Multiline EPR Signal Research Articles

Abnormal redox reactions in photosynthetic O 2-evolving centers in NaCl/EDTA-washed PS II. A dark-stable EPR multiline signal and an unknown positive charge accumulator

Redox events that occur in photosynthetic O 2-evolving centers in NaCl/EDTA-washed PS II membranes were investigated by means of low temperature EPR and thermoluminescence. The following results have been obtained: (i) In the washed membranes, O 2 centers could maintain the S 2 state more than 3 h in darkness at room temperature. This dark-stable S 2 was modified as seen by a multiline EPR signal with reduced hyperfine line spacing and also by a thermoluminescence band with upshifted peak temperature. (ii) This modified S 2 state had an abnormally long life of t 1 2 = 7 h at 20°C, and its appearance required the presence of EDTA in the medium. On addition of exogenous Ca 2+, the modified S 2 was converted in darkness to normal S 2, and then decayed rapidly to be undetectable. (iii) On illuminating this modified S 2, an EPR signal centering at aroung g = 2 was newly induced at no expense of the dark-stable EPR multiline signal. This EPR signal was accompanied by a new thermoluminescence band peaking at around 5°C, suggesting the presence of a new redox component whose oxidized form is capable of providing a positive charge for thermoluminescence in place of Mn. (iv) This new component was efficiently oxidized by illumination at −5°C but much less at −60°C, showing a half-inhibition temperature at around −40°C. (v) Addition of various divalent cations in place of Ca 2+ variously affected both thermoluminescence glow peaks arising from the dark-stable S 2 or from the new redox component, suggesting a cation-species-dependent modulation of the redox properties of both components. (vi) Both of these two thermoluminescence bands showed no dependency on flash number, suggesting interruption of further oxidation beyond their respective abnormal states. On addition of Ca 2+, all these abnormal properties were abolished and normal period-four flash pattern was restored. These abnormal properties of the redox events in NaCl/EDTA-washed PS II membranes were discussed in relation to the demand for exogenous Ca 2+ in recovery of normal properties.

Purification of highly active oxygen-evolving photosystem II from Chlamydomonas reinhardtii

A method is described for the isolation and purification of active oxygen-evolving photosystem II (PS II) membranes from the green alga Chlamydomonas reinhardtii. The isolation procedure is a modification of methods evolved for spinach (Berthold et al. 1981). The purity and integrity of the PS II preparations have been assesssed on the bases of the polypeptide pattern in SDS-PAGE, the rate of oxygen evolution, the EPR multiline signal of the S2 state, the room temperature chlorophyll a fluorescence yield, the 77 K emission spectra, and the P700 EPR signal at 300 K. These data show that the PS II characteristics are increased by a factor of two in PS II preparations as compared to thylakoid samples, and the PS I concentration is reduced by approximately a factor ten compared to that in thylakoids.

Studies on calcium depletion of PS II by pH 8.3 treatment

We have investigated by EPR the hypothesis that the S 1 state of the oxygen-evolving complex (OEC) becomes unstable at pH 8.3 and is converted to the S 0 state in the dark (Plitjer et al. (1986) FEBS Lett. 195, 313–318). Treatment at pH 8.3 removed the ability to generate the S 2 multiline EPR signal, but this was restored by returning the sample to pH 6.3. The depletion of calcium by an NaCl-wash procedure at pH 8.3 in the dark inhibited oxygen evolution and the formation of the multiline EPR signal, even on returning to pH 6.3. The characteristic 25% reduction of the electron donor, D +, over 4 h in untreated samples was absent in pH 8.3 NaCl-washed samples. Calcium and vanadyl ions, VO 2+, restore the normal dark stable S 1 state, as measured by the large decrease in D + and ability to form the S 2 state multiline EPR signal after 200 K illumination. Calcium, strontium and vanadyl ions also partially restore oxygen evolution in calcium-depleted PS II. These results are interpreted as indicating the loss of the S 1 state at pH 8.3 in the dark and that there is a calcium requirement for S 0 to S 1 at pH 6.3.

