) radical on the D2 protein was also studied, and found to vary between the S-0 and the S-2 states of the enzyme in a manner similar to the EPR signals from the OEC, Furthermore, we found that the S-2 multiline signal in the second turnover of the enzyme is significantly more difficult to saturate than in the first turnover. This suggests differences in the OEC between the first and second cycles of the enzyme. The increased relaxation rate may be caused by the appearance of a relaxation enhancer, or it may be due to subtle structural changes as the OEC is brought into an active state.
The Mn-4-cluster of photosystem II (PSII) from Synechococcus elongatus was studied by electron paramagnetic resonance (EPR) spectroscopy after a series of saturating laser flashes given in the presence of either methanol or ethanol. Results were compared to those obtained in similar experiments done on PSII isolated from plants. The flash-dependent changes in amplitude of the EPR multiline signals were virtually identical in all samples. In agreement with earlier work [Messinger, J., Nugent, J, H. A., and Evans, M. C. W. (1997) Biochemistry, 36, 11055-11060; Ahrling, K. A., Peterson, S., and Styring, S, (1997) Biochemistry 36, 13148-13152], detection of an EPR multiline signal from the So state in PSII from plants was only possible with methanol present. In PSII from S. elongatus, it is shown that the S-0 state exhibits an EPR multiline signal in the absence of methanol (however, ethanol was present as a solvent for the artificial electron acceptor). The hyperfine lines are better resolved when methanol is present. The S-0 multiline signals detected in plant PSII and in S, elongatus were similar but not identical. Unlike the situation seen in plant PSII, the S-2 state in S. elongatus is not affected by the addition of methanol in that (i) the S-2 multiline EPR signal is not modified by methanol and (ii) the spin state of the S-2 state is affected by infrared light when methanol is present. It is also shown that the magnetic relaxation properties of an oxidized low-spin heme, attributed to cytochrome c(550), vary with the S states. This heme then is in the magnetic environment of the Mn-4 cluster.
Illuminating a photosystem II sample at low temperatures (here 5-10 K) yields so called split signals detectable with CW-EPR. These signals reflect the oxidized, deprotonated radical of D1-Tyr161 (YZ(•)) in a magnetic interaction with the CaMn4 cluster in a particular S state. The intensity of the split EPR signals are affected by the addition of the water substrate analogue methanol. This was previously shown by the induction of split EPR signals from the S1, S3 and S0 states [Su, J-H. et al. (2006) Biochemistry 45, 7617-7627.]. Here, we use two split EPR signals induced from photosystem II trapped in the S2 state to further probe the binding of methanol in an S state dependent manner. The signals are induced with either visible or near-infrared light illumination provided at 5-10 K where methanol cannot bind or un-bind from its site. The results imply that the binding of methanol not only changes the magnetic properties of the CaMn4 cluster but also the hydrogen bond network in the OEC, thereby affecting the relative charge of the S2 state. The induction mechanisms for the two split signals are different resulting in two different redox states, S2YZ(•) and S1YZ(•) respectively. The two states show different methanol dependence for their induction. This indicates the existence of two binding sites for methanol in the CaMn4 cluster. It is proposed that methanol binds to MnA with high affinity and to MnD with lower affinity. The molecular nature and S-state dependence of the methanol binding to each respective site is discussed.
The light-induced oxidation of water to O2 is catalyzed by a four-manganese atom cluster associated with Photosystem II (PS II). This chapter summarizes ongoing investigations
of the oxidation state, the structure and the associated cofactors calcium and chloride of the catalytic Mn cluster using
X-ray and electron paramagnetic resonance (EPR) spectroscopy. Manganese K-edge X-ray spectroscopy, Kβ X-ray emission spectroscopy
(XES), and extended X-ray absorption fine structure (EXAFS) studies have not only determined the oxidation states and structural
features, but also changes that occur in oxidation state of the Mn cluster and in its structural organization during the accumulation
of oxidizing equivalents leading to O2 formation. Combining X-ray spectroscopy information with X-ray diffraction studies, and consistent with the available EPR
data, we have succeeded in limiting the range of likely structures of the Mn cluster. EXAFS studies at the strontium and calcium
K-edges have provided evidence that the catalytic center is a Mn/Ca heteronuclear complex. Based on the X-ray spectroscopy
data, models for the structure and a mechanism for O2 evolution are presented.
