Protons bound to the Mn cluster in photosystem II oxygen evolving complex detected by proton matrix ENDOR

Protons in the vicinity of the oxygen-evolving manganese cluster in photosystem II were studied by proton matrix ENDOR. Six pairs of proton ENDOR signals were detected in both the S(0) and S(2) states of the Mn-cluster. Two pairs of signals that show hyperfine constants of 2.3/2.2 and 4.0 MHz, respectively, disappeared after D(2)O incubation in both states. The signals with 2.3/2.2 MHz hyperfine constants in S(0) and S(2) state multiline disappeared after 3 h of D(2)O incubation in the S(0) and S(1) states, respectively. The signal with 4.0 MHz hyperfine constants in S(0) state multiline disappeared after 3 h of D(2)O incubation in the S(0) state, while the similar signal in S(2) state multiline disappeared only after 24 h of D(2)O incubation in the S(1) state. The different proton exchange rates seem to be ascribable to the change in affinities of water molecules to the variation in oxidation state of the Mn cluster during the water oxidation cycle. Based on the point dipole approximation, the distances between the center of electronic spin of the Mn cluster and the exchangeable protons were estimated to be 3.3/3.2 and 2.7 A, respectively. These short distances suggest the protons belong to the water molecules ligated to the manganese cluster. We propose a model for the binding of water to the manganese cluster based on these results.     

Protons bound to the Mn cluster in photosystem II oxygen evolving complex detected by proton matrix ENDOR

Protons in the vicinity of the oxygen-evolving manganese cluster in photosystem II were studied by proton matrix ENDOR. Six pairs of proton ENDOR signals were detected in both the S₀ and S₂ states of the Mn-cluster. Two pairs of signals that show hyperfine constants of 2.3/2.2 and 4.0 MHz, respectively, disappeared after D₂O incubation in both states. The signals with 2.3/2.2 MHz hyperfine constants in S₀ and S₂ state multiline disappeared after 3 h of D₂O incubation in the S₀ and S₁ states, respectively. The signal with 4.0 MHz hyperfine constants in S₀ state multiline disappeared after 3 h of D₂O incubation in the S₀ state, while the similar signal in S₂ state multiline disappeared only after 24 h of D₂O incubation in the S₁ state. The different proton exchange rates seem to be ascribable to the change in affinities of water molecules to the variation in oxidation state of the Mn cluster during the water oxidation cycle. Based on the point dipole approximation, the distances between the center of electronic spin of the Mn cluster and the exchangeable protons were estimated to be 3.3/3.2 and 2.7 Å, respectively. These short distances suggest the protons belong to the water molecules ligated to the manganese cluster. We propose a model for the binding of water to the manganese cluster based on these results.     

Interaction of nitric oxide with the oxygen evolving complex of photosystem II and manganese catalase: a comparative study

Thermus thermophilus catalase. Flash fluorescence studies indicate that the S₃ state of the OEC in the presence of ca. 0.6 mM NO is reduced to the S₁ with an apparent halftime of ca. 0.4 s at about 18 °C, compared with a biphasic decay, with approximate halftimes of 28 s for S₃ to S₂ and 140 s for S₂ to S₁ in the absence of NO. Under similar conditions the S₂ state is reduced by NO to the S₁ state with an approximate halftime of 2 s. These results extend a recent study indicating a slow reduction of the S₁ state at −30°C, via the S₀ and S₋₁ states, to a Mn(II)-Mn(III) state resembling the corresponding state in catalase. The reductive mode of action of NO is repeated with the di-Mn cluster of catalase: the Mn(III)-Mn(III) redox state is reduced to the Mn(II)-Mn(II) state via the intermediate Mn(II)-Mn(III) state. The kinetics of this reduction suggest a decreasing reduction potential with decreasing oxidation state, similar to what is observed with the active states of the OEC. What is unique about the OEC is the rapid interaction of NO with the S₃ state of the OEC, which is compatible with a metalloradical character of this state. Received: 16 June 1999 / Accepted: 28 February 2000     

