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.     

Characterization of the manganese O2-evolving complex and the iron-quinone acceptor complex in photosystem II from a thermophilic cyanobacterium by electron paramagnetic resonance and X-ray absorption spectroscopy

The Mn donor complex in the S1 and S2 states and the iron-quinone acceptor complex (Fe2+-Q) in O2-evolving photosystem II (PS II) preparations from a thermophilic cyanobacterium, Synechococcus sp., have been studied with X-ray absorption spectroscopy and electron paramagnetic resonance (EPR). Illumination of these preparations at 220-240 K results in formation of a multiline EPR signal very similar to that assigned to a Mn S2 species observed in spinach PS II, together with g = 1.8 and 1.9 EPR signals similar to the Fe2+-QA- acceptor signals seen in spinach PS II. Illumination at 110-160 K does not produce the g = 1.8 or 1.9 EPR signals, nor the multiline or g = 4.1 EPR signals associated with the S2 state of PS II in spinach; however, a signal which peaks at g = 1.6 appears. The most probable assignment of this signal is an altered configuration of the Fe2+-QA- complex. In addition, no donor signal was seen upon warming the 140 K illuminated sample to 215 K. Following continuous illumination at temperatures between 140 and 215 K, the average X-ray absorption Mn K-edge inflection energy changes from 6550 eV for a dark-adapted (S1) sample to 6551 eV for the illuminated (S2) sample. The shift in edge inflection energy indicates an oxidation of Mn, and the absolute edge inflection energies indicate an average Mn oxidation state higher than Mn(II). Upon illumination a significant change was observed in the shape of the features associated with 1s to 3d transitions. The S1 spectrum resembles those of Mn(III) complexes, and the S2 spectrum resembles those of Mn(IV) complexes. The extended X-ray absorption fine structure (EXAFS) spectrum of the Mn complex is similar in the S1 and S2 states. Simulations indicate O or N ligands at 1.75 +/- 0.05 A, transition metal neighbor(s) at 2.73 +/- 0.05 A, which are assumed to be Mn, and terminal ligands which are probably N and O at a range of distances around 2.2 A. The Mn-O bond length of 1.75 A and the transition metal at 2.7 A indicate the presence of a di-mu-oxo-bridged Mn structure. Simulations indicate that a symmetric tetranuclear cluster is unlikely to be present, while binuclear, trinuclear, or highly distorted tetranuclear structures are possible. The striking similarity of these results to those from spinach PS II suggests that the structure of the Mn complex is largely conserved across evolutionarily diverse O2-evolving photosynthetic species.     

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)     

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.     

Decay products of the S(3) state of the oxygen-evolving complex of photosystem II at cryogenic temperatures. Pathways to the formation of the S = 7/2 S(2) state configuration

The water-oxidizing complex of photosystem II cycles through five oxidation states, denoted S(i)() (i = 0-4), during water oxidation to molecular oxygen, which appears at the (transient) S(4) state. The recent detection of bimodal EPR signals from the S(3) state [Matsukawa, T., Mino, H., Yoneda, D., Kawamori, A. (1999) Biochemistry 38, 4072-4077] has drawn significant attention to this critical state. An interesting property of the S(3) state is the sensitivity to near-IR (NIR) light excitation. Excitation of the S(3) state by near-IR light at cryogenic temperatures induces among other signals a derivative-shaped EPR signal at g= 5 [Ioannidis, N., and Petrouleas, V. (2000) Biochemistry 39, 5246-5254]. The signal bears unexpected similarities to a signal observed earlier in samples that had undergone multiple turnovers and subsequently had been stored at 77 K for a week or longer [Nugent, J. H. A., Turconi, S., and Evans, M. C. W. (1997) Biochemistry 36, 7086-7096]. Recently, both signals were assigned to an S = 7/2 configuration of the Mn cluster [Sanakis, Y., Ioannidis, N., Sioros, G., and Petrouleas, V. (2001) J. Am. Chem. Soc. 123, 10766-10767]. In the present study, we employ bimodal EPR spectroscopy to investigate the pathways of formation of this unusual state. The following observations are made: (i) The g = 5 signal evolves in apparent correlation with the diminution of the S(3) state signals during the slow (tens of hours to several days range) charge recombination of S(3) with Q(A)(-) at 77 K. The tyrosyl radical D* competes with S(3) for recombination with Q(A)(-), the functional redox couple at cryogenic temperatures inferred to be D*/D(-). Transfer to -50 degrees C and above results in the relaxation of the g = 5 to the multiline and g = 4.1 signals of the normal S(2) state. (ii) The transition of S(3) to the state responsible for the g = 5 signal can be reversed by visible light illumination directly at -30 degrees C or by illumination at 4.2 K followed by brief (2 min) transfer to -50 degrees C in the dark. The latter step is required in order to overcome an apparent thermal activation barrier (charge recombination appears to be faster than forward electron transfer at 4.2 K). (iii) The "g = 5" state can be reached in a few tens of minutes at 4.2 K by near-IR light excitation of the S(3) state. This effect is attributed to the transfer of the positive hole from the Mn cluster to a radical (probably tyr Z), which recombines much faster than the Mn cluster with Q(A)(-). (iv) The above properties strongly support the assignment of the configuration responsible for the g = 5 signal to a modified S(2) state, denoted S(2)'. Evidence supporting the assignment of the S(2)' to a proton-deficient S(2) configuration is provided by the observation that the spectrum of S(2) at pH 8.1 (obtained by illumination of the S(1) state at -30 degrees C) contains a g = 5 contribution.     