The structures of the functional sites in photosystem II in higher plants have been studied by electron-nuclear double resonance
(ENDOR), electron spin echo envelope modulation and pulsed electron-electron double resonance (PELDOR). On the acceptor side
the distances from the primary acceptor quinone to P680 and triplet chlorophyll were determined on the basis of the dipolar
interaction between the radical pair. On the donor side the microenvironment of tyrosine Z radical (Y2
.) was studied by ENDOR to elucidate the specific electron transfer function. PELDOR of the Mn4 cluster in the S0 and S2 states and tyrosine D radical (YD
.) revealed that spin projections on the four manganese atoms are necessary to estimate the distance between them.
Recent spectroscopic studies on the water oxidizing complex (WOC) of Photosystem II are discussed in terms of the possible
nature and structures for the Mn containing catalytic site. Emphasis is given to the various electron paramagnetic resonance
techniques which have been increasingly employed, as well as examination of complementary data from optical and X-ray absorption
spectroscopies. All of the quasi-stable intermediate states of the catalytic turnover cycle of the WOC (S0 to S3) are now accessible spectroscopically. We show that the available data may be rationalized by a scheme in which the ‘active,’
catalytically cycling component of the water oxidizing site contains a pair of coupled, oxo bridged Mn ions, closely associated
with a Ca ion. The structure of the active Mn site resembles the structure of the dinuclear Mn catalase. During functional
turnover, oxidizing equivalents are stored both in Mn ions and ligand groups and one Mn ion has a unique, low symmetry ligand
environment.
The redox-active tyrosine residue (YZ) plays a crucial role in the mechanism of the water oxidation. Metalloradical electron paramagnetic resonance (EPR) signals
reflecting the light-induced YZ· in magnetic interaction with the CaMn4-cluster in the particular S-state, YZ·SX intermediates, have been found in intact photosystem II. These so-called split EPR signals are induced by illumination at
cryogenic temperatures and provide means to both study the otherwise transient YZ· and to probe the S-states with EPR spectroscopy. The illumination used for signal induction grouped the observed split EPR signals in two categories:
(i) YZ in the lower S-states was oxidized by P680+ formed via charge separation, while (ii) YZ in the higher S-states was oxidized by an excited, highly oxidizing Mn species. Applied mechanistic studies of the YZ·SX intermediates in the different S-states are reviewed and compared to investigations in photosystem II at physiological temperature. Addition of methanol induced
S-state characteristic changes in the split signals’ formation which reflect changes in the magnetic coupling within the CaMn4-cluster due to methanol binding. The pH titration of the split EPR signals, on the other hand, could probe the proton-coupled
electron transfer properties of the YZ oxidation. The apparent pK
as found for decreased split signal induction were interpreted in the fate of the phenol proton.
Dinuclear and tetranuclear complexes of spin-coupled manganese-manganese, manganese-iron, and iron-iron ions occur in enzymes
which are of importance in biology and photosynthesis. These multinuclear systems contain higher oxidation states of manganese
and iron ions than those of the commonly occurring Mn(II) and Fe(III) mononuclear ions, in which there has been a lot of interest
recently with regard to their electron paramagnetic resonance (EPR) spectra. In order to aid the interpretation of EPR spectra,
the spin Hamiltonians, and the resulting energy levels, characterizing binuclear and tetranuclear manganese ions will be discussed
in this presentation. These will include, in particular, Mn(II)−Mn(III), Mn(III)−Mn(IV), and Mn(IV)−Mn(V) systems for the
binuclear situation, and the Mn4O2 “butterfly”, cubane, dimer of dimer systems, and diamond structure for the tetramer situation. These coupled systems are
characterized by a variety of exchange interactions whose magnitudes and nature, ferromagnetic or antiferromagnetic, affect
profoundly the energy levels. In addition, the hyperfine interactions amongst the various55Mn nuclei produce a complex EPR spectrum consisting of a large number of hyperfine lines. In order to interpret EPR spectra
properly, one needs to simulate them as accurately as possible. Details of a rigorous technique for the simulation of EPR
spectra of these systems with matrix diagonalization and homotopy technique will be provided.
