Assignment of the g = 4.1 ERP signal to manganese in the S2 state of the photosynthetic oxygen-evolving complex: An X-ray absorption edge spectroscopy study

X-ray absorption spectroscopy at the Mn K-edge has been utilized to study the origin of the g = 4.1 EPR signal associated with the Mn-containing photosynthetic O₂-evolving complex. Formation of the g = 4.1 signal by illumination of Photosystem II preparations at 140 K is associated with a shift of the Mn edge inflection point to higher energy. This shift is similar to that observed upon formation of the S₂ multiline EPR signal by 190 K illumination. The g = 4.1 signal is assigned to the Mn complex in the S₂ state.     

The g = 2 multiline EPR signal of the S2 state of the photosynthetic oxygen-evolving complex originates from a ground spin state.

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.     

The manganese site of the photosynthetic oxygen-evolving complex probed by EPR spectroscopy of oriented photosystem II membranes: the g = 4 and g = 2 multiline signals.

The g = 4 and g = 2 multiline EPR signals arising from the Mn cluster of the photosynthetic oxygen-evolving complex (OEC) in the S2 state were studied in preparations of oriented photosystem II (PSII) membranes. The ammonia-modified forms of these two signals were also examined. The g = 4 signal obtained in oriented PSII membranes treated with NH4Cl at pH 7.5 displays at least 16 partially resolved Mn hyperfine transitions with a regular spacing of 36 G [Kim, D.H., Britt, R.D., Klein, M.P., & Sauer, K. (1990) J. Am. Chem. Soc. 112, 9389-9391]. The observation of this g = 4 "multiline signal" provides strong spectral evidence for a tetranuclear Mn origin for the g = 4 signal and is strongly suggestive of a model in which different spin state configurations of a single exchange-coupled Mn cluster give rise to the g = 4 and g = 2 multiline signals. A simulation shows the observed spectrum to be consistent with an S = 3/2 or S = 5/2 state of a tetranuclear Mn complex. The resolution of hyperfine structure on the NH3-modified g = 4 signal is strongly dependent on sample orientation, with no resolved hyperfine structure when the membrane normal is oriented perpendicular to the applied magnetic field. The dramatic NH3-induced changes in the g = 4 signal resolved in the spectra of oriented samples are suggestive that NH3 binding at the Cl- site of the OEC may represent direct coordination of NH3 to the Mn cluster.(ABSTRACT TRUNCATED AT 250 WORDS)     

Q-band EPR of the S2 state of photosystem II confirms an S = 5/2 origin of the X-band g = 4.1 signal

Disagreement has remained about the spin state origin of the g = 4.1 EPR signal observed at X-band (9 GHz) from the S2 oxidation state of the Mn cluster of Photosystem II. In this study, the S2 state of PSII-enriched membrane fragments was examined at Q-band (34 GHz), with special interest in low-field signals. Light-induced signals at g = 3.1 and g = 4.6 were observed. The intensity of the signal at g = 3.1 was enhanced by the presence of F- and suppressed by the presence of 5% ethanol, indicating that it was from the same spin system as the X-band signal at g = 4.1. The Q-band signal at g = 4.6 was also enhanced by F-, but not suppressed by 5% ethanol, making its identity less clear. Although it can be accounted for by the same spin system, other sources for the signal are considered. The observation of the signal at g = 3.1 agrees well with a previous study at 15.5 GHz, in which the X-band g = 4.1 signal was proposed to arise from the middle Kramers doublet of a near rhombic S = 5/2 system. Zero-field splitting values of D = 0.455 cm(-1) and E/D = 0.25 are used to simulate the spectra.     

