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.     

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).     

Intermediates of the S(3) state of the oxygen-evolving complex of photosystem II

The S(3) state of the water-oxidizing complex (WOC) of photosystem II (PSII) is the last state that can be trapped before oxygen evolution occurs at the transient S(4) state. A number of EPR-detectable intermediates are associated with this critical state. The preceding paper examined mainly the decay of S(3) at cryogenic temperatures leading to the formation of a proton-deficient configuration of S(2) termed S(2)'. This second paper examines all intermediates formed by the near-IR light (NIR) excitation of the S(3) state and compares these with the light-excitation products of the S(2)' state. The rather complex set of observations is organized in a comprehensive flowchart, the central part of which is the S(3)...Q(A)(-) state. This state can be converted to various intermediates via two main pathways: (A) Excitation of S(3) by NIR light at temperatures below 77 K results presumably in the formation of an excited S(3) state, S(3), which decays via either of two pathways. Slowly at liquid helium temperatures but much faster at 77 K, S(3) decays to an EPR-silent state, denoted S(3)' ', which by raising the temperature to ca. 190 K converts to a spin configuration of the Mn cluster, characterized by g = 21, 3.7 in perpendicular and g = 23 in parallel mode EPR, denoted S(3)'. Upon further warming to 220 K, S(3)' relaxes to the untreated S(3) state. Below about 77 K and more favorably at liquid helium temperatures, an alternative pathway of S(3) decay via the metallo-radical intermediate S(2)'Z*...Q(A)(-) can be traced. This leads to the metastable state S(2)'Z...Q(A) via charge recombination. S(2)'Z* is characterized by a split-radical signal at g = 2, while all S(2)' transients are characterized by the same g = 5/2.9 (S = (7)/(2)) configuration of the Mn cluster with small modifications, reflecting an influence of the tyr Z oxidation state on the crystal-field symmetry at the Mn cluster. (B) S(2)'...Q(A) can be reached alternatively by the slow charge recombination of S(3) and Q(A)(-) at 77 K. White-light illumination of S(2)'.Q(A) below about 20 K results in charge separation, reforming the intermediate S(2)'Z*...Q(A)(-). Thermally activated branches to the main pathways are also described, e.g., at elevated temperatures tyr Z* reoxidizes S(2)' to the S(3) state. The above observations are discussed in terms of a molecular model of the S(3) state of the OEC. Main aspects of the model are the following. Intermediates, isoelectronic to S(3), are attributed to the NIR-induced translocation of the positive hole to different Mn ligands, or to tyr Z. On the basis of a comparison of the electron-donating efficiency of tyr Z and tyr D at cryogenic temperatures, it is inferred that the Mn cluster acts as the main proton acceptor from tyr Z. Water associated with the Mn cluster is assumed to be in hydrogen-bonding equilibrium with tyr Z, and an array comprising this water and adjacent water (or OH or O) ligands to Mn followed by a sequence of proton acceptors is proposed to act as an efficient proton translocation pathway. Oxidation of the tyrosine by P(680)(+) repels protons to and out from the Mn cluster. This proposed role of tyr Z in the water-splitting process is described as a proton repeller/electron abstractor.     

Low-temperature photochemistry in photosystem II from Thermosynechococcus elongatus induced by visible and near-infrared light

The active site for water oxidation in photosystem II (PSII) consists of a Mn4Ca cluster close to a redox-active tyrosine residue (TyrZ). The enzyme cycles through five sequential oxidation states (S0 to S4) in the water oxidation process. Earlier electron paramagnetic resonance (EPR) work showed that metalloradical states, probably arising from the Mn4 cluster interacting with TyrZ., can be trapped by illumination of the S0, S1 and S2 states at cryogenic temperatures. The EPR signals reported were attributed to S0TyrZ., S1TyrZ. and S2TyrZ., respectively. The equivalent states were examined here by EPR in PSII isolated from Thermosynechococcus elongatus with either Sr or Ca associated with the Mn4 cluster. In order to avoid spectral contributions from the second tyrosyl radical, TyrD., PSII was used in which Tyr160 of D2 was replaced by phenylalanine. We report that the metalloradical signals attributed to TyrZ. interacting with the Mn cluster in S0, S1, S2 and also probably the S3 states are all affected by the presence of Sr. Ca/Sr exchange also affects the non-haem iron which is situated approximately 44 A units away from the Ca site. This could relate to the earlier reported modulation of the potential of QA by the occupancy of the Ca site. It is also shown that in the S3 state both visible and near-infrared light are able to induce a similar Mn photochemistry.     