The solar water-splitting protein complex, photosystem II, catalyzes one of the most energetically demanding reactions in nature by using light energy to drive water oxidation. The four-electron water oxidation reaction occurs at the tetranuclear manganese-calcium-oxo cluster that is present in the oxygen-evolving complex of photosystem II. The tetranuclear manganese-calcium-oxo cluster is comprised of mixed-valence Mn(III) and Mn(IV) ions in the ground state. The oxo-manganese dimer, [H(2)O(terpy)Mn(III)(μ-O)(2)Mn(IV)(terpy)OH(2)](NO(3))(3) (terpy = 2,2':6',2″-terpyridine) (1), is an excellent biomimetic model that has been extensively used to gain insight on the molecular structure and mechanism of water oxidation in photosystem II. In this work, weak magnetic interactions between the protons of the two terminal water ligands and the paramagnetic dimanganese "di-μ-oxo" core of 1 are quantitatively characterized using two-dimensional hyperfine sublevel correlation (HYSCORE) spectroscopy. For the water molecule that is directly coordinated at the Mn(III) ion, the two protons are found to be magnetically equivalent and exhibit near axial hyperfine anisotropy. In contrast, for the first time, we demonstrate that the two protons of the water molecule that is directly coordinated at the Mn(IV) ion are inequivalent. We obtain the isotropic and anisotropic components of the hyperfine interaction for each proton. A comparison of the HYSCORE spectra measured in the presence and absence of acetate ions provides unambiguous evidence that only one molecule of acetate binds to 1 by replacing a terminal water molecule that is coordinated at the Mn(III) ion.
The photosystem II core complex (PSIIcc) is the key enzyme of oxygenic photosynthesis, as it catalyzes the light-induced oxidation of water to form dioxgyen and protons. It is located in the thylakoid membrane of cyanobacteria, algae, and plants and consists of 20 protein subunits binding about 100 cofactors. In this review, we discuss what is presently known about the "donor side" of PSIIcc, covering the photosynthetic reaction center and the water oxidase part. The focus is on the catalytic Mn4Ca cluster and its protein environment. An attempt is made to connect recent crystallographic data (up to 2.9 Å resolution) with the wealth of information about Nature's water oxidation device from spectroscopic, biochemical and theoretical work.
Large size (228 atom, 229 atom for protonated form) molecular models of the oxygen evolving complex of photosystem II (OEC), with a complete set of ligating aminoacids, the redox-active tyrosine Y(Z), and proton/water transfer channels terminating at the water oxidizing Mn/Ca cluster, are constructed based on the highest available resolution X-ray diffraction structures of the protein and our previous density functional theory (DFT) studies of isolated metal cluster model structures. Geometries optimized using the general gradient approximation (GGA) or hybrid density functionals are compared with high-resolution extended X-ray absorption fine structure (EXAFS) spectroscopic data and show that an antiferromagnetic configuration of the Mn centers in the cluster gives computed metal-metal distances in excellent agreement with experiment. The excitation energies predicted by time-dependent density functional theory (TDDFT) calculations for truncated 106 atom and 78 atom structures derived from the large models show that a previously proposed III-III-III-II oxidation pattern of the Mn atoms agrees very well with the X-ray absorption near-edge structure (XANES) observed for the S(0) state of the OEC. This supports a "low" Mn oxidation state paradigm for the OEC, when a realistic protein imposed environment for the catalytic metal cluster is used in calculations. The probable protonation sites in the cluster and roles of the proton/water transfer channels are discussed in light of the computational results.
Succinate:quinone reductases (SQRs) and quinol:fumarate reductases (QFRs) each contain a bi-, a tri- and a tetra-nuclear iron-sulfur cluster. The C-terminal half of the iron-sulfur protein subunit of these enzymes shows two fully conserved motifs of cysteine residues, stereotypical for ligands of [3Fe-4S] and [4Fe-4S] clusters. To analyze the functional role of the trinuclear cluster S3 in Bacillus subtilis SQR, a fourth cysteine residue was introduced into the putative ligation motif to that cluster. A corresponding mutation in Escherichia coli QFR results in a tri- to tetranuclear conversion (Manodori et al. (1992) Biochemistry 31, 2703-2731). We have found that presence of the extra cysteine in B. subtilis SQR does not result in cluster conversion. It does, however, affect the EPR properties of the cluster S3, whereas those of the other two clusters remain normal. The results strongly support the view that residues in the most C-terminal cysteine motif in the iron-sulfur protein subunit of SQRs and QFRs ligate the trinuclear cluster. Compared to wild-type SQR, S3 in the B. subtilis mutant enzyme is not sensitive to methanol and the midpoint redox potential is close to normal. The quinone reductase activity of the mutant enzyme is only 35% of normal. Thus, the architecture around cluster S3 plays a role in electron transfer to quinone or in the binding of quinone to the enzyme.
Electron paramagnetic resonance of spinach chloroplasts given a series of laser flashes, n = 0, 1,..., 6, at room temperature and rapidly cooled to -140 degrees C reveals a signal possessing at least 16 and possibly 21 or more hyperfine lines when observed below 35 K. The spectrum is consistent with a pair of antiferromagnetically coupled Mn ions, or possibly a tetramer of Mn ions, in which Mn(III) and Mn(IV) oxidation states are present. The intensity of this signal peaks on the first and fifth flashes, suggesting a cyclic change in oxidation state of period 4. The multiline signal produced on the first flash is not affected by the electron transport inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea but is abolished by agents that influence the state of bound manganese, such as incubation with alkaline Tris, or dithionite, and by extraction with cholate detergent in the presence of ammonium sulfate. These results indicate that the paramagnetic signal is monitoring oxidation state changes in the enzyme involved in oxidation of water.
The authors report the detection of a new electron paramagnetic resonance (EPR) signal that demonstrates the presence of a paramagnetic intermediate in the resting (S{sub 1}) state of the photosynthetic oxygen-evolving complex. The signal was detected using the method of parallel polarization EPR, which is sensitive to {Delta}m = 0 transitions in high spin systems. The properties of the parallel polarization EPR signal in the S{sub 1} state are consistent with an S=1 spin state of and exchange-coupled manganese center that corresponds to the reduced form of the species giving rise to the multiline EPR signal in the light-induced S{sub 2} state. The implications for the electronic structure of the oxygen-evolving complex are discussed. 36 refs., 2 fig., 1 tab.
Fe+ + + and Fe+ + ions in glass have been studied by means of paramagnetic resonance. The correlation of the Fe+ + + concentration with an intense resonance at g=4.27 shows the resonance is due to Fe+ + +. A theory is proposed explaining the observed g value and the implications for the atomic surroundings of Fe+ + + are discussed. It is also proposed that the g=6 resonance observed in glass by Sands is due to Fe+ + +.
Microwave power saturation studies have been performed over the range 4–20 K on EPR signals photogenerated in PS II particles by low-temperature illumination (180–240 K). In the presence of 3% methanol (+ MeOH), with no g 4.1 signal present, the multiline signal intensity (extrapolated to zero power) shows strict Curie law behaviour over the 4–20 K range. With no MeOH present in the suspension buffer (−MeOH), both the multiline and 4.1 signals show complementary deviations from Curie law behaviour. These are consistent with the signals arising respectively from the ground, S = 1/2, and first excited S = 3/2, states of a total spin = 7/2 multiplet, such as could occur in an MnIV-MnIII antiferromagnetically coupled pair. The deduced height of the 3/2 state above the ½ state is 9.0 K. An inferential estimate, from relaxation data, of this height for the +MeOH case is about 40 K. A broad, featureless component around g = 2 appears to underlie the multiline pattern in the presence of the 4.1 signal, and has a similar temperature behaviour to the latter. A possible exchange coupling model, involving four Mn centres, is presented to accommodate these and other findings on the S2 state signals.
In Photosystem II preparations at low temperature we were able to generate and trap an intermediate state between the S1 and S2 states of the Kok scheme for photosynthetic oxygen evolution. Illumination of dark-adapted, oxygen-evolving Photosystem II preparations at 140 K produces a 320-G-wide EPR signal centered near g = 4.1 when observed at 10 K. This signal is superimposed on a 5-fold larger and somewhat narrower background signal; hence, it is best observed in difference spectra. Warming of illuminated samples to 190 K in the dark results in the disappearance of the light-induced g = 4.1 feature and the appearance of the multiline EPR signal associated with the S2 state. Low-temperature illumination of samples prepared in the S2 state does not produce the g = 4.1 signal. Inhibition of oxygen evolution by incubation of PS II preparations in 0.8 M NaCl buffer or by the addition of 400 μM NH2OH prevents the formation of the g = 4.1 signal. Samples in which oxygen evolution is inhibited by replacement of Cl− with F− exhibit the g = 4.1 signal when illuminated at 140 K, but subsequent warming to 190 K neither depletes the amplitude of this signal nor produces the multiline signal. The broad signal at g = 4.1 is typical for a spin system in a rhombic environment, suggesting the involvement of non-heme Fe in photosynthetic oxygen evolution.