Comparative study of the g=4.1 EPR signals in the S(2) state of photosystem II

The Mn(4) complex which is involved in water oxidation in photosystem II is known to exhibit three types of EPR signals in the S(2) state, one of the five redox states of the enzyme cycle: a multiline signal (spin 1/2), signals at g5 (spin 5/2) and a signal at g=4.1 (or g=4.25). The g=4.1 signal could be generated under two distinct sets of conditions: either by illumination at room temperature or at 200 K in certain experimental conditions (g4(S) signal) or by near-infrared illumination between approximately 77 and approximately 160 K of the S(2)-multiline state (g4(IR) signal). The two g=4.1 signals arise from states which have quite different stability in terms of temperature. In the present work we have compared these two signals in order to test if they originate from the same or from different chemical origins. The microwave power saturation properties of the two signals measured at 4.2 K were found to be virtually identical. Their temperature dependencies measured at non-saturating powers were also identical. The presence of Curie law behavior for the g4(S) and g4(IR) signals indicates that the states responsible for both signals are ground states. The orientation dependence, anisotropy and resolved hyperfine structure of the two g4 signals were also found to be virtually indistinguishable. We have been unable to confirm the behavior reported earlier indicating that the g4(S) signal is an excited state, nor were we able to confirm the presence of signal from a higher excited state in samples containing the g4(S), nor a radical signal in samples containing the g4(IR). These findings are best interpreted assuming that the two signals have a common origin i.e. a spin 5/2 ground state arising from a magnetically coupled Mn-cluster of 4 Mn ions.     

Comparison of the structure of the manganese complex in the S1 and S2 states of the photosynthetic O2-evolving complex: an x-ray absorption spectroscopy study

A Mn-containing enzyme complex is involved in the oxidation of H2O to O2 in algae and higher plants. X-ray absorption spectroscopy is well suited for studying the structure and function of Mn in this enzyme complex. Results of X-ray K-edge and extended X-ray absorption fine structure (EXAFS) studies of Mn in the S1 and S2 states of the photosynthetic O2-evolving complex in photosystem II preparations from spinach are presented in this paper. The S2 state was prepared by illumination at 190 K or by illumination at 277 K in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU); these are protocols that limit the photosystem II reaction center to one turnover. Both methods produce an S2 state characterized by a multiline electron paramagnetic resonance (EPR) signal. An additional protocol, illumination at 140 K, produces as a state characterized by the g = 4.1 EPR signal. We have previously observed a shift to higher energy in the X-ray absorption K-edge energy of Mn upon advancement from the dark-adapted S1 state to the S2 state produced by illumination at 190 K [Goodin, D. B., Yachandra, V. K., Britt, R. D., Sauer, K., & Klein, M. P. (1984) Biochim. Biophys. Acta 767, 209-216]. The Mn K-edge spectrum of the 277 K illuminated sample is similar to that produced at 190 K, indicating that the S2 state is similar when produced at 190 or 277 K.(ABSTRACT TRUNCATED AT 250 WORDS)     

EPR properties of a g=2 broad signal trapped in an S1 state in Ca2+-depleted Photosystem II

We investigated a new EPR signal that gives a broad line shape around g=2 in Ca(2+)-depleted Photosystem (PS) II. The signal was trapped by illumination at 243 K in parallel with the formation of Y(Z)*. The ratio of the intensities between the g=2 broad signal and the Y(Z)* signal was 1:3, assuming a Gaussian line shape for the former. The g=2 broad signal and the Y(Z)* signal decayed together in parallel with the appearance of the S(2) state multiline at 243 K. The g=2 broad signal was assigned to be an intermediate S(1)X* state in the transition from the S(1) to the S(2) state, where X* represents an amino acid radical nearby manganese cluster, such as D1-His337. The signal is in thermal equilibrium with Y(Z)*. Possible reactions in the S state transitions in Ca(2+)-depleted PS II were discussed.     

Electron spin-lattice relaxation studies of different forms of the S2 state multiline EPR signal of the Photosystem II oxygen-evolving complex

The pulsed EPR inversion recovery sequence has been utilized to monitor the temperature dependence of the electron spin-lattice relaxation rate of the Mn cluster of the Photosystem II oxygen evolving complex poised in a variety of S ₂ state forms giving rise to g = 2 multiline EPR signals. A previous study (Lorigan and Britt (1994) Biochemistry 33: 12072–12076) showed that for PS II membranes treated with 5% ethanol, the S ₂ state Mn cluster relaxes via the Orbach spin-lattice relaxation mechanism, where the relaxation is enhanced via phonon scattering off an excited state spin manifold, in this case at an energy of Δ = 36.5 cm⁻¹ above the S = 1/2 ground state giving rise to the multiline EPR signal. Parallel experiments are reported for PS II membranes with 5% methanol, treated with ammonia, and following short and long term dark adaptation. In each case, the temperature dependence of the electron spin-lattice relaxation rate is consistent with Orbach relaxation, and the range of excited state energies is relatively narrow (33.8 cm⁻¹ ≤ Δ ≤ 39.7 cm⁻¹). In addition, short term dark adapted (6 min, ‘active state’) PS II membranes show biphasic recovery traces which indicate that a minority fraction of the oxygen evolving complexes are trapped in a form with greatly slowed spin-lattice relaxation. This revised version was published online in June 2006 with corrections to the Cover Date.     