The EPR spectrum of tyrosine Z* and its decay kinetics in O2-evolving photosystem II preparations

The O2-evolving complex of photosystem II, Mn 4Ca, cycles through five oxidation states, S0,..., S4, during its catalytic function, which involves the gradual abstraction of four electrons and four protons from two bound water molecules. The direct oxidant of the complex is the tyrosine neutral radical, YZ(*), which is transiently produced by the highly oxidizing power of the photoexcited chlorophyll species P680. EPR characterization of YZ(*) has been limited, until recently, to inhibited (non-oxygen-evolving) preparations. A number of relatively recent papers have demonstrated the trapping of YZ(*) in O2-evolving preparations at liquid helium temperatures as an intermediate of the S0 to S1, S1 to S2, and S2 to S3 transitions. The respective EPR spectra are broadened and split at g approximately 2 by the magnetic interaction with the Mn cluster, but this interaction collapses at temperatures higher than about 100K [Zahariou et al. (2007) Biochemistry 46, 14335 -14341]. We have conducted a study of the Tyr Z(*) transient in the temperature range 77-240 K by employing rapid or slow EPR scans. The results reveal for the first time high-resolution X-band spectra of Tyr Z(*) in the functional system and at temperatures close to the onset of the S-state transitions. We have simulated the S 2Y Z(*) spectrum using the simulation algorithm of Svistunenko and Cooper [(2004) Biophys. J. 87, 582 -595]. The small g(x) = 2.00689 value inferred from the analysis suggests either a H-bonding of Tyr Z (*) (presumably with His190) that is stronger than what has been assumed from studies of Tyr D(*) or Tyr Z(*) in Mn-depleted preparations or a more electropositive environment around Tyr Z(*). The study has also yielded for the first time direct information on the temperature variation of the YZ(*)/QA(-) recombination reaction in the various S states. The reaction follows biphasic kinetics with the slow phase dominating at low temperatures and the fast phase dominating at high temperatures. It is tentatively proposed that the slow phase represents the action of the YZ(*)/YZ(-) redox couple while the fast phase represents that of the YZ(*)/YZH couple; it is inferred that Tyr Z at elevated temperatures is protonated at rest. It is also proposed that YZ(*)/YZH is the couple that oxidizes the Mn cluster during the S1-S2 and S2-S3 transitions. A simple mechanism ensuring a rapid (concerted) protonation of Tyr Z upon oxidation of the Mn cluster is discussed, and also, a structure-based molecular model suggesting the participation of His190 into two hydrogen bonds is proposed.     

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.     

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.     

Spectral resolution of the split EPR signals induced by illumination at 5 K from the S1, S3, and S0 states in photosystem II

S-State-dependent split EPR signals that are induced by illumination at cryogenic temperatures (5 K) have been measured in spinach photosystem II without interference from the Y(D)* radical in the g approximately 2 region. This allows us to present the first decay-associated spectra for the split signals, which originate from the CaMn4 cluster in magnetic interaction with a nearby radical, presumably Y(Z)*. The three split EPR signals that were investigated, "Split S1", "Split S3", and Split S0", all exhibit spectral features at g approximately 2.0 together with surrounding characteristic peaks and troughs. From microwave relaxation studies we can reach conclusions about which parts of the complex spectra belong together. Our analysis strