Characterization of the tyrosine-Z radical and its environment in the spin-coupled S2TyrZ* state of photosystem II from Thermosynechococcus elongatus

The Mn4Ca cluster of photosystem II (PSII) goes through five sequential oxidation states (S0-S4) in the water oxidation process that also involves a tyrosine radical intermediate (TyrZ*). An S2TyrZ* state in which the Mn4Ca cluster and TyrZ* are magnetically coupled to each other and which is characterized by a distinct "split-signal" EPR spectrum can be generated in acetate-treated PSII. This state was examined by high-field EPR (HFEPR) in PSII from Thermosynechococcus elongatus isolated from a D2-Tyr160Phe mutant to avoid spectral contributions from TyrD*. In contrast to the same state in plants, both antiferromagnetic and ferromagnetic spin-spin couplings were observed. The intrinsic g values of TyrZ* in the coupled state were directly measured from the microwave frequency dependence of the HFEPR spectrum. The TyrZ* gx value in the antiferromagnetic centers was 2.0083, indicating that the coupled radical was in a less electropositive environment than in Mn-depleted PSII. Two gx values were found in the ferromagnetically coupled centers, 2.0069 and 2.0079. To put these values in perspective, the second redox-active tyrosine, TyrD*, was examined in various electrostatic environments. The TyrD* gx value changed from 2.0076 in the wild type to 2.0095 when the hydrogen bond from histidine 189 to TyrD* was removed using the D2-His189Leu mutant, indicating a change to a significantly less electropositive environment. BLY3P/6-31+G** density functional calculations on the hydrogen-bonded p-ethylphenoxy radical-imidazole supermolecular model complex showed that the entire range of Tyr* gx values, from 2.0065 to 2.0095, could be explained by the combined effects of hydrogen bonding and the dielectric constant of the local protein environment.     

Biosynthetic Ca2+/Sr2+ exchange in the photosystem II oxygen-evolving enzyme of Thermosynechococcus elongatus

The thermophilic cyanobacterium, Thermosynechococcus elongatus, has been grown in the presence of Sr2+ instead of Ca2+ with the aim of biosynthetically replacing the Ca2+ of the oxygen-evolving enzyme with Sr2+. Not only were the cells able to grow normally with Sr2+, they actively accumulated the ion to levels higher than those of Ca2+ in the normal cultures. A protocol was developed to purify a fully active Sr(2+)-containing photosystem II (PSII). The modified enzyme contained a normal polypeptide profile and 1 strontium/4 manganese, indicating that the normal enzyme contains 1 calcium/4 manganese. The Sr(2+)- and Ca(2+)-containing enzymes were compared using EPR spectroscopy, UV-visible absorption spectroscopy, and O2 polarography. The Ca2+/Sr2+ exchange resulted in the modification of the EPR spectrum of the manganese cluster and a slower turnover of the redox cycle (the so-called S-state cycle), resulting in diminished O2 evolution activity under continuous saturating light: all features reported previously by biochemical Ca2+/Sr2+ exchange in plant PSII. This allays doubts that these changes could be because of secondary effects induced by the biochemical treatments themselves. In addition, the Sr(2+)-containing PSII has other kinetics modifications: 1) it has an increased stability of the S3 redox state; 2) it shows an increase in the rate of electron donation from TyrD, the redox-active tyrosine of the D2 protein, to the oxygen-evolving complex in the S3-state forming S2; 3) the rate of oxidation of the S0-state to the S1-state by TyrD* is increased; and 4) the release of O2 is slowed down to an extent similar to that seen for the slowdown of the S3TyrZ* to S0TyrZ transition, consistent with the latter constituting the limiting step of the water oxidation mechanism in Sr(2+)-substituted enzyme as well as in the normal enzyme. The replacement of Ca2+ by Sr2+ appears to have multiple effects on kinetics properties of the enzyme that may be explained by S-state-dependent shifts in the redox properties of both the manganese complex and TyrZ as well as structural effects.     

Near-infrared-induced transitions in the manganese cluster of photosystem II: action spectra for the S2 and S3 redox states

The Mn4Ca complex that is involved in water oxidation in PSII is affected by near-infrared (NIR) light in certain redox states and these phenomena can be monitored by electron paramagnetic resonance (EPR) at low temperature. Here we report the action spectra of the NIR effects in the S2 and S3 states in PSII from plants and the thermophilic cyanobacterium Thermosynechococcus elongatus. The action spectra obtained are very similar in both S states, indicating the presence of the same photoactive form of the Mn4Ca complex in both states. Since the chemical nature of the photoactive species is not known, an unequivocal interpretation of this result cannot be made; however, it appears to be more easily reconciled with the view that the redox state of the Mn4Ca cluster does not change from the S2 to the S3 transition, at least in those centers sensitive to NIR light. The temperature dependence of the NIR effect and the action spectra for S2 indicate the presence of structural heterogeneity in the Mn4Ca cluster.     

Glutamate-354 of the CP43 polypeptide interacts with the oxygen-evolving Mn4Ca cluster of photosystem II: a preliminary characterization of the Glu354Gln mutant

In the recent X-ray crystallographic structural models of photosystem II, Glu354 of the CP43 polypeptide is assigned as a ligand of the O2-evolving Mn4Ca cluster. In this communication, a preliminary characterization of the CP43-Glu354Gln mutant of the cyanobacterium Synechocystis sp. PCC 6803 is presented. The steady-state rate of O2 evolution in the mutant cells is only approximately 20% compared with the wild-type, but the kinetics of O2 release are essentially unchanged and the O2-flash yields show normal period-four oscillations, albeit with lower overall intensity. Purified PSII particles exhibit an essentially normal S2 state multiline electron paramagnetic resonance (EPR) signal, but exhibit a substantially altered S2-minus-S1 Fourier transform infrared (FTIR) difference spectrum. The intensities of the mutant EPR and FTIR difference spectra (above 75% compared with wild-type) are much greater than the O2 signals and suggest that CP43-Glu354Gln PSII reaction centres are heterogeneous, with a minority fraction able to evolve O2 with normal O2 release kinetics and a majority fraction unable to advance beyond the S2 or S3 states. The S2-minus-S1 FTIR difference spectrum of CP43-Glu354Gln PSII particles is altered in both the symmetric and asymmetric carboxylate stretching regions, implying either that CP43-Glu354 is exquisitely sensitive to the increased charge that develops on the Mn4Ca cluster during the S1-->S2 transition or that the CP43-Glu354Gln mutation changes the distribution of Mn(III) and Mn(IV) oxidation states within the Mn4Ca cluster in the S1 and/or S2 states.     

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.     

Probing the role of chloride in Photosystem II from Thermosynechococcus elongatus by exchanging chloride for iodide

The active site for water oxidation in Photosystem II (PSII) goes through five sequential oxidation states (S(0) to S(4)) before O(2) is evolved. It consists of a Mn(4)CaO(5) cluster and Tyr(Z), a redox-active tyrosine residue. Chloride ions have been known for long time to be required for the function of the enzyme. However, X-ray data have shown that they are located about 7? away from the Mn(4)CaO(5) cluster, a distance that seems too large to be compatible with a direct involvement of chloride in the water splitting chemistry. We have investigated the role of this anion by substituting I(-) for Cl(-) in the cyanobacterium Thermosynechococcus elongatus with either Ca(2+) or Sr(2+) biosynthetically assembled into the Mn(4) cluster. The electron transfer steps affected by the exchanges were investigated by time-resolved UV-visible absorption spectroscopy, time-resolved EPR at room temperature and low temperature cw-EPR spectroscopy. In both Ca-PSII and Sr-PSII, the Cl(-)/I(-) exchange considerably slowed down the two S(3)Tyr(Z)(?)→(S(3)Tyr(Z)(?))'→S(0) reactions in which the fast phase, S(3)Tyr(Z)(?)→(S(3)Tyr(Z)(?))', reflects the electrostatically triggered expulsion of one proton from the catalytic center caused by the positive charge near/on Tyr(Z)(?) and the slow phase corresponds to the S(0) and O(2) formations and to a second proton release. The t(1/2) for S(0) formation increased from 1.1ms in Ca/Cl-PSII to ≈6ms in Ca/I-PSII and from 4.8ms in Sr/Cl-PSII to ≈45ms in Sr/I-PSII. In all cases the Tyr(Z)(?) reduction was the limiting step. The kinetic effects are interpreted by a model in which the Ca(2+) binding site and the Cl(-) binding site, although spatially distant, interact. This interaction is likely mediated by the H-bond and/or water molecules network(s) connecting the Cl(-) and Ca(2+) binding sites by which proton release may be channelled.     

Formation spectra of the EPR split signals from the S0, S1, and S3 states in photosystem II induced by monochromatic light at 5 K

The interaction EPR split signals from photosystem II (PSII) have been reported from the S0, S1, and S3 states. The signals are induced by illumination at cryogenic temperatures and are proposed to reflect the magnetic interaction between YZ* and the Mn4Ca cluster. We have investigated the formation spectra of these split EPR signals induced in PSII enriched membranes at 5 K using monochromatic laser light from 400 to 900 nm. We found that the formation spectra of the split S0, split S1, and split S3 EPR signals were quite similar, but not identical, between 400 and 690 nm, with maximum formation at 550 nm. The major deviations were found between 440 and 480 nm and between 580 and 680 nm. In the regions around 460 and 680 nm the amplitudes of the formation spectra were 25-50% of that at 550 nm. A similar formation spectrum was found for the S2-state multiline EPR signal induced at 0 degrees C. In general, the formation spectra of these signals in the visible region resemble the reciprocal of the absorption spectra of our PSII membranes. This reflects the high chlorophyll concentration necessary for the EPR measurements which mask the spectral properties of other absorbing species. No split signal formation was found by the application of infrared laser illumination between 730 and 900 nm from PSII in the S0 and S1 states. However, when such illumination was applied to PSII membranes poised in the S3 state, formation of the split S3 EPR signal was observed with maximum formation at 740 nm. The quantum yield was much less than in the visible region, but the application of intensive illumination at 830 nm resulted in accumulation of the signal to an amplitude comparable to that obtained with illumination with visible light. The split S3 EPR signal induced by NIR light was much more stable at 5 K (no observable decay within 60 min) than the split S3 signal induced by visible light (50% of the signal decayed within 30 min). The split S3 signals induced by each of these light regimes showed the same EPR spectral features and microwave power saturation properties, indicating that illumination of PSII in the S3 state by visible light or by NIR light produces a similar configuration of YZ* and the Mn4Ca cluster.     

The collapse of the tyrosine Z*-Mn spin-spin interaction above approximately 100 K reveals the spectrum of tyrosine Z*. An application of rapid-scan EPR to the study of intermediates of the water splitting mechanism of photosystem II

Tyr Z of photosystem II mediates electron transfer from the water splitting site, a Mn4Ca cluster, to the specialized chlorophyll assembly P680. Due to its proton-limited redox properties and the proximity to the Mn cluster, it is thought to play a critical role in the proton-coupled electron transfer reactions that constitute the four-step oxidation mechanism (so-called S-state transitions) of water to molecular oxygen. Spectroscopic evidence for the Tyr Z radical has been scarce in intact preparations (it is difficult to probe it optically, and too short-lived for EPR characterization) until recently. Advances in recent years have allowed the trapping at liquid helium temperatures and EPR characterization of metalloradical intermediates, attributed to tyrosyl Z* magnetically interacting with the Mn cluster. We have extended these studies and examined the evolution of the spectra of five intermediates: S0YZ*, S0YZ* (with 5% MeOH), S1YZ*, S2YZ*, and S2YZ* (with 5% MeOH) in the temperature range of 11-230 K. A rapid-scan EPR method has been applied at elevated temperatures. The tyrosyl radical decouples progressively from Mn, as the Mn relaxation rate increases with an increase in temperature. Above approximately 100 K, the spectra collapse to the unperturbed spectrum of Tyr Z*, which is found to be somewhat broader than that of the stable Tyr D* radical. This study provides a simple means for recording the spectrum of Tyr Z* and extends earlier observations that link the photochemistry at liquid helium temperatures to the photochemistry at temperatures that support S-state transitions.     

EPR spectroscopy of the manganese cluster of photosystem II

Electron paramagnetic resonance (EPR) spectroscopy is a valuable tool for understanding the oxidation state and chemical environment of the Mn₄Ca cluster of photosystem II. Since the discovery of the multiline signal from the S₂ state, EPR spectroscopy has continued to reveal details about the catalytic center of oxygen evolution. At present EPR signals from nearly all of the S-states of the Mn₄Ca cluster, as well as from modified and intermediate states, have been observed. This review article describes the various EPR signals obtained from the Mn₄Ca cluster, including the metalloradical signals due to interaction of the cluster with a nearby organic 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.     

Detection of an electron paramagnetic resonance signal in the S0 state of the manganese complex of photosystem II from Synechococcus elongatus.

The Mn(4)-cluster of photosystem II (PSII) from Synechococcus elongatus was studied by electron paramagnetic resonance (EPR) spectroscopy after a series of saturating laser flashes given in the presence of either methanol or ethanol. Results were compared to those obtained in similar experiments done on PSII isolated from plants. The flash-dependent changes in amplitude of the EPR multiline signals were virtually identical in all samples. In agreement with earlier work [Messinger, J., Nugent, J. H. A., and Evans, M. C. W. (1997) Biochemistry 36, 11055-11060; Ahrling, K. A., Peterson, S., and Styring, S. (1997) Biochemistry 36, 13148-13152], detection of an EPR multiline signal from the S(0) state in PSII from plants was only possible with methanol present. In PSII from S. elongatus, it is shown that the S(0) state exhibits an EPR multiline signal in the absence of methanol (however, ethanol was present as a solvent for the artificial electron acceptor). The hyperfine lines are better resolved when methanol is present. The S(0) multiline signals detected in plant PSII and in S. elongatus were similar but not identical. Unlike the situation seen in plant PSII, the S(2) state in S. elongatus is not affected by the addition of methanol in that (i) the S(2) multiline EPR signal is not modified by methanol and (ii) the spin state of the S(2) state is affected by infrared light when methanol is present. It is also shown that the magnetic relaxation properties of an oxidized low-spin heme, attributed to cytochrome c(550), vary with the S states. This heme then is in the magnetic environment of the Mn(4) cluster.     

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.     

Split EPR signals from photosystem II are modified by methanol, reflecting S state-dependent binding and alterations in the magnetic coupling in the CaMn4 cluster

Methanol binds to the CaMn4 cluster in photosystem II (PSII). Here we report the methanol dependence of the split EPR signals originating from the magnetic interaction between the CaMn4 cluster and the Y(Z)* radical in PSII which are induced by illumination at 5 K. We found that the magnitudes of the "split S1" and "split S3" signals induced in the S1 and S3 states of PSII centers, respectively, are diminished with an increase in the methanol concentration. The methanol concentrations at which half of the respective spectral changes had occurred ([MeOH](1/2)) were 0.12 and 0.57%, respectively. By contrast, the "split S0" signal induced in the S0 state is broadened, and its amplitude is enhanced. [MeOH](1/2) for this change was found to be 0.54%. We discuss these observations with respect to the location and nature of the methanol binding site. Furthermore, by comparing this behavior with methanol effects reported for other EPR signals in the different S states, we propose that the observed methanol-dependent changes in the split S1 and split S0 EPR signals are caused by an increase in the extent of magnetic coupling within the cluster.     

Probing the functional role of Ca2+ in the oxygen-evolving complex of photosystem II by metal ion inhibition

Photosynthetic oxygen evolution in photosystem II (PSII) takes place in the oxygen-evolving complex (OEC) that is comprised of a tetranuclear manganese cluster (Mn4), a redox-active tyrosine residue (YZ), and Ca2+ and Cl- cofactors. The OEC is successively oxidized by the absorption of 4 quanta of light that results in the oxidation of water and the release of O2. Ca2+ is an essential cofactor in the water-oxidation reaction, as its depletion causes the loss of the oxygen-evolution activity in PSII. In recent X-ray crystal structures, Ca2+ has been revealed to be associated with the Mn4 cluster of PSII. Although several mechanisms have been proposed for the water-oxidation reaction of PSII, the role of Ca2+ in oxygen evolution remains unclear. In this study, we probe the role of Ca2+ in oxygen evolution by monitoring the S1 to S2 state transition in PSII membranes and PSII core complexes upon inhibition of oxygen evolution by Dy3+, Cu2+, and Cd2+ ions. By using a cation-exchange procedure in which Ca2+ is not removed prior to addition of the studied cations, we achieve a high degree of reversible inhibition of PSII membranes and PSII core complexes by Dy3+, Cu2+, and Cd2+ ions. EPR spectroscopy is used to quantitate the number of bound Dy3+ and Cu2+ ions per PSII center and to determine the proximity of Dy3+ to other paramagnetic centers in PSII. We observe, for the first time, the S2 state multiline electron paramagnetic resonance (EPR) signal in Dy3+- and Cd2+-inhibited PSII and conclude that the Ca2+ cofactor is not specifically required for the S1 to S2 state transition of PSII. This observation provides direct support for the proposal that Ca2+ plays a structural role in the early S-state transitions, which can be fulfilled by other cations of similar ionic radius, and that the functional role of Ca2+ to activate water in the O-O bond-forming reaction that occurs in the final step of the S state cycle can only be fulfilled by Ca2+ and Sr2+, which have similar Lewis acidities.     

Biosynthetic exchange of bromide for chloride and strontium for calcium in the photosystem II oxygen-evolving enzymes

The active site for water oxidation in photosystem II goes through five sequential oxidation states (S(0) to S(4)) before O(2) is evolved. It consists of a Mn(4)Ca cluster close to a redox-active tyrosine residue (Tyr(Z)). Cl(-) is also required for enzyme activity. To study the role of Ca(2+) and Cl(-) in PSII, these ions were biosynthetically substituted by Sr(2+) and Br(-), respectively, in the thermophilic cyanobacterium Thermosynechococcus elongatus. Irrespective of the combination of the non-native ions used (Ca/Br, Sr/Cl, Sr/Br), the enzyme could be isolated in a state that was fully intact but kinetically limited. The electron transfer steps affected by the exchanges were identified and then investigated by using time-resolved UV-visible absorption spectroscopy, time-resolved O(2) polarography, and thermoluminescence spectroscopy. The effect of the Ca(2+)/Sr(2+) and Cl(-)/Br(-) exchanges was additive, and the magnitude of the effect varied in the following order: Ca/Cl < Ca/Br < Sr/Cl < Sr/Br. In all cases, the rate of O(2) release was similar to that of the S(3)Tyr(Z)(.) to S(0)Tyr(Z) transition, with the slowest kinetics (i.e. the Sr/Br enzyme) being approximately 6-7 slower than in the native Ca/Cl enzyme. This slowdown in the kinetics was reflected in a decrease in the free energy level of the S(3) state as manifest by thermoluminescence. These observations indicate that Cl(-) is involved in the water oxidation mechanism. The possibility that Cl(-) is close to the active site is discussed in terms of recent structural models.     

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)     

An evaluation of structural models for the photosynthetic water-oxidizing complex derived from spectroscopic and X-ray diffraction signatures

Four of the five intermediate oxidation states (S-states) in the catalytic cycle of water oxidation used by O2-evolving photoautotrophs have been previously characterized by EPR and/or ENDOR spectroscopy, with the first reports for the S0, S1, and S3 states available in just the last three years. The first electron density map of the Mn cluster derived from X-ray diffraction measurements of single crystals of photosystem II at 3.8-4.2 A resolution has also appeared this year. This wealth of new information has provided significant insight into the structure of the inorganic core (Mn4OxCa1Cl1-2), the Mn oxidation states, and the location and function of the essential Ca2+ cofactor within the water-oxidizing complex (WOC). We summarize these advances and provide a unified interpretation of debated structural proposals and Mn oxidation states, based on an integrated analysis of the published data, particularly from Mn X-ray absorption spectroscopy (XAS) and EPR/ENDOR data. Only three magnetic spin-exchange models for the inter-manganese interactions are possible from consideration of the EPR data for the S0, S1, S2 and S(-N) (NO-reduced) states. These models fall into one of three types denoted butterfly, funnel, or tetrahedron. A revised set of eight allowed chemical structures for the Mn4Ox core can be deduced that are shown to be consistent with both EPR and XAS. The popular "dimer-of-dimers" structural model is not compatible with the possible structural candidates. EPR data have identified two inter-manganese couplings that are sensitive to the S-state, suggesting two possible bridging sites for substrate water molecules. Spin densities derived from 55Mn hyperfine data together with Mn K-edge energies from Ca-depleted samples provide an internally consistent assignment for the Mn oxidation states of Mn4(3III,IV) for the S2 state. EPR and XAS data also provide a consistent picture, locating Ca2+ as an integral part of the inorganic core, probably via shared bridging ligands with Mn (aqua/hydroxo/carboxylato/chloro). XAS data reveal that the Ca2+ cofactor increases the Mn(1s-->4p) transition energy by 0.6-1 eV with minimal structural perturbation versus the Ca-depleted WOC. Thus, calcium binding appears to increase the Mn-ligand covalency by increasing electron transfer from shared ligands to Mn, suggesting a direct role for Ca2+ in substrate water oxidation. Consideration of both the XAS and the EPR data, together with reactivity studies on two model complexes that evolve O2, suggest two favored structure types as feasible models for the reactive S4 state that is precursor to the O2 evolution step. These are a calcium-capped "cuboidal" core and a calcium-capped "funnel" core.     