The multiline EPR signals arising from manganese in the S2 state of the oxygen-evolving system of spinach and the cyanobacterium Anacystis nidulans have very similar properties and are affected identically by NH3, suggesting that the system is highly conserved. The temperature dependence of the signal amplitude follows Curie behavior down to sub-helium temperatures. This is in contrast to previous reports, which were taken as evidence for a tetrameric manganese cluster. Thus, it seems that it is not yet possible from EPR data alone to distinguish between this model and a dimeric structure.
— Using isolated chloroplasts and techniques as described by Joliot and Joliot[6] we studied the evolution of O2 in weak light and light flashes to analyze the interactions between light induced O2 precursors and their decay in darkness. The following observations and conclusions are reported: 1. Light flashes always produce the same number of oxidizing equivalents either as precursor or as O2. 2. The number of unstable precursor equivalents present during steady state photosynthesis is ∼ 1.2 per photochemical trapping center. 3. The cooperation of the four photochemically formed oxidizing equivalents occurs essentially in the individual reaction centers and the final O2 evolution step is a one quantum process. 4. The data are compatible with a linear four step mechanism in which a trapping center, or an associated catalyst, (S) successively accumulates four + charges. The S4+ state produces O2 and returns to the ground state S0. 5. Besides S0 also the first oxidized state S+ is stable in the dark, the two higher states, S2+ and S3+ are not. 6. The relaxation times of some of the photooxidation steps were estimated. The fastest reaction, presumably S*1←S2, has a (first) half time ≤ 200 μsec. The S*2 state and probably also the S*0 state are processed somewhat more slowly (˜ 300–400 μsec).
Interactions of water and methanol with a mixed valence Mn(III)Mn(IV) complex are explored with 1H electron spin echo (ESE)-electron nuclear double resonance (ENDOR) and 1H and 2H ESE envelope modulation (ESEEM). Derivatives of the (2-OH-3,5-Cl2-SALPN)2 Mn(III)Mn(IV) complex are ideal for structural and spectroscopic modeling of water binding to multinuclear Mn complexes in metalloproteins, specifically photosystem II (PSII) and manganese catalase (MnCat). Using ESE-ENDOR and ESEEM techniques, 1H hyperfine parameters are determined for both water and methanol ligated to the Mn(III) ion of the complex. The protons of water directly bound to Mn(III) are inequivalent and exhibit roughly axial dipolar hyperfine interactions (Tdip = 8.4 MHz and Tdip = 7.4 MHz), permitting orientations and radial distances to be determined using a model where the proton experiences a point dipole interaction with each Mn ion. General equations are given for the components of the rhombic dipolar hyperfine interaction between a proton and a spin coupled dinuclear metal cluster. The observed ENDOR pattern is from water protons 2.65 and 2.74 Å from the Mn(III) which make an Mn(IV)−Mn(III)−H angle of 160°. For the alcohol proton in the analogous methanol bound complex, a 2.65 Å Mn(III)−H distance is observed. Three pulse 2H ESEEM gives best fit Mn(III)−2H(1H) radial distances of 3.0, 3.5, and 4.0 Å for the three methyl deuterons in this complex.
The microwave power for half-saturation (P1/2) for the radical in photosystem II giving rise to signal IIslow (SIIs) has been measured by EPR in samples illuminated by a series of flashes. The charge storage state of the oxygen-evolving complex (S0-S4) was monitored by measuring the multiline EPR signal arising from the S2 state. The following results were obtained: (1) SIIs becomes easier to saturate after tris(hydroxymethyl)aminomethane (Tris) washing, a treatment that partially removes the Mn cluster. (2) P1/2 for SIIs oscillates with the flash number. P1/2 is lower in S1 (in dark-adapted material and after four flashes) than in S2, S3, or S0. (3) P1/2(S2) = P1/2(S3). (4) At 8 K P1/2(S2) > P1/2(S2), but at 20 K P1/2(S0) < P1/2(S2). (5) P1/2 for SIIs increases with temperature (8-70 K) in the S1 state. SHs is more difficult to saturate in S2, S3, and S0 than in S1 over the investigated temperature range. In addition, the increase in P1/2 is complex around 20-30 K in S2, S3, and S0. (6) In S0, P1/2 for SIIs decreases with time (decay half-time 30-60 s) to a stable level significantly above the dark level. The data are explained in terms of cross relaxation between the radical giving rise to SIIs and an efficient relaxer, which is suggested to be the Mn cluster. This relaxes more slowly in S1 than in the other S states. Since it is known that a mixed-valence Mn cluster is present in S2, and because P1/2 of SIIs in S3 and S0 is comparable to that in S2, it is suggested that mixed-valence Mn clusters are present in the S3 and S0 states also. Different models with these features can be proposed, the simplest of which is the following: S0 [Mn(H)-Mn(III)], S1 [Mn(III)-Mn(III)], S2 [Mn(III)-Mn(IV)], and S3 [Mn(III)-Mn(IV)].