S-state dependent Fourier transform infrared difference spectra for the photosystem II oxygen evolving complex

Vibrational spectroscopy provides a means to investigate molecular interactions within the active site of an enzyme. We have applied difference FTIR spectroscopy coupled with a flash turnover protocol of photosystem II (PSII) to study the oxygen evolving complex (OEC). Our data show two overlapping oscillatory patterns as the sample is flashed through the four-step S-state cycle that produces O(2) from two H(2)O molecules. The first oscillation pattern of the spectra shows a four-flash period four oscillation and reveals a number of new vibrational modes for each S-state transition, indicative of unique structural changes involved in the formation of each S-state. Importantly, the first and second flash difference spectra are reproduced in the 1800-1200 cm(-)(1) spectral region by the fifth and sixth flash difference spectra, respectively. The second oscillation pattern observed is a four-flash, period-two oscillation associated with changes primarily to the amide I and II modes and reports on changes in sign of these modes that alternate 0:0:1:1 during S-state advance. This four-flash, period-two oscillation undergoes sign inversion that alternates during the S(1)-to-S(2) and S(3)-to-S(0) transitions. Underlying this four-flash period two is a small-scale change in protein secondary structure in the PSII complex that is directly related to S-state advance. These oscillation patterns and their relationships with other PSII phenomena are discussed, and future work can initiate more detailed vibrational FTIR studies for the S-state transitions providing spectral assignments and further structural and mechanistic insight into the photosynthetic water oxidation reaction.     

Digalactosyldiacylglycerol is required for stabilization of the oxygen-evolving complex in photosystem II

The galactolipid digalactosyldiacylglycerol (DGDG) is present in the thylakoid membranes of oxygenic photosynthetic organisms such as higher plants and cyanobacteria. Recent x-ray crystallographic analysis of protein-cofactor supercomplexes in thylakoid membranes revealed that DGDG molecules are present in the photosystem II (PSII) complex (four molecules per monomer), suggesting that DGDG molecules play important roles in folding and assembly of subunits in the PSII complex. However, the specific role of DGDG in PSII has not been fully clarified. In this study, we identified the dgdA gene (slr1508, a ycf82 homolog) of Synechocystis sp. PCC6803 that presumably encodes a DGDG synthase involved in the biosynthesis of DGDG by comparison of genomic sequence data. Disruption of the dgdA gene resulted in a mutant defective in DGDG synthesis. Despite the lack of DGDG, the mutant cells grew as rapidly as the wild-type cells, indicating that DGDG is not essential for growth in Synechocystis. However, we found that oxygen-evolving activity of PSII was significantly decreased in the mutant. Analyses of the PSII complex purified from the mutant cells indicated that the extrinsic proteins PsbU, PsbV, and PsbO, which stabilize the oxygen-evolving complex, were substantially dissociated from the PSII complex. In addition, we found that heat susceptibility but not dark-induced inactivation of oxygen-evolving activity was notably increased in the mutant cells in comparison to the wild-type cells, suggesting that the PsbU subunit is dissociated from the PSII complex even in vivo. These results demonstrate that DGDG plays important roles in PSII through the binding of extrinsic proteins required for stabilization of the oxygen-evolving complex.     

Inhibition of photosystem II photochemistry by Cr is caused by the alteration of both D1 protein and oxygen evolving complex

The effect of chromium (Cr) on photosystem II (PSII) electron transport and the change of proteins content within PSII complex were investigated. When Lemna gibba was exposed to Cr during 96 h, growth inhibition was found to be associated with an alteration of the PSII electron transport at both PSII oxidizing and reducing sides. Investigation of fluorescence yields at transients K, J, I, and P suggested for Cr inhibitory effect to be located at the oxygen-evolving complex and Q_A reduction. Those Cr-inhibitory effects were related to the change of the turnover of PSII D1 protein and the alteration of 24 and 33 kDa proteins of the oxygen-evolving complex. The inhibition of the PSII electron transport and the formation of reactive oxygen species induced by Cr were highly correlated with the decrease in the content of D1 protein and the amount of 24 and 33 kDa proteins. Therefore, functional alteration of PSII activity by Cr was closely related with the structural change within PSII complex.     

Relative stability of the S2 isomers of the oxygen evolving complex of photosystem II

Photosynthesis Research - The oxidation of water to O2 is catalyzed by the Oxygen Evolving Complex (OEC), a Mn4CaO5 complex in Photosystem II (PSII). The OEC is sequentially oxidized from state S0...     

LPA2 is required for efficient assembly of photosystem II in Arabidopsis thaliana

To elucidate the molecular mechanism of photosystem II (PSII) assembly, we characterized the low psii accumulation2 (lpa2) mutant of Arabidopsis thaliana, which is defective in the accumulation of PSII supercomplexes. The levels and processing patterns of the RNAs encoding the PSII subunits are unaltered in the mutant. In vivo protein-labeling experiments showed that the synthesis of CP43 (for chlorophyll a binding protein) was greatly reduced, but CP47, D1, and D2 were synthesized at normal rates in the lpa2-1 mutant. The newly synthesized CP43 was rapidly degraded in lpa2-1, and the turnover rates of D1 and D2 were higher in lpa2-1 than in wild-type plants. The newly synthesized PSII proteins were assembled into PSII complexes, but the assembly of PSII was less efficient in the mutant than in wild-type plants. LPA2 encodes an intrinsic thylakoid membrane protein, which is not an integral subunit of PSII. Yeast two-hybrid assays indicated that LPA2 interacts with the PSII core protein CP43 but not with the PSII reaction center proteins D1 and D2. Moreover, direct interactions of LPA2 with Albino3 (Alb3), which is involved in thylakoid membrane biogenesis and cell division, were also detected. Thus, the results suggest that LPA2, which appears to form a complex with Alb3, is involved in assisting CP43 assembly within PSII.     

PsbT polypeptide is required for efficient repair of photodamaged photosystem II reaction center.

PsbT is a small chloroplast-encoded hydrophobic polypeptide associated with the photosystem II (PSII) core complex. A psbT-deficient mutant (Delta psbT) of the green alga Chlamydomonas reinhardtii grows photoautotrophically, whereas its growth is significantly impaired in strong light. To understand the photosensitivity of Delta psbT, we have studied the effect of strong illumination on PSII activity and proteins. It is shown that the level of PSII activity and proteins is reduced in the Delta psbT more significantly than in wild type under strong light. When recovery of the photodamaged PSII is inhibited by a chloroplast protein synthesis inhibitor, the light-induced inactivation and degradation of PSII occur similarly in wild-type and mutant cells. On the contrary, the recovery of PSII activity after partial photoinactivation is remarkably delayed in the Delta psbT cells, suggesting that PsbT is required for efficient recovery of the photodamaged PSII complex. These results therefore present the first evidence for involvement of this small PSII polypeptide in the recovery process. Partial disintegration of the purified PSII core complex and localization of PSII proteins in the resulting PSII subcore complexes have revealed that PsbT is associated with D1/D2 heterodimer. A possible role of PsbT in the recovery process is discussed.     

EPR study of the oxygen evolving complex in His-tagged photosystem II from the cyanobacterium Synechococcus elongatus

The Mn(4)-cluster and the cytochrome c(550) in histidine-tagged photosystem II (PSII) from Synechococcus elongatus were studied using electron paramagnetic resonance (EPR) spectroscopy. The EPR signals associated with the S(0)-state (spin = 1/2) and the S(2)-state (spin = 1/2 and IR-induced spin = 5/2 state) were essentially identical to those detected in the non-His-tagged strain. The EPR signals from the S(3)-state, not previously reported in cyanobacteria, were detectable both using perpendicular (at g = 10) and parallel (at g = 14) polarization EPR, and these signals are similar to those found in plant PSII. In the S(3)-state, near-infrared illumination at 50 K induced a 176-G-wide split signal at g = 2 and signals at g = 5.20 and g = 1.51. These signals differ slightly from those reported in plant PSII [Ioannidis, N., and Petrouleas, V. (2000) Biochemistry 39, 5246-5254]. In accordance with the cited work, the split signal presumably reflects a radical interacting with the Mn(4)-cluster in a fraction of centers, while the g = 5.20 and g = 1.51 signals are tentatively attributed to a high-spin state of the Mn(4)-cluster with zero field splitting parameters different from those in plant PSII, reflecting minor changes in the environment of the Mn(4)-cluster. Biochemical modifications (Sr(2+)/Ca(2+) substitution, acetate and NH(3) treatments) were also investigated. In Sr(2+)-reconstituted PSII, in addition to the expected modified S(2) multiline signal, a signal at g = 5.2 was present instead of the g approximately 4 signal seen in plant PSII. In NH(3)-treated samples, in addition to the expected modified S(2)-multiline signal, a g approximately 4 signal was detected in a small proportion of the reaction centers. This is of note since g approximately 4 spectra arising from the Mn(4)-cluster in the S(2) state have not yet been published in cyanobacterial PSII. The detection of modified S(3)-signals in both perpendicular (at g = 7.5) and parallel (at g = 12) polarization EPR from NH(3)-treated PSII indicate that NH(3) is still bound in the S(3)-state. The acetate-treated PSII behaves essentially as in plant PSII. A study using oriented samples indicated that the heme plane of the oxidized low spin Cytc(550) was perpendicular to the plane of the membrane.     

D1-Asp170 is structurally coupled to the oxygen evolving complex in photosystem II as revealed by light-induced Fourier transform infrared difference spectroscopy

We report both mid-frequency (1800-1200 cm(-)(1)) and low-frequency (670-350 cm(-)(1)) S(2)/S(1) FTIR difference spectra of photosystem II (PSII) particles isolated from wild-type and D1-D170H mutant cells of the cyanobacterium Synechocystis sp. PCC 6803. Both mid- and low-frequency S(2)/S(1) spectra of the Synechocystis wild-type PSII particles closely resemble those from spinach PSII samples, which confirms an earlier result by Noguchi and co-workers [Noguchi, T., Inoue, Y., and Tang, X.-S. (1997) Biochemistry 36, 14705-14711] and indicates that the coordination environment of the oxygen evolving complex (OEC) in Synechocystis is very similar to that in spinach. We also found that there is no appreciable difference between the mid-frequency S(2)/S(1) spectra of wild-type and of D1-D170H mutant PSII particles, from which we conclude that D1-Asp170 does not undergo a significant structural change during the S(1) to S(2) transition. This result also suggests that, if D1-Asp170 ligates Mn, it does not ligate the Mn ion that is oxidized during the S(1) to S(2) state transition. Finally, we found that a mode at 606 cm(-)(1) in the low-frequency wild-type S(2)/S(1) spectrum shifts to 612 cm(-)(1) in the D1-D170H mutant spectrum. Because this 606 cm(-)(1) mode has been previously assigned to an Mn-O-Mn cluster mode of the OEC [Chu, H.-A., Sackett, H., and Babcock, G. T. (2000) Biochemistry 39, 14371-14376], we conclude that D1-Asp170 is structurally coupled to the Mn-O-Mn cluster structure that gives rise to this band. Our results suggest that D1-Asp170 either directly ligates Mn or Ca(2+) or participates in a hydrogen bond to the Mn(4)Ca(2+) cluster. Our results demonstrate that combining FTIR difference spectroscopy with site-directed mutagenesis has the potential to provide insights into structural changes in Mn and Ca(2+) coordination environments in the different S states of the OEC.     

A Psb27 homologue in Arabidopsis thaliana is required for efficient repair of photodamaged photosystem II

Psb27 has been identified as a lumenal protein associated with photosystem II (PSII). To gain insight into the function of Psb27, we isolated a mutant Arabidopsis plant with a loss of psb27 function. The quantity of PSII complexes and electron transfer within PSII remained largely unaffected in the psb27 mutant. Our results also showed that under high-light-illumination, PSII activity and the content of the PSII reaction center protein D1 decreased more significantly in the psb27 mutant than in wild-type (WT) plant. Treatment of leaves with a chloroplast protein synthesis inhibitor resulted in similar light-induced PSII inactivation levels and D1 protein degradation rates in the WT and psb27 mutant plants. Recovery of PSII activity after photoinhibition was delayed in the psb27 mutant, suggesting that Psb27 is required for efficient recovery of the photodamaged PSII complex. Overall, these results demonstrated that Psb27 in Arabidopsis is not essential for oxygenic photosynthesis and PSII formation. Instead, our results provide evidence for the involvement of this lumenal protein in the recovery process of PSII. Hua Chen and Dongyuan Zhang contribute equally to this work.     

Structure of the manganese complex in photosystem II: insights from X-ray spectroscopy

We have used Mn K-edge absorption and Kbeta emission spectroscopy to determine the oxidation states of the Mn complex in the various S states. We have started exploring the new technique of resonant inelastic X-ray scattering spectroscopy; this technique can be characterized as a Raman process that uses K-edge energies (1s to 4p, ca. 6550 eV) to obtain L-edge-like spectra (2p to 3d, ca. 650 eV). The relevance of these data to the oxidation states and structure of the Mn complex is presented. We have obtained extended X-ray absorption fine structure data from the S(0) and S(3) states and observed heterogeneity in the Mn-Mn distances leading us to conclude that there may be three rather than two di-mu-oxo-bridged units present per tetranuclear Mn cluster. In addition, we have obtained data using Ca and Sr X-ray spectroscopy that provide evidence for a heteronuclear Mn-Ca cluster. The possibility of three di-mu-oxo-bridged Mn-Mn moieties and the proximity of Ca is incorporated into developing structural models for the Mn cluster. The involvement of bridging and terminal O ligands of Mn in the mechanism of oxygen evolution is discussed in the context of our X-ray spectroscopy results.     

A cluster of carboxylic groups in PsbO protein is involved in proton transfer from the water oxidizing complex of Photosystem II

The hypothesis presented here for proton transfer away from the water oxidation complex of Photosystem II (PSII) is supported by biochemical experiments on the isolated PsbO protein in solution, theoretical analyses of better understood proton transfer systems like bacteriorhodopsin and cytochrome oxidase, and the recently published 3D structure of PS II (Pdb entry 1S5L). We propose that a cluster of conserved glutamic and aspartic acid residues in the PsbO protein acts as a buffering network providing efficient acceptors of protons derived from substrate water molecules. The charge delocalization of the cluster ensures readiness to promptly accept the protons liberated from substrate water. Therefore protons generated at the catalytic centre of PSII need not be released into the thylakoid lumen as generally thought. The cluster is the beginning of a localized, fast proton transfer conduit on the lumenal side of the thylakoid membrane. Proton-dependent conformational changes of PsbO may play a role in the regulation of both supply of substrate water to the water oxidizing complex and the resultant proton transfer.     

Subsequent events to GTP binding by the plant PsbO protein: structural changes, GTP hydrolysis and dissociation from the photosystem II complex

Besides an essential role in optimizing water oxidation in photosystem II (PSII), it has been reported that the spinach PsbO protein binds GTP [C. Spetea, T. Hundal, B. Lundin, M. Heddad, I. Adamska, B. Andersson, Proc. Natl. Acad. Sci. U.S.A. 101 (2004) 1409-1414]. Here we predict four GTP-binding domains in the structure of spinach PsbO, all localized in the beta-barrel domain of the protein, as judged from comparison with the 3D-structure of the cyanobacterial counterpart. These domains are not conserved in the sequences of the cyanobacterial or green algae PsbO proteins. MgGTP induces specific changes in the structure of the PsbO protein in solution, as detected by circular dichroism and intrinsic fluorescence spectroscopy. Spinach PsbO has a low intrinsic GTPase activity, which is enhanced fifteen-fold when the protein is associated with the PSII complex in its dimeric form. GTP stimulates the dissociation of PsbO from PSII under light conditions known to also release Mn(2+) and Ca(2+) ions from the oxygen-evolving complex and to induce degradation of the PSII reaction centre D1 protein. We propose the occurrence in higher plants of a PsbO-mediated GTPase activity associated with PSII, which has consequences for the function of the oxygen-evolving complex and D1 protein turnover.     

Probing the N-terminal sequence of spinach PsbO: evidence that essential threonine residues bind to different functional sites in eukaryotic photosystem II

The N-terminal 1E-?L domain of the manganese-stabilizing protein (PsbO) from spinach prevents non-specific binding of the subunit to photosystem II (PSII) and deletions of the 1E-?T or 1E-1?T sequences from the PsbO N-terminus reduce or impair, respectively, functional binding of PsbO to PSII (Popelkova et al., Biochemistry 42:6193-6200, 2003). The work presented here provides deeper insights into the interaction of PsbO with PSII. The data show that a single mutation, 1?T → A in mature PsbO from spinach reduces the stoichiometry of its functional binding from two to one subunit per PSII and decreases reconstitution of activity to about 45 % of the wild-type control. Replacement of the 1E-?L domain with ?M in the T15A PsbO mutant has no additional negative effect on recovery of O? evolution activity, but it significantly weakens both functional and nonspecific binding of the truncated mutant to PSII. These results suggest that the 1?T side-chain by itself is essential for binding of one of two PsbO subunits to eukaryotic PSII and that specific PSII-binding sites for PsbO are distinguishable; one PSII-binding site does not require PsbO-1?T and probably interacts with the other N-terminal domain of PsbO. Identity of the latter domain is revealed by a requirement for the presence of the 1E-?L sequence that is shown here to be necessary for high-affinity binding of PsbO to PSII. When combined with previous results, the data presented here lead to a more detailed model for PsbO binding in eukaryotic PSII.     

Protection against the photo-induced inactivation of the photosystem II complex by abscisic acid

To examine the effect of abscisic acid (ABA) on the photo‐induced inactivation of the photosystem II (PSII) complex, a suspension culture of Chlamydomonas reinhardtii was treated with ABA for 24 h in darkness and then, after removal of ABA, the cells were exposed to strong light at a photon flux density of 2000 μ mol m ^{? 2} s ^{? 1} at various temperatures. The activity of PSII, as estimated in terms of chlorophyll fluorescence and the evolution of oxygen, decreased significantly during the exposure of cells to the strong light, and the extent of the photo‐induced decrease in PSII activity was much greater at lower temperatures. Irrespective of temperature, the decrease in PSII activity in ABA‐treated cells was significantly smaller than that in control cells. Moreover, the recovery of PSII activity from the photo‐inactivated state in ABA‐treated cells was significantly faster than that in control cells. The recovery of PSII activity in both ABA‐treated and control cells was almost entirely prevented by the presence of chloramphenicol. These results indicate that ABA protects the PSII complex in C. reinhardtii against photo‐induced inactivation by accelerating the recovery of this complex.     