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.     

Binding of amines to the O2-evolving center of photosystem II

The binding of several primary amines to the O2-evolving center (OEC) of photosystem II (PSII) has been studied by using low-temperature electron paramagnetic resonance (EPR) spectroscopy of the S2 state. Spinach PSII membranes treated with NH4Cl at pH 7.5 produce a novel S2-state multiline EPR spectrum with a 67.5-G hyperfine line spacing when the S2 state is produced by illumination at 0 degrees C [Beck, W. F., de Paula, J. C., & Brudvig, G. W. (1986) J. Am. Chem. Soc. 108, 4018-4022]. The altered hyperfine line spacing and temperature dependence of the S2-state multiline EPR signal observed in the presence of NH4Cl are direct spectroscopic evidence for coordination of one or more NH3 molecules to the Mn site in the OEC. In contrast, the hyperfine line pattern and temperature dependence of the S2-state multiline EPR spectrum in the presence of tris(hydroxymethyl)aminomethane, 2-amino-2-ethyl-1,3-propanediol, or CH3NH2 at pH 7.5 were the same as those observed in untreated PSII membranes. We conclude that amines other than NH3 do not readily bind to the Mn site in the S2 state because of steric factors. Further, NH3 binds to an additional site on the OEC, not necessarily located on Mn, and alters the stability of the S2-state g = 4.1 EPR signal species. The effects on the intensities of the g = 4.1 and multiline EPR signals as the NH3 concentration was varied indicate that both EPR signals arise from the same paramagnetic site and that binding of NH3 to the OEC affects an equilibrium between two configurations exhibiting the different EPR signals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Low-temperature electron transfer in photosystem II: a tyrosyl radical and semiquinone charge pair

The states induced by illumination at 7 K in the oxygen-evolving enzyme (PSII) from Thermosynechococcus elongatus were studied by EPR. In the S(0) and S(1) redox states, two g approximately 2 EPR signals, a split signal and a g = 2.03 signal, respectively, were generated by illumination with visible light. These signals were comparable to those already reported in plant PSII in terms of their g value, shape, and stability at low temperatures. We report that the formation and decay of these signals correlate with EPR signals from the semiquinone of the first quinone electron acceptor, Q(A)(-). The light-induced EPR signals from oxidized side-path electron donors (Cyt b(559), Car, and Chl(Z)) were also measured, and from these and the signals from Q(A)(-), estimates were made of the proportion of centers involved in the formation of the g approximately 2 signals (approximately 50% in S(0) and 40% in S(1)). Comparisons with the signals generated in plant PSII indicated approximately similar yields for the S(0) split signal. A single laser flash at 7 K induced more than 75% of the maximum split and g = 2.03 EPR signal observed by continuous illumination, with no detectable oxidation of side-path donors. The matching electron acceptor side reactions, the high quantum yield, and the relatively large proportion of centers involved support earlier suggestions that the state being monitored is Tyr(Z)(*)Q(A)(-), with the g approximately 2 EPR signals arising from Tyr(Z)(*) interacting magnetically with the Mn complex. The current picture of the photochemical reactions occurring in PSII at low temperatures is reassessed.     

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.     

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.     

Active and resting states of the O2-evolving complex of photosystem II

During dark adaptation, a change in the O2-evolving complex (OEC) of spinach photosystem II (PSII) occurs that affects both the structure of the Mn site and the chemical properties of the OEC, as determined from low-temperature electron paramagnetic resonance (EPR) spectroscopy and O2 measurements. The S2-state multiline EPR signal, arising from a Mn-containing species in the OEC, exhibits different properties in long-term (4 h at 0 degrees C) and short-term (6 min at 0 degree C) dark-adapted PSII membranes or thylakoids. The optimal temperature for producing this EPR signal in long-term dark-adapted samples is 200 K compared to 170 K for short-term dark-adapted samples. However, in short-term dark-adapted samples, illumination at 170 K produces an EPR signal with a different hyperfine structure and a wider field range than does illumination at 160 K or below. In contrast, the line shape of the S2-state EPR signal produced in long-term dark-adapted samples is independent of the illumination temperature. The EPR-detected change in the Mn site of the OEC that occurs during dark adaptation is correlated with a change in O2 consumption activity of PSII or thylakoid membranes. PSII membranes and thylakoid membranes slowly consume O2 following illumination, but only when a functional OEC and excess reductant are present. We assign this slow consumption of O2 to a catalytic reduction of O2 by the OEC in the dark. The rate of O2 consumption decreases during dark adaptation; long-term dark-adapted PSII or thylakoid membranes do not consume O2 despite the presence of excess reductant.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electron transfer in photosystem II at cryogenic temperatures

The photochemistry in photosystem II of spinach has been characterized by electron paramagnetic resonance (EPR) spectroscopy in the temperature range of 77-235 K, and the yields of the photooxidized species have been determined by integration of their EPR signals. In samples treated with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), a single stable charge separation occurred throughout the temperature range studied as reflected by the constant yield of the Fe(II)-QA-EPR signal. Three distinct electron donation pathways were observed, however. Below 100 K, one molecule of cytochrome b559 was photooxidized per reaction center. Between 100 and 200 K, cytochrome b559 and the S1 state competed for electron donation to P680+. Photooxidation of the S1 state occurred via two intermediates: the g = 4.1 EPR signal species first reported by Casey and Sauer [Casey, J. L., & Sauer, K. (1984) Biochim. Biophys. Acta 767, 21-28] was photooxidized between 100 and 160 K, and upon being warmed to 200 K in the dark, this EPR signal yielded the multiline EPR signal associated with the S2-state. Only the S1 state donated electrons to P680+ at 200 K or above, giving rise to the light-induced S2-state multiline EPR signal. These results demonstrate that the maximum S2-state multiline EPR signal accounts for 100% of the reaction center concentration. In samples where electron donation from cytochrome b559 was prevented by chemical oxidation, illumination at 77 K produced a radical, probably a chlorophyll cation, which accounted for 95% of the reaction center concentration. This electron donor competed with the S1 state for electron donation to P680+ below 100 K.(ABSTRACT TRUNCATED AT 250 WORDS)     

Abnormal S-state turnovers in NH3-binding Mn centers of photosynthetic O2 evolving system

The inhibitory effects of NH3 on S-state turnovers were studied by curve fitting and deconvolution of thermoluminescence glow curves and low-temperature EPR spectroscopy. The following results were found: (i) High concentrations of NH3 upshifted the recombination temperatures of both S2QB- and S2QA- charge pairs, indicating formation of an abnormal S2 state having a lowered oxidation potential. (ii) The abnormal S2 was correlated to alterations in EPR multiline signal: high concentrations of NH3 induced the modified multiline signal having reduced hyperfine line spacing, accompanied by disappearance of the g = 4.1 signal, while low concentrations of NH3 reduced the line width of the g = 4.1 signal with a slight shift in its g value to 4.2 concomitant with suppression in amplitude of the normal multiline signal, both suggesting coordination of NH3 to the Mn center. (iii) More than half of the NH3-binding abnormal S2 centers underwent S-state turnover to yield S3QB- and S3QA- pairs having normal thermoluminever, the NH3-binding S3 was unable to undergo further S-state turnovers. (iv) The interruption of S-state turnover at S3 was assumed to be due to the inability of electron abstraction from the S3 state. Based on these, the mechanism of NH3 inhibition was discussed.     

Structural and functional modifications of the manganese cluster in Ca(2+)-depleted S1 and S2 states: electron paramagnetic resonance and X-ray absorption spectroscopy studies.

The effect of extraction of weakly bound Ca2+ by low-pH treatment on the O2-evolving apparatus was studied by use of low-temperature electron paramagnetic resonance (EPR) and X-ray absorption spectroscopy. In low-pH-treated PSII membranes, an S2 EPR multiline signal with modified line shape was induced by illumination at 0 degrees C, but its signal amplitude decreased upon lowering the excitation temperature with concomitant oxidation of cytochrome (cyt) b-559 in place of Mn. The half-inhibition temperature for formation of the modified multiline signal was found at -33 degrees C, which was much higher than that for formation of the normal S2 state in untreated control membranes. Signal IIf was normally induced down to -30 degrees C, but its dependence on excitation temperature was different from that for modified S2. This was interpreted as indicating that the low-temperature blockage of modified S2 formation is due to the incapability of electron abstraction from the Mn cluster. The Mn K-edge of X-ray absorption near-edge structure (XANES) spectrum shifted to lower energy by 0.8 eV after low-pH treatment, but the shift was reversed by addition of Ca2+. Upon illumination at 0 degrees C of treated membranes, the K-edge energy was up-shifted by 0.8 eV, but was not upon illumination at 210 K. These results were interpreted as indicating that extraction of weakly bound Ca2+ by low-pH treatment gives rise to structural and functional modulations of the Mn cluster.     

Near-IR irradiation of the S2 state of the water oxidizing complex of photosystem II at liquid helium temperatures produces the metalloradical intermediate attributed to S1Y(Z*)

Near-IR (NIR) excitation at liquid He temperatures of photosystem II (PSII) membranes from the cyanobacterium Synechococcus vulcanus or from spinach poised in the S2 state results in the production of a g = 2.035 EPR resonance, reminiscent of metalloradical signals. The signal is smaller in the spinach preparations, but it is significantly enhanced by the addition of exogenous quinones. Ethanol (2-3%, v/v) eliminates the ability to trap the signal. The g = 2.035 signal is identical to the one recently obtained by Nugent et al. by visible-light illumination of the S1 state, and preferably assigned to S1Y(Z*) [Nugent, J. H. A., Muhiuddin, I. P., and Evans, M. C. W. (2002) Biochemistry 41, 4117-4126]. The production of the g = 2.035 signal by liquid He temperature NIR excitation of the S2 state is paralleled by a significant reduction (typically 40-45% in S. vulcanus) of the S2 state multiline signal. This is in part due to the conversion of the Mn cluster to higher spin states, an effect documented by Boussac et al. [Boussac, A., Un, S., Horner, O., and Rutherford, A. W. (1998) Biochemistry 37, 4001-4007], and in part due to the conversion to the g = 2.035 configuration. Following the decay of the g = 2.035 signal at liquid helium temperatures (decay halftimes in the time range of a few to tens of minutes depending on the preparation), annealing at elevated temperatures (-80 degrees C) results in only partial restoration of the S2 state multiline signal. The full size of the signal can be restored by visible-light illumination at -80 degrees C, implying that during the near-IR excitation and subsequent storage at liquid helium temperatures recombination with Q(A-) (and therefore decay of the S2 state to the S1 state) occurred in a fraction of centers. In support of this conclusion, the g = 2.035 signal remains stable for several hours (at 11 K) in centers poised in the S2...Q(A) configuration before the NIR excitation. The extended stability of the signal under these conditions has allowed the measurement of the microwave power saturation and the temperature dependence in the temperature range of 3.8-11 K. The signal intensity follows Curie law temperature dependence, which suggests that it arises from a ground spin state, or a very low-lying excited spin state. The P1/2 (microwave power at half-saturation) value is 1.7 mW at 3.8 K and increases to 96 mW at 11 K. The large width of the g = 2.035 signal and its relatively fast relaxation support the assignment to a radical species in the proximity of the Mn cluster. The whole phenomenology of the g = 2.035 signal production is analogous to the effects of NIR excitation on the S3 state [Ioannidis, N., Nugent, J. H. A., and Petrouleas, V. (2002) Biochemistry 41, 9589-9600] producing an S2'Y(Z*) intermediate. In the present case, the intermediate is assigned to S1Y(Z*). The NIR-induced increase in the oxidative capability of the Mn cluster is discussed in relation to the photochemical properties of a Mn(III) ion that exists in both S2 and S3 states. The EPR properties of the S1Y(Z*) intermediate cannot be reconciled easily with our current understanding of the magnetic properties of the S1 state. It is suggested that oxidation of tyr Z alters the magnetic properties of the Mn cluster via exchange of a proton.     