The S3 state of photosystem II: differences between the structure of the manganese complex in the S2 and S3 states determined by X-ray absorption spectroscopy

O2-evolving photosystem II (PSII) membranes from spinach have been cryogenically stabilized in the S3 state of the oxygen-evolving complex. The cryogenic trapping of the S3 state was achieved using a double-turnover illumination of dark-adapted PSII preparations maintained at 240 K. A double turnover of PSII was accomplished using the high-potential acceptor, Q400, which is the high-spin iron of the iron-quinone acceptor complex. EPR spectroscopy was the principal tool establishing the S-state composition and defining the electron-transfer events associated with a double turnover of PSII. The inflection point energy of the Mn X-ray absorption K-edge of PSII preparations poised in the S3 state is the same as for those poised in the S2 state. This is surprising in light of the loss of the multiline EPR signal upon advancing to the S3 state. This indicates that the oxidative equivalent stored within the oxygen-evolving complex (OEC) during this transition resides on another intermediate donor which must be very close to the manganese complex. An analysis of the Mn extended X-ray absorption fine structure (EXAFS) of PSII preparations poised in the S2 and S3 states indicates that a small structural rearrangement occurs during this photoinduced transition. A detailed comparison of the Mn EXAFS of these two S states with the EXAFS of four multinuclear mu-oxo-bridged manganese compounds indicates that the photosynthetic manganese site most probably consists of a pair of binuclear di-mu-oxo-bridged manganese structures. However, we cannot rule out, on the basis of the EXAFS analysis alone, a complex containing a mononuclear center and a linear trinuclear complex. The subtle differences observed between the S states are best explained by an increase in the spread of Mn-Mn distances occurring during the S2----S3 state transition. This increased disorder in the manganese distances suggests the presence of two inequivalent di-mu-oxo-bridged binuclear structures in the S3 state.     

The S1YZ* metalloradical EPR signal of photosystem II contains two distinct components that advance respectively to the multiline and g = 4.1 conformations of S2

The S2 state of the oxygen-evolving complex (OEC) of photosystem II is heterogeneous, exhibiting two main EPR spectral forms, the multiline and the g = 4.1 signal. It is not clearly established whether this heterogeneity develops during the S1 to S2 transition or is already present in the precursor states. We have compared the spectra of the S1YZ* intermediate, obtained by visible light excitation (induction of charge separation) of the S1 state at liquid He temperatures, (S1YZ*)vis, or by near-infrared (NIR) light excitation of the S2 state (utilization of the unusual property of the Mn cluster to act as an oxidant of Yz when excited by NIR), (S1YZ*)NIR. The decay kinetics of the (S1YZ*)vis spectrum at 11 K was also studied by the application of rapid-scan EPR. The two spectra share in common a signal with a characteristic feature at g = 2.035, but the (S1YZ*)vis spectrum contains in addition a fast decaying component 26 G wide. The analysis of the surface of the rapid-scan spectra yielded 270 +/- 35 and 90 +/- 15 s for the respective half-times of the two components of the (S1YZ*)vis spectrum at 11 K. (S1YZ*)vis advances efficiently to S2 when annealed at 200 K; notably the g = 2.035 signal advances to the multiline while the 26 G component advances to the g = 4.1 conformation. The "26 G" component is absent or very small, respectively, in thermophilic cyanobacteria or glycerol-containing spinach samples, in correlation to vanishing or very small amounts of the g = 4.1 component in the S2 spectrum. The results validate the assignment of S1YZ* to a true S1 to S2 intermediate and imply that the heterogeneity observed in S2 is already present in S1. Tentative valences are assigned to the individual Mn ions of the OEC in the two heterogeneous conformations of S1.     

Multifrequency EPR investigations into the origin of the S2-state signal at g = 4 of the O2-evolving complex.

The low-temperature S2-state EPR signal at g = 4 from the oxygen-evolving complex (OEC) of spinach Photosystem-II-enriched membranes is examined at three frequencies, 4 GHz (S-band), 9 GHz (X-band) and 16 GHz (P-band). While no hyperfine structure is observed at 4 GHz, the signal shows little narrowing and may mask underlying hyperfine structure. At 16 GHz, the signal shows g-anisotropy and a shift in g-components. The middle Kramers doublet of a near rhombic S = 5/2 system is found to be the only possible origin consistent with the frequency dependence of the signal. Computer simulations incorporating underlying hyperfine structure from an Mn monomer or dimer are employed to characterize the system. The low zero field splitting (ZFS) of D = 0.43 cm-1 and near rhombicity of E/D = 0.25 lead to the observed X-band g value of 4.1. Treatment with F- or NH3, which compete with Cl- for a binding site, increases the ZFS and rhombicity of the signal. These results indicate that the origin of the OEC signal at g = 4 is either an Mn(II) monomer or a coupled Mn multimer. The likelihood of a multimer is favored over that of a monomer.     

Manganese K-edge X-ray absorption spectra of the cyclic S-states in the photosynthetic oxygen-evolving system

A set of Mn K-edge XANES spectra due to the redox states S₀–S₃ of the OEC were determined by constructing a highly-sensitive X-ray detection system for use with physiologically native PS II membranes capable of cycling under a series of saturating laser-flashes. The spectra showed almost parallel upshifts with relatively high K-edge half-height energies given by 6550.9±0.2 eV, 6551.7±0.2 eV, 6552.5±0.2 eV and 6553.6±0.2 eV for the S₀, S₁, S₂ and S₃ states, respectively. The successive difference spectra between S₀ and S₁, S₁ and S₂, and S₂ and S₃ states were found to exhibit a similar peak around 6552–6553 eV, indicating that one Mn(III) ion or its direct ligand is univalently oxidized upon each individual S-state transition from S₀ to S₃. The present data, together with other observations of EPR and pre-edge XANES spectroscopy, suggest that the oxidation state of the Mn cluster undergoes a periodic change; S₀: Mn(III,III,III,IV) S₁: Mn(III,IV,III,IV) S₂: Mn(III,IV,IV,IV) S₃: Mn(IV,IV,IV,IV) or Mn(III,IV,IV,IV)·L⁺ with L being a direct ligand of a Mn(III) ion.Abbreviations Chl chlorophyll - D tyrosine 160 on the D2 protein, an accessory electron donor in PS II - D⁺ the oxidized form of D - EDTA ethylene-diaminetetraacetic acid - EPR electron paramagnetic resonance - EXAFS extended X-ray absorption fine structure - HL py-2,6-bis[bis(2-pyridylmethyl)aminomethyl]-4-methylphenol - Mes 2-(N-morpholino)ethanesulfonic acid - N4 py-tris(2-pyridylmethyl)amine - OEC oxygen evolving complex - P680 primary electron donor of PS II - PS II Photosystem II - Q₄₀₀ a high spin Fe³⁺ of the iron-quinone acceptor complex in PS II - SSD solid state detector - XAFS X-ray absorption fine structure - XANES X-ray absorption near edge structure     

Functional and structural study on chelator-induced suppression of S2/S1 FTIR spectrum in photosynthetic oxygen-evolving complex

Chelating agents have been shown to induce characteristic changes in the light-minus-dark Fourier transform infrared (FTIR) difference spectrum for the S(2)/S(1) difference in the oxygen-evolving complex (OEC). Addition of various ethylenediamine-N,N,N',N'-tetraacetic acid (EDTA)-type chelators, such as EDTA, O,O'-bis(2-aminoethyl)ethyleneglycol-N,N,N',N'-tetraacetic acid (EGTA), trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CyDTA), or N-(2-hydroxyethyl)ethylenediamine-N,N',N'-triacetic acid (HEDTA), to Ca(2+)-depleted PS II membranes resulted in the suppression of typical S(2)/S(1) vibrational features, including the symmetric (1365(+)/1404(-) cm(-1)) and the asymmetric (1587(+)/1566(-) cm(-1)) carboxylate stretching vibrations, as well as the amide I and II modes of the backbone polypeptides. In contrast, the addition of ethylenediamine-N,N'-diacetic acid (EDDA) showed less inhibitory effects. The effects of the chelators depended on the number of the carboxylate groups; chelators with more than two carboxymethyl groups were effective in altering the FTIR spectrum. The bridging structure that connects the two nitrogen atoms also influenced the inhibitory effects. However, the effects were not necessarily correlated with the stability constants of the chelators to Mn(2+). The vibrational modes that were suppressed by EDTA were almost completely restored by subsequent washing with Chelex-treated Ca(2+)-free buffer medium, indicating that the spectral changes are attributable to the reversible association of chelators with the Ca(2+)-depleted OEC. Nevertheless, prolonged incubation with chelators led to the impairment of the O(2)-evolving capability, with differences in the effectiveness, in the order that is consistent with that for the suppression effects on FTIR spectra. Chelators with carboxylate and/or carboxymethyl groups bound to a single nitrogen [nitrilotriacetic acid (NTA) and iminodiacetic acid (IDA)] or carbon (citric acid) were relatively ineffective for the suppression. A chelator that includes four phosphate groups, ethylenediamine-N,N,N',N'-tetrakis(methylenephosphonic) acid (EDTPO), also showed suppression effects on both the carboxylate and amide modes. Based on these findings, a possible mode of interaction between the chelators and the Mn cluster is discussed.     

The state of manganese in the photosynthetic apparatus. 3. Light-induced changes in X-ray absorption (K-edge) energies of manganese in photosynthetic membranes

Photosynthetic water oxidation by higher plants proceeds as though five intermediates, S₀-S₄, operate in a cyclic fashion. In this study of the manganese involvement in the process, a low temperature EPR signal is used as an indicator of S-state composition for manganese X-ray absorption K-edge measurements of a spinach Photosystem II preparation. A dramatic change is observed in the edge properties between samples prepared in states S₁ and either S₂ or S₃, establishing a direct relation between the local environment of Mn and the S-state composition. Samples in S₂ or S₃ exhibit a broadening of the principal absorption peak and a shift to higher energy by as much as 2.5 eV relative to S₁ samples. The magnitude of these changes is directly related to the EPR signal intensity induced by illumination. Models are discussed in which these data may be interpreted in terms of a conformation-induced change in Mn ligation and/or oxidation during the S₁ to S₂ transition.     

Inhomogeneity of the EPR multiline signal from the S2-state of the photosystem II oxygen-evolving enzyme

The manganese complex (Mn₄) which is responsible for water oxidation in photosystem II is EPR detectable in the S₂-state, one of the five redox states of the enzyme cycle. The S₂-state is observable at 10?K either as an EPR multiline signal (spin S?=?1/2) or as a signal at g?=?4.1 (spin S?=?3/2 or 5/2). It has recently been shown that the state responsible for the multiline signal is converted to that responsible for the g?=?4.1 signal upon the absorption of near-infrared light [Boussac A, Girerd J-J, Rutherford AW (1996) Biochemistry 35?:?6984–6989]. It is shown here that the yield of the spin interconversion may be variable and depends on the photosystem II (PSII) preparations. The EPR multiline signal detected after near-infrared illumination, and which originates from PSII centers not susceptible to the near-infrared light, is shown to be different from that which originates from infrared-susceptible PSII centers. The total S₂-multiline signal results from the superposition of the two multiline signals which originate from these two PSII populations. One S₂ population gives rise to a "narrow" multiline signal characterized by strong central lines and weak outer lines. The second population gives rise to a "broad" multiline signal in which the intensity of the outer lines, at low and high field, are proportionally larger than those in the narrow multiline signal. The larger the relative amplitude of the outer lines at low and high field, the higher is the proportion of the near-infrared-susceptible PSII centers and the yield of the multiline to g?=?4.1 signal conversion. This inhomogeneity of the EPR multiline signal is briefly discussed in terms of the structural properties of the Mn₄ complex.     

Dual-mode EPR study of new signals from the S3-state of oxygen-evolving complex in photosystem II

The light-induced new EPR signals at g = 12 and 8 were observed in photosystem II (PS II) membranes by parallel polarization EPR. The signals were generated after two flashes of illumination at room temperature, and the signal intensity had four flashes period oscillation, indicating that the signal origin could be ascribed to the S3-state. Successful simulations were obtained assuming S = 1 spin for the values of the zero-field parameters, D = +/-0.435 +/- 0. 005 cm-1 and E/D = -0.317 +/- 0.002. Orientation dependence of the g =12 and 8 signal intensities shows that the axial direction of the zero-field interaction of the manganese cluster is nearly parallel to the membrane normal.     