EPR signals from modified charge accumulation states of the oxygen evolving enzyme in Ca2+-deficient photosystem II

Photosystem II enriched membranes were depleted of Ca2+ and the 17- and 23-kDa polypeptides by treatment with NaCl and EGTA. The 17- and 23-kDa polypeptides were then reconstituted. This preparation was incapable of O2 evolution until Ca2+ was added. An EPR study revealed the presence of two new EPR signals. One of these is a modified S2 multiline signal with an isotropic g value of 1.96 with at least 26 hyperfine peaks (average spacing 55 G) distributed over approximately 1600 G. The other is a near-Gaussian signal with an isotropic g value of 2.004, which is attributed to a formal S3 state. Experiments involving the interconversion of these signals and the effect of Ca2+ and Sr2+ rebinding provide evidence for these assignments. From these results the following conclusions are drawn: (1) These results are consistent with our earlier demonstration that charge accumulation is blocked after formation of S3 when Ca2+ is deficient. (2) Binding of the 17- and 23-kDa polypeptides to photosystem II in the absence of Ca2+ results in the perturbation of the Mn cluster. This is taken as a further indication that the Ca2+-binding site is close to or even an integral part of the Mn cluster. (3) The S3 signal may arise from an organic free radical interacting magnetically with the Mn cluster. However, other possible origins for this signal, including the Mn cluster itself, must also be considered.     

Does aspartate 170 of the D1 polypeptide ligate the manganese cluster in photosystem II? An EPR and ESEEM Study

Aspartate 170 of the D1 polypeptide provides part of the high-affinity binding site for the first Mn(II) ion that is photooxidized during the light-driven assembly of the (Mn)(4) cluster in photosystem II [Campbell, K. A., Force, D. A., Nixon, P. J., Dole, F., Diner, B. A., and Britt, R. D. (2000) J. Am. Chem. Soc. 122, 3754-3761]. However, despite a wealth of data on D1-Asp170 mutants accumulated over the past decade, there is no consensus about whether this residue ligates the assembled (Mn)(4) cluster. To address this issue, we have conducted an EPR and ESEEM (electron spin-echo envelope modulation) study of D1-D170H PSII particles purified from the cyanobacterium Synechocystis sp. PCC 6803. The line shapes of the S(1) and S(2) state multiline EPR signals of D1-D170H PSII particles are unchanged from those of wild-type PSII particles, and the signal amplitudes correlate approximately with the lower O(2) evolving activity of the mutant PSII particles (40-60% compared to that of the wild type). These data provide further evidence that the assembled (Mn)(4) clusters in D1-D170H cells function normally, even though the assembly of the (Mn)(4) cluster is inefficient in this mutant. In the two-pulse frequency domain ESEEM spectrum of the 9.2 GHz S(2) state multiline EPR signal of D1-D170H PSII particles, the histidyl nitrogen modulation observed at 4-5 MHz is unchanged from that of wild-type PSII particles and no significant new modulation is observed. Three scenarios are presented to explain this result. (1) D1-Asp170 ligates the assembled (Mn)(4) cluster, but the hyperfine couplings to the ligating histidyl nitrogen of D1-His170 are too large or anisotropic to be detected by ESEEM analyses conducted at 9.2 GHz. (2) D1-Asp170 ligates the assembled (Mn)(4) cluster, but D1-His170 does not. (3) D1-Asp170 does not ligate the assembled (Mn)(4) cluster.