Electron transfer from the water oxidizing complex at cryogenic temperatures: the S1 to S2 step

We report the detection of a "split" electron paramagnetic resonance (EPR) signal during illumination of dark-adapted (S(1) state) oxygen-evolving photosystem II (PSII) membranes at <20 K. The characteristics of this signal indicate that it arises from an interaction between an organic radical and the Mn cluster of PSII. The broad radical signal decays in the dark following illumination either by back-reaction with Qa*- or by forward electron transfer from the Mn cluster. The forward electron transfer (either from illumination at 11 K followed by incubation in the dark at 77 K or by illumination at 77 K) results in the formation of a multiline signal similar to, but distinct from, other well-characterized multiline forms found in the S0 and S2 states. The relative yield of the "S1 split signal", which we provisionally assign to S1X*, where X could be YZ* or Car*+, and that of the 77 K multiline signal indicate a relationship between the two states. An approximate quantitation of the yield of these signals indicates that up to 40-50% of PSII centers can form the S1 split signal. Ethanol addition removes the ability to observe the S1 split signal, but the multiline signal is still formed at 77 K. The multiline forms with <700 nm light and is not affected by near-infrared (IR) light, showing that we are detecting electron transfer in centers not responsive to IR illumination. The results provide important new information about the mechanism of electron abstraction from the water oxidizing complex (WOC).     

Electron paramagnetic resonance signals from the S(3) state of the oxygen-evolving complex. A broadened radical signal induced by low-temperature near-infrared light illumination

The tetranuclear manganese cluster responsible for the oxidation of water in photosystem II cycles through five redox states denoted S(i)() (i = 0, 1, 2, 3, 4). Progress has been made recently in the detection of weak low-field EPR absorptions in both the perpendicular and parallel modes, associated with the integer spin state S(3) [Matsukawa, T., Mino, H., Yoneda, D., and Kawamori, A. (1999) Biochemistry 38, 4072-4077]. We confirm observation of these signals and have obtained them in high yield by illumination of photosystem II membranes, in which the non-heme iron was chemically preoxidized. It is shown that a split g = 4 signal accompanies the S(3) state signals. The signals diminish in the presence of ethanol and vanish in the presence of methanol. This effect is similar to that exerted by these alcohols to the high-spin component (g = 4.1) of the S(2) state and suggests that the latter spin configuration is the precursor of the S(3) state low-field signals. The S(3) state shows similar sensitivity to infrared illumination as has been observed previously in the S(2) state [Boussac, A., Un, S., Horner, O., and Rutherford, A. W. (1998) Biochemistry 37, 4001-4007]. Illumination of the S(3) state with near-infrared light (700-900 nm), at temperatures around 50 K, results in the modification of the low-field signals and most notably to the appearance of a broad (DeltaH approximately 200 G) radical-type signal centered at g = 2. The signal is tentatively assigned to the interaction of the Mn cluster in a modified S(2) state with a radical.     

Trapping of the S2 to S3 state intermediate of the oxygen-evolving complex of photosystem II

Photosystem II preparations poised in the S(2)...Q(A) state produce no detectable intermediate during straightforward illumination at liquid helium temperatures. However, upon flash illumination in the range of 77-190 K, they produce a transient state which at -10 degrees C advances to S(3) or after rapid cooling to 10 K gives rise to a 116 G wide metalloradical EPR signal. The latter decays with half-times on the order of a few minutes, presumably by charge recombination, and can be regenerated repeatedly by illumination at 10 K. The constraints for Tyr Z oxidation are attributed to the presence of excess positive charge in S(2). Elevated temperatures are required presumably to overcome a thermal barrier in the deprotonation of Tyr Z(+) or most likely to allow secondary proton transfer away from the base partner of Tyr Z. Treatment with 5% (v/v) MeOH appears to remove the constraints for Tyr Z oxidation, and a 160 G wide metalloradical EPR signal is produced by illumination at 10 K, which decays with a half-time of ca. 80 s. Formation of the metalloradical signals is accompanied by reversible changes in the Mn multiline signal. The intermediates are assigned to Tyr Z(*) magnetically interacting with the Mn cluster in S(2), S(2)Y(Z)(*). A molecular model which extends an earlier suggestion and provides a plausible explanation of a number of observations, including the binding of small molecules to the Mn cluster, is presented.     

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.