A study of electron paramagnetic resonance (EPR) signals from components on the electron donor side of photosystem II has been performed. By measurement of EPR signal IIslow (D+) it is shown that, after three flashes, D+ decays slowly in the dark at room temperature in the fraction of the centers that was in the S0 state (t1/2 of 20 min in thylakoid membranes and 50 min in photosystem II enriched membranes). This reaction is accompanied by a conversion of S0 to S1. The concentration of S1 was estimated from the amplitude of the S2-state multiline EPR signal that could be generated by illumination at 200 K. These observations indicate that D+ accepts an electron from S0 in a dark reaction in which D and S1 are formed. In addition, the reactions by which D donates an electron to S2 or S3 have been directly measured by monitoring both signal IIslow and the multiline signal. The redox interactions between the D/D+ couple and the S states are explained in terms of a model in which D/D+ has a midpoint potential between those of S0/S1, and S1/S2. In addition, this model provides explanations for a number of previously unrelated phenomena, and the proposal is put forward that the reaction between D+ and Mn2+ is involved in the so-called photoactivation process.
The light-induced EPR multiline signal is studied in O2-evolving PS II membranes. The following results are reported: (1) Its amplitude is shown to oscillate with a period of 4, with respect to the number of flashes given at room temperature (maxima on the first and fifth flashes). (2) Glycerol enhances the signal intensity. This effect is shown to come from changes in relaxation properties rather than an increase in spin concentration. (3) Deactivation experiments clearly indicate an association with the S2 state of the water-oxidizing enzyme. A signal at g = 4.1 with a linewidth of 360 G is also reported and it is suggested that this arises from an intermediate donor between the S states and the reaction centre. This suggestion is based on the following observations: (1) The g = 4.1 signal is formed by illumination at 200 K and not by flash excitation at room temperature, suggesting that it arises from an intermediate unstable under physiological conditions. (2) The formation of the g = 4.1 signal at 200 K does not occur in the presence of DCMU, indicating that more than one turnover is required for its maximum formation. (3) The g = 4.1 signal decreases in the dark at 220 K probably by recombination with Q−AFe. This recombination occurs before the multiline signal decreases, indicating that the g = 4.1 species is less stable than S2. (4) At short times, the decay of the g = 4.1 signal corresponds with a slight increase in the multiline S2 signal, suggesting that the loss of the g = 4.1 signal results in the disappearance of a magnetic interaction which diminishes the multiline signal intensity. (5) Tris-washed PS II membranes illuminated at 200 K do not exhibit the signal.
The spin-lattice relaxation time of the EPR signal of the tyrosine radical D+ has been measured with electron spin echo spectroscopy in the range 5–30 K in Photosystem II preparations with intact oxygen evolving complex (OEC). The charge storage state of the OEC was set by illumination with a series of flashes and monitored by measuring the multiline EPR signal of the S2-state. The OEC was synchronized to 100% S1 initial state by dark-adaptation and one preflash. Agreeing with previous work (De Groot, A., Plijter, J.J., Evelo, R., Babcock, G.T. and Hoff, A.J. (1986) Biochim. Biophys. Acta 848, 8–15), the spin-lattice relaxation curves were foundto be bi-phasic. The average relaxation time τ¯ of each S-state was calculated from the data obtained for the 0—3 flash sample and the known S-state distribution,τ¯was found to be maximal in the S1-state. It decreased about 40% for the S2-state, was essentially the same for the S2- and the S3-states and decreased again by about 55% for the S0-state. These results are similar to those obtained earlier by cw EPR (Styring, S. and Rutherford, A.W. (1988) Biochemistry 27, 4915–4923). At 5 K the two exponentials describing the relaxation curves had characteristic timesτf, and τs that differed by an order of magnitude. Their amplitude was about equal, except for S0 where the faster process predominated. At 20 K the characteristic time of both the fast and the slow process was reduced by a factor of about five;their amplitudes were again about equal. The observed relaxation times τf and τs were deconvoluted as a function of S-state by an approximate method. At 5 K it was found that τf was about twice as fast for S0 and S3 than for S, and S2 (1.3 vs. 2.6 ms) and τ sabout twice as fast for S0, S2, S3 than for S1 (13.7–14.5 vs. 28.6 ms). The same trend was observed at higher temperatures. Interpreting the results with relaxation enhancement theory and integrating them with the results from cw EPR, NMR and EXAFS spectroscopy the following model for the OEC is presented, (i) To explain the biphasic relaxation of D+ it is suggested that two Mn are close to D+ at different distances, enhance the relaxation of D+and are not magnetically coupled. Their oxidation state differs by 1 unit, is probably Mn3+ and Mn4+, and does not change during the S0 → S3 sequence. It is postulated that at high temperature there is a charge resonance between the two Mn ions that is frozen out when cooling to cryogenic temperature, (ii) Two of the four Mn of the OEC form an antiferromagnetically coupled binuclear cluster in the oxidation state Mn2+·Mn3+, Mn3+·Mn3+, Mn3+·Mn4+, Mn3+·Mn4+ in the S0, S1, S2 and S3-sta respectively, (iii) From the temperature dependence of the relaxation of D+ in the S0-state, it is estimated that the distance between the Mn cluster and D+ is 30–40 .