A marked upshift in threshold temperature for the S 1-to-S 2 transition induced by low pH treatment of PS II membranes

The temperature dependence of the S 1-to-S 2 transition in low-pH-treated PS II membranes was investigated by means of low-temperature EPR spectroscopy and thermoluminescence. (1) continuous illumination at −60°C (213 K) of low-pH-treated PS II induced neither the EPR multiline and g = 4.1 signals nor the thermoluminescence glow peak (B-band), but the same illumination at −5°C appreciably induced both EPR multiline signal and glow peak, although the multiline signal was modified in its fine structures and the g = 4.1 signal was largely suppressed. (2) Temperature dependence analysis by use of thermoluminescence revealed that the half-inhibition temperature for S 1-to-S 2 transition is markedly upshifted (by about 70°C) in low-pH-treated PS II. (3) The upshifted threshold temperature was returned to normal by the addition of exogenous Ca 2+. These results are discussed in relation to the roles of Ca in S-state transition, and in comparison with the abnormal S 2 state reported for Cl −-depleted PS II.

The S0 state of photosystem II induced by hydroxylamine: differences between the structure of the manganese complex in the S0 and S1 states determined by x-ray absorption spectroscopy

Hydroxylamine at low concentrations causes a two-flash delay in the first maximum flash yield of oxygen evolved from spinach photosystem II (PSII) subchloroplast membranes that have been excited by a series of saturating flashes of light. Untreated PSII membrane preparations exhibit a multiline EPR signal assigned to a manganese cluster and associated with the S2 state when illuminated at 195 K, or at 273 K in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). We used the extent of suppression of the multiline EPR signal observed in samples illuminated at 195 K to determine the fraction of PSII reaction centers set back to a hydroxylamine-induced S0-like state, which we designate S0*. The manganese K-edge X-ray absorption edges for dark-adapted PSII preparations with or without hydroxylamine are virtually identical. This indicates that, despite its high binding affinity to the oxygen-evolving complex (OEC) in the dark, hydroxylamine does not reduce chemically the manganese cluster within the OEC in the dark. After a single turnover of PSII, a shift to lower energy is observed in the inflection of the Mn K-edge of the manganese cluster. We conclude that, in the presence of hydroxylamine, illumination causes a reduction of the OEC, resulting in a state resembling S0. This lower Mn K-edge energy of S0*, relative to the edge of S1, implies the storage and stabilization of an oxidative equivalent within the manganese cluster during the S0----S1 state transition. An analysis of the extended X-ray absorption fine structure (EXAFS) of the S0* state indicates that a significant structural rearrangement occurs between the S0* and S1 states. The X-ray absorption edge position and the structure of the manganese cluster in the S0* state are indicative of a heterogeneous mixture of formal valences of manganese including one Mn(II) which is not present in the S1 state.

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.

A calcium-specific site influences the structure and activity of the manganese cluster responsible for photosynthetic water oxidation

EPR studies have revealed that removal of calcium using citric acid from the site in spinach photosystem II which is coupled to the photosynthetic O2-evolving process produces a structural change in the manganese cluster responsible for water oxidation. If done in the dark, this yields a modified S1' oxidation state which can be photooxidized above 250 K to form a structurally altered S2' state, as seen by formation of a "modified" multiline EPR signal. Compared to the "normal" S2 state, this new S2'-state EPR signal has more lines (at least 25) and 25% narrower 55Mn hyperfine splittings, indicative of disruption of the ligands to manganese. The calcium-depleted S2' oxidation state is greatly stabilized compared to the native S2 oxidation state, as seen by a large increase in the lifetime of the S2' EPR signal. Calcium reconstitution results in the reduction of the oxidized tyrosine residue 161YD+ (Em approximately 0.7-0.8 V, NHE) within the reaction center D1 protein in both the S1' and S2' states, as monitored by its EPR signal intensity. We attribute this to reduction by Mn. Thus a possible structural role which calcium plays is to bring YD+ into redox equilibrium with the Mn cluster. Photooxidation of S2' above 250 K produces a higher S state (S3 or S4) having a new EPR signal at g = 2.004 +/- 0.003 and a symmetric line width of 163 +/- 3 G, suggestive of oxidation of an organic donor, possibly an amino acid, in magnetic contact with the Mn cluster. This EPR signal forms in a stoichiometry of 1-2 relative to YD+.(ABSTRACT TRUNCATED AT 250 WORDS)

Ammonia binds to the catalytic manganese of the oxygen-evolving complex of photosystem II. Evidence by electron spin-echo envelope modulation spectroscopy

The mechanism of ammonia inhibition of photosynthetic oxygen evolution has been examined by the pulsed EPR technique of electron spin-echo envelop modulation (ESEEM), revealing the direct coordination of an ammonia-derived ligand to the catalytic Mn complex during the S{sub 1} {yields}S{sub 2} transition of the oxygen evolution S-state cycle. ESEEM experiments were performed on the multiline Mn EPR signal observed in photosystem II enriched spinach thylakoid membranes which were treated with either {sup 14}NH{sub 4}Cl or {sup 15}NH{sub 4}Cl (100 mM, pH 7.5). {sup 15}NH{sub 4}Cl treatment produced modulation of the electron spin-echo signal which arises from an I = 1/2 {sup 15}N nucleus with an isotropic hyperfine coupling A({sup 15}N) = 3.22 MHz. {sup 14}NH{sub 4}Cl treatment produced a different ESEEM pattern resulting from an I = 1 {sup 14}N nucleus with A({sup 14}N) = 2.29 MHz, and with electric quadrupole parameters e{sup 2}qQ = 1.61 MHz and {eta} = 0.59. The {sup 14}n electric quadrupole parameters are interpreted with respect to possible chemical structures for the ligand. An amido (NH{sub 2}) bridge between metal ions is proposed as the molecular identity of the ammonia-derived ligand to the catalytic Mn of photosystem II.

EPR relaxation measurements of Photosystem II reaction centers: influence of S-state oxidation and temperature

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 .

Isnitrogen liganded to manganese in the photosynthetic oxygen-evolving system? EPR studies after isotopic replacement with 15N

The nature of the ligands to manganese in the photosynthetic oxygen-evolving system was investigated by the effect of isotopic substitution with 15N on the spectral properties of the multiline EPR signal of the S2 state. No changes were observed in the general shape of the signal or in the linewidth. The results show that the resolved fine structure in the multiline signal cannot be explained as nitrogen superhyperfine structure but most likely arises from manganese hyperfine interaction. The large difference between the highest value for the nitrogen coupling constant consistent with the results and that earlier observed in a binuclear, antiferromagnetically coupled manganese model complex strongly favors non-nitrogen ligands such as oxygen in carboxylic amino acid side chains.

The interaction of ammonia with the photosynthetic oxygen-evolving system

The reaction of ammonia with the oxygen-evolving system was investigated using EPR. Two sites with distinct binding properties were found. One site, previously known to be responsible for the modification by ammonia of the multiline EPR signal from the S 2 state and believed to be accessible in this state only, was found to bind ammonia also in the S 1 state although weaker. The second binding site, identified by the effect of bound ammonia on the shape and position of the g = 4.1 EPR signal, was also found to be accessible in both the S 1 and S 2 states. The apparent dissociation constants for ammonia at the two sites in the S 1 and S 2 states were determined. In neither state did the binding the ammonia account for the observed inhibition of oxygen evolution, suggesting that binding to other S states plays an important role in the inhibition. Chloride, which is known to interfere with ammonia-induced inhibition of oxygen evolution, was found to compete with ammonia at the site associated with the modification of the g = 4.1 EPR signal. The broadening of the hyperfine lines of the multiline EPR signal, seen in the presence of 17O-labeled water, was still observed after the modification of the signal by ammonia. This indicates that ammonia has not completely displaced water bound to the catalytic site in the S 2 state. The results of the binding studies are interpreted in terms of a two state — two site model, where the two states are identified by their EPR signals, the multiline and the g = 4.1 signal, respectively, and the two sites identified by the effects of ammonia on these signals and where the equilibrium between the two states is regulated by the binding of ligands to the sites.

Photosystem II disorganization and manganese release after photoinhibition of isolated spinach thylakoid membranes

The activity, the protein content and the manganese properties of photosystem II have been compared after photoinhibition of isolated thylakoid membranes. The results show a concomitant disappearance of the oxygen evolving activity and the ability to form the S 2-state multiline EPR signal. The D 1-protein is degraded in a subsequent event which closely correlates to release of manganese from the thylakoid membranes.