Investigation of the origin of the "S3" EPR signal from the oxygen-evolving complex of photosystem 2: the role of tyrosine Z.

The origin of the "S3" EPR signal from calcium-depleted photosystem 2 samples has been investigated. This signal is observed after freezing samples under illumination and has been assigned to an interaction between the manganese cluster and an oxidized histidine radical [Boussac et al. (1990) Nature 347; 303-306]. In calcium-depleted samples prepared by three different methods, we observed the trapping of the tyrosine radical YZ+ under conditions which also formed the "S3" signal. An "S3"-type signal and YZ+ were also formed in PS2 samples treated with the water analogue ammonia. Following illumination at 277 K, the "S3" and YZ+ signals decayed at the same rate at 273 K in the dark. Both the YZ+ and "S3" signals decayed on storage at 77 K and could be subsequently regenerated by illumination at 8-77 K. No evidence to support histidine oxidation was found. The effects of DCMU, chelators, and alkaline pH on the dark-stable multiline S2 and the "S3" signals from calcium-depleted samples were determined. Both signals required the presence of EGTA or citrate for maximum yield. The addition of DCMU caused a reduction in the yield of "S3" generated by freezing under illumination. Incubation at pH 7.5 resulted in the loss of both signals. We propose that a variety of treatments which affect calcium and chloride binding cause a stabilization of the S2 state and slow the reduction of YZ+. This allows the trapping of YZ+, the interaction with the manganese cluster (probably in the S2 state) resulting in the "S3" signal. The data allow the position of the manganese cluster to be estimated as within 10 A of tyrosine Z (D1-161).     

The two substrate-water molecules are already bound to the oxygen-evolving complex in the S2 state of photosystem II

The first direct evidence which shows that both substrate-water molecules are bound to the O(2)-evolving catalytic site in the S(2) state of photosystem II (PSII) is presented. Rapid (18)O isotope exchange measurements between H(2)(18)O incubated in the S(2) state of PSII-enriched membrane samples and the photogenerated O(2) reveal a fast and a slow phase of exchange at m/e 34 (which measures the level of the (16)O(18)O product). The rate constant for the slow phase of exchange ((34)k(1)) equals 1.9 +/- 0.3 s(-1) at 10 degrees C, while the fast phase of exchange is unresolved by our current experimental setup ((34)k(2) >or= 175 s(-1)). The unresolvable fast phase has left open the possibility that the second substrate-water molecule binds to the catalytic site only after the formation of the S(3) state [Hillier, W., and Wydrzynski, T. (2000) Biochemistry 39, 4399-4405]. However, for PSII samples depleted of the 17 and 23 kDa extrinsic proteins (Ex-depleted PSII), two completely resolvable phases of (18)O exchange are observed in the S(2) state of the residual activity, with the following rate constants: (34)k(1) = 2.6 +/- 0.3 s(-1) and (34)k(2) = 120 +/- 14 s(-1) at 10 degrees C. Upon addition of 15 mM CaCl(2) to Ex-depleted PSII, the O(2) evolution activity increases to approximately 80% of the control level, while the two resolvable phases of exchange remain the same. In measurements of Ex-depleted PSII at m/e 36 (which measures the level of the (18)O(18)O product), only a single phase of exchange is observed in the S(2) state, with a rate constant ((36)k(1) = 2.5 +/- 0.2 s(-1)) that is identical to the slow rate of exchange in the m/e 34 data. Taken together, these results show that the fast phase of (18)O exchange is specifically slowed by the removal of the 17 and 23 kDa extrinsic proteins and that the two substrate-water molecules must be bound to independent sites already in the S(2) state. In contrast, the (18)O exchange behavior in the S(1) state of Ex-depleted PSII is no different from what is observed for the control, with or without the addition of CaCl(2). Since the fast phase of exchange in the S(1) state is unresolved (i.e., (34)k(2) > 100 s(-1)), the possibility remains that the second substrate-water molecule binds to the catalytic site only after the formation of the S(2) state. The role of the 17 and 23 kDa extrinsic proteins in establishing an asymmetric dielectric environment around the substrate binding sites is discussed.     

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.     

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.