A cluster of carboxylic groups in PsbO protein is involved in proton transfer from the water oxidizing complex of Photosystem II

The hypothesis presented here for proton transfer away from the water oxidation complex of Photosystem II (PSII) is supported by biochemical experiments on the isolated PsbO protein in solution, theoretical analyses of better understood proton transfer systems like bacteriorhodopsin and cytochrome oxidase, and the recently published 3D structure of PS II (Pdb entry 1S5L). We propose that a cluster of conserved glutamic and aspartic acid residues in the PsbO protein acts as a buffering network providing efficient acceptors of protons derived from substrate water molecules. The charge delocalization of the cluster ensures readiness to promptly accept the protons liberated from substrate water. Therefore protons generated at the catalytic centre of PSII need not be released into the thylakoid lumen as generally thought. The cluster is the beginning of a localized, fast proton transfer conduit on the lumenal side of the thylakoid membrane. Proton-dependent conformational changes of PsbO may play a role in the regulation of both supply of substrate water to the water oxidizing complex and the resultant proton transfer.     

Subsequent events to GTP binding by the plant PsbO protein: Structural changes, GTP hydrolysis and dissociation from the photosystem II complex

Besides an essential role in optimizing water oxidation in photosystem II (PSII), it has been reported that the spinach PsbO protein binds GTP [C. Spetea, T. Hundal, B. Lundin, M. Heddad, I. Adamska, B. Andersson, Proc. Natl. Acad. Sci. U.S.A. 101 (2004) 1409-1414]. Here we predict four GTP-binding domains in the structure of spinach PsbO, all localized in the β-barrel domain of the protein, as judged from comparison with the 3D-structure of the cyanobacterial counterpart. These domains are not conserved in the sequences of the cyanobacterial or green algae PsbO proteins. MgGTP induces specific changes in the structure of the PsbO protein in solution, as detected by circular dichroism and intrinsic fluorescence spectroscopy. Spinach PsbO has a low intrinsic GTPase activity, which is enhanced fifteen-fold when the protein is associated with the PSII complex in its dimeric form. GTP stimulates the dissociation of PsbO from PSII under light conditions known to also release Mn²⁺ and Ca²⁺ ions from the oxygen-evolving complex and to induce degradation of the PSII reaction centre D1 protein. We propose the occurrence in higher plants of a PsbO-mediated GTPase activity associated with PSII, which has consequences for the function of the oxygen-evolving complex and D1 protein turnover.     

Theoretical study of the multiline EPR signal from the S2 state of the oxygen evolving complex of photosystem II: Evidence for a magnetic tetramer

The Oxygen evolving complex of plant photosystem II is made of a manganese cluster that gives rise to a low temperature EPR multiline signal in the S₂ oxidation state. The origin of this EPR signal has been addressed with respect to the question of the magnetic couplings between the electron and nuclear spins of the four possible Mn ions that make up this complex. Considering Mn(III) and Mn(IV) as the only possible oxidation states present in the S₂ state, and no large anisotropy of the magnetic tensors, the breadths of the EPR spectra calculated for dimers and trimers with S = ½ have been compared with that of the biological site. It is concluded that neither a dinuclear nor a trinuclear complex made of Mn(III) and Mn(IV) can be responsible for the multiline signal; but that, by contrast, a tetranuclear Mn complex can be the origin of this signal. The general shape of the experimental spectrum, its particular hyperfine pattern, the positions of most of the hyperfine lines and their relative intensities can be fit by a tetramer model described by the following six fitting parameters: g ≈ 1.987, A₁ ≈ 122.4 10^-4 cm^-1, A₂ ≈ 87.2 10^-4 cm^-1, A₃ ≈ 81.6 10^-4 cm^-1, A₄ ≈ 19.1 10^-4 cm^-1 and δH = 24.5 G. A second model described by parameters very close to those given above except for A₄ ≈ 77.5 10^-4 cm^-1 gives an equally good fit. However, no other set of parameters gives an EPR spectrum that reproduces the hyperfine pattern of the S₂ multiline signal. This demonstrates that in the S₂ state of the oxygen evolving complex, the four manganese ions are organized in a magnetic tetramer.