Deactivation kinetics and temperature dependence of the S-state transitions in the oxygen-evolving system of Photosystem II measured by EPR spectroscopy

The decay kinetics for the S 2 and S 3 states of the oxygen-evolving complex in Photosystem II have been measured in the presence of an external electron acceptor. The S 2- and S 3-states decay monophasically with half-decay times at 18°C of 3–3.5 min and 3.5–4 min, respectively. The results also show that S 3 decays via S 2 under these circumstances. The temperature dependence of the individual S-state transitions has been measured in single flash experiments in which the multiline EPR signal originating from the S 2 state has been used as spectroscopic probe. The half-inhibition temperatures are for S 0 to S 1 220–225 K, for S 1 to S 2 135–140 K, for S 2 to S 3 230 K and for the S 3-to-S 0 transition 235 K.

Characterisation of the low-fluorescent (LF1) mutant of Scenedesmus by EPR

An EPR study of the low fluorescent mutant, LF1, of the green alga, Scenedesmus obliquus, has been performed and the following results were obtained. (1) Stable photoinduced charge separation occurs at low temperature forming the characteristic EPR signals from Q − AFe 2+, the primary semiquinone-iron complex, indicating no major structural modification of the electron acceptor complex in the LF1 mutant. (2) The source of the electron in this reaction at 200 K is a component, probably a chlorophyll molecule, which gives rise to an EPR signal at g ≈ 2.0026 when photooxidized; in the wild type the S 2 manganese multiline EPR signal is generated under these conditions. (3) Contrary to a previous report (Biochim. Biophys. Acta 682 (1982) 106–114), Signal II is present in the dark in LF1; however, it exhibits different microwave power saturation characteristics from those of Signal II in the wild type. These results show that the LF1 mutant has a functional reaction centre which lacks the redox active manganese of the O 2-evolving enzyme. In addition, as reported previously, cytochrome b-559 is in the low-potential, oxidized form in LF1, while it is largely in the high-potential, reduced form in the wild-type. This effect is thought to be only indirectly related to the 34 kDa to the 36 kDa change in D 1 polypeptide size, which is assumed to be responsible for the lack of Mn binding in the LF1 mutant.

A Comparison of the Manganese Center Responsible for Photosynthetic Water Oxidation in O2‐Evolving Core Particles and Photosystem II Enriched Membranes: EPR of the S2 State

AbstractWe have characterized the electron donors to Photosystem II (PS II) in an O2‐evolving reaction center core preparation from spinach (Ghanotakis, D.F.; Yocum, C.F. FEBS Lett., 1986, 197: 244–248) using EPR spectroscopy of the manganese center involved in water oxidation and of the tyrosine donor responsible for signal II. Both the 16‐line and the 19‐line form of the S2 multiline EPR signal can be observed in the core particles under conditions similar to those which produce these signals in PS II membranes. Consequently, the structure of the coupled cluster of three or four Mn ions remains intact in the core particles. The Kok parameters which characterize the number of reaction centers capable of advancing by 0, 1, or 2 equivalents in response to a short laser pulse are found to be the same in PS II membranes and in the core particles. Also, there is no evidence for partially inactivated centers which reach the S2 or S3 states, but which do not advance to O2 release. Thus the new class of highly resolved O2‐evolving particles appears to be well suited for biophysical studies directed at the mechanism of water oxidation.

Mn2+/Mn3+ and Mn3+/Mn4+ mixed valence binuclear manganese complexes of biological interest

Binuclear and multinuclear manganese containing enzymes which occur in biology are involved in functions such as hydrogen peroxide decomposition in bacteria and the oxidation of water to oxygen during photosynthesis, respectively. In the case of the water-oxidizing complex, the so-called S/sub 2/ oxidation state has been characterized by EPR spectroscopy to be a mixed valence manganese cluster. This can be produced in two forms. The native form, which is found in active O/sub 2/-evolving samples, is considered to be either tetranuclear or trinuclear in Mn, based upon interpretation of its broad 1500 G-wide 19-line multiline EPR signal and unusual temperature dependence. A partially decoupled form is found in inactivated samples having a narrow width (1345 G), with only 16 lines and a more Curie-like temperature dependence, all features which are found in typical Mn/sup 3 +//M/sup 4 +/ binuclear centers (16-line EPR, spin S = 1/2 ground state). For the native form both different oxidation states and different numbers for the manganese ions in the cluster have been proposed in order to account for the larger spectral width due to the hyperfine interaction with /sup 55/Mn. Both binuclear Mn/sup 2 +//Mn/sup 3 +/ and tetranuclear/sup 4/ 3Mn/sup 3 +//Mn/sup 4more » +/ states have been considered. Notably lacking in this analysis has been EPR data for binuclear complexes in which both types of mixed valence oxidation states, Mn/sup 3 +//Mn/sup 4 +/ and Mn/sup 2 +//Mn/sup 3 +/, are compared within the same ligand system. The present report gives the first experimental evidence showing how /sup 55/Mn hyperfine data can easily distinguish between binuclear manganese complexes having these mixed valence oxidation states.« less

A comparison between the multiline EPR signals of spinach and Anacystis nidulans and their temperature dependence

The multiline EPR signals arising from manganese in the S 2 state of the oxygen-evolving system of spinach and the cyanobacterium Anacystis nidulans have very similar properties and are affected identically by NH 3, 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.

Electron transfer in the water-oxidizing complex of Photosystem II.

An overview is presented of secondary electron transfer at the electron donor side of Photosystem II, at which ultimately two water molecules are oxidized to molecular oxygen, and the central role of manganese in catalyzing this process is discussed. A powerful technique for the analysis of manganese redox changes in the water-oxidizing mechanism is the measurement of ultraviolet absorbance changes, induced by single-turnover light flashes on dark-adapted PS II preparations. Various interpretations of these ultraviolet absorbance changes have been proposed. Here it is shown that these changes are due to a single spectral component, which presumably is caused by the oxidation of Mn(III) to Mn(IV), and which oscillates with a sequence +1, +1, +1, -3 during the so-called S0----S1----S2----S3----S0 redox transitions of the oxygen-evolving complex. This interpretation seems to be consistent with the results obtained with other techniques, such as those on the multiline EPR signal, the intervalence Mn(III)-Mn(IV) transition in the infrared, and EXAFS studies. The dark distribution of the S states and its modification by high pH and by the addition of low concentrations of certain water analogues are discussed. Finally, the patterns of proton release and of electrochromic absorbance changes, possibly reflecting the change of charge in the oxygen-evolving system, are discussed. It is concluded that nonstoichiometric patterns must be considered, and that the net electrical charge of the system probably is the highest in state S2 and the lowest in state S1.

Purification and properties of an oxygen-evolving Photosystem II reaction-center complex from spinach

A chlorophyll-protein complex, capable of photochemical water oxidation and consisting of only one extrinsic protein of 33 kDa in addition to six intrinsic proteins of the Photosystem II reaction center, has been isolated from spinach thylakoids by digitonin extraction, performed at pH 6.0, followed by chromatographic separations using DEAE-Toyopearl 650S as described briefly (Tang, X.S. and Satoh, K. (1985) FEBS Lett. 179, 60–64). The protein complex contained approx. 3–4 manganese atoms, 2 mol plastoquinone-9 and 2 mol low-potential forms of cytochrome b-559 heme per mol of the photoactive primary acceptor, Q A. The oxygen evolution of the complex was highly stimulated by the presence of CaCl 2 and stabilized by glycerol; the typical rate of 400–500 μmol O 2 per mg Chl per h was attained with 2,5-dichlorobenzoquinone and potassium ferricyanide as electron acceptors in the presence of 50 mM CaCl 2. The protein complex exhibited a dark-stable EPR Signal II; the microwave power saturation profile of the signal was almost identical with that of oxygen-evolving membrane preparations. The multiline EPR signal ascribable to Kok's S 2-state was elicited in this protein complex by illumination at 200 K, as in membrane preparations. These results indicate that the basic machinery of photosynthetic water oxidation is preserved in an almost intact state in the isolated chlorophyll-protein complex.

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 O 2-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 S 2 multiline EPR signal by 190 K illumination. The g = 4.1 signal is assigned to the Mn complex in the S 2 state.