The electron-spin relaxation of iron-sulphur centres in a range of simple proteins (ferredoxin, high-potential iron-sulphur protein and rubredoxin) was investigated by means of the temperature dependence and microwave power saturation of the EPR signal. The proteins containing [2Fe-2S] centres all showed temperature optima higher than those for [4Fe-4S] centres, but the difference between the slowest-relaxing [4Fe-4S] protein (Chromatium high-potential iron-sulphur protein) and the fastest-relaxing [2Fe-2S] protein (Halobacterium halobium ferredoxin) was small. A greater distinction was seen in the power saturation behaviour at low temperature (10--20 K). The behaviour of the signal intensity as a function of microwave power was analyzed in terms of the power for half saturation P 1/2 and the degree of homogeneous/inhomogeneous broadening. The effect of distorting the protein structure by salts, organic solvents and urea was to decrease the electron-spin relaxation rate as shown by a decreased value of P 1/2. The addition of Ni2+ as a paramagnetic perturbing agent caused an increase in the electron-spin relaxation rate of all the proteins, with the exception of adrenal ferredoxin, as shown by an increased P 1/2 and, in a few cases, broadening of the linewidth. Ferricyanide, a commonly used oxidizing agent, has similar effects. These results are discussed in relation to the use of paramagnetic probes to determine whether iron-sulphur centres are near to a membrane surface. Spin-spin interactions between two paramagnetic centres in a protein molecule such as a 2[4Fe-4S] ferredoxin, lead to more rapid electron-spin relaxation. This method was used to detect a spin-spin interaction between molybdenum V and centre Fe-SI in xanthine oxidase.
The amplitude of the g = 2 Mn 'multiline' EPR signal of the S2 state of the photosynthetic oxygen-evolving complex varies inversely with temperature, indicating that this signal arises from a ground spin state. Electron spin echo experiments at temperatures of 4.2 K and 1.4 K show such Curie-law behavior of the g = 2 multiline EPR signal, as do continuous-wave EPR experiments performed at a non-saturating microwave power in the range from 15.0 K to 4.2 K.
This guide is intended to aid in the detection and identification of paramagnetic species in Photosystem II membranes, by electron paramagnetic resonance spectroscopy. The spectral features and occurrence of each of the electron paramagnetic resonance signals from Photosystem II are discussed, in relation to the nature of the moiety giving rise to the signal and the role of that species in photosynthetic electron transport. Examples of most of the signals discussed are shown. The electron paramagnetic resonance signals produced by the cytochrome b6f and Photosystem I complexes, as well as the signals from other common contaminants, are also reviewed. Furthermore, references to seminal experiments on bacterial reaction centers are included. By reviewing both the spectroscopic and biochemical bases for the electron paramagnetic resonance signals of the cofactors that mediate photosynthetic electron transport, this paper provides an introduction to the use and interpretation of electron paramagnetic resonance spectroscopy in the study of Photosystem II.
A sensitive method for obtaining the zero-field crystalline field splitting is described and applied to ferrimyoglobin (H2O) and ferrimyoglobin (F−). The high accuracy (1–2%) with which the splitting can be determined should make it possible to explore the environmental (conformatinal) changes at the Fe3+ site.
Magnetic properties of the S1-state manganese cluster in the oxygen-evolving photosystem II were studied by parallel polarization electron paramagnetic resonance spectroscopy. Dark minus light spectra gave rise to a broad S1-state signal with a g value of about 4.9 [Dexheimer, S. L., Klein, M. P. (1992) J. Am. Chem. Soc. 114, 2821-2826]. Temperature variation of the signal intensity between 1.9 and 10 K observed in PS II with a sucrose buffer indicates that the signal originates from an excited state with a spin S of 1 with separation from the ground state (S = 0) of about 2.5 K. The S1-state signal was also observed in the sucrose buffer supplemented by 50% glycerol. However, no S1-state signal was detected by addition of 3% methanol or 30% ethylene glycol in the sucrose buffer, although illumination at 200 K in the presence of these alcohols induced the normal multiline S2 signal. Furthermore, modification of the Mn cluster by Cl- or Ca2+ depletion from PS II membranes failed to produce a detectable S1-state signal. A possible magnetic structure of the Mn cluster responsible for the generation of the S1-state signal is discussed on the basis of these observations.
The S0* state was generated by incubation of dark-adapted (S1 state) photosystem II membranes either with the exogenous two electron reductant hydrazine and subsequent 273 K illumination in the presence of DCMU or by dark incubation with low amounts of the one electron reductant hydroxylamine. In agreement with earlier reports, the S1 and S-1 states were found to be electron paramagnetic resonance (EPR) silent. However, in the presence of 0.5-1.5% methanol, a weak EPR multiline signal centered around g = 2.0 was observed at 7 K for the S0* states generated by both procedures. This signal has a similar average line splitting to the well-characterized S2 state multiline EPR signal, but can be clearly distinguished from that and other modified S2 multiline signals by differences in line position and intensities. In addition, at 4 K it can be seen that the S0* multiline has a greater spectral breadth than the S2 multilines and is composed of up to 26 peaks. The S0* signal is not seen in the absence of methanol and is not affected by 1 mM EDTA in the buffer medium. We assign the S0* multiline signal to the manganese cluster of the oxygen evolving complex in a mixed valence state of the form MnIIMnIIIMnIIIMnIII,MnIIMnIIIMnIVMnIV, or MnIIIMnIIIMnIIIMnIV. Addition of methanol may be helpful in future to find an EPR signal originating form the natural S0 state.
Photosynthesis produces the oxygen necessary for all aerobic life. During this process, the manganese-containing oxygen evolving complex (OEC) in photosystem II (PSII), cycles through five oxidation states, S0-S4. One of these, S2, is known to be paramagnetic and gives rise to electron paramagnetic resonance (EPR) signals used to probe the catalytic structure and function of the OEC. The S0 states has long been thought to be paramagnetic. We report here a Mn EPR signal from the previously EPR invisible S0 state. The new signal oscillates with a period of four, indicating that it originates from fully active PSII centers. Although similar to the S2 state multiline signal, the new signal is wider (2200 gauss compared with 1850 gauss in samples produced by flashing), with different peak intensity and separation (82 gauss compared with 89 gauss). These characteristics are consistent with the S0 state EPR signal arising from a coupled MnII-MnIII intermediate. The new signal is more stable than the S2 state signal and its decay in tens of minutes is indicative of it originating from the S0 state. The S0 state signal will provide invaluable information toward the understanding of oxygen evolution in plants.
The essential involvement of manganese in photosynthetic water oxidation was implicit in the observation by Pirson in 1937 that plants and algae deprived of Mn in their growth medium lost the ability to evolve Oâ. Addition of this essential element to the growth medium resulted in the restoration of water oxidation within 30 min. There is increased interest in the study of Mn in biological chemistry and dioxygen metabolism in the last two decades with the discovery of several Mn redox enzymes. The list of enzymes where Mn is required for redox activity includes a Mn superoxide dismutase, a binuclear Mn-containing catalase, a binuclear Mn-containing ribonucleotide reductase, a proposed binuclear Mn site in thiosulfate oxidase, a Mn peroxidase that is capable of oxidative degradation of lignin, and perhaps the most complex and important, the tetranuclear Mn-containing oxygen-evolving complex in photosystem II (Mn-OEC). Mn is well suited for the redox role with accessible oxidation states of II, III, and IV, and possibly V: oxidation states that have all been proposed to explain the mechanisms of the Mn redox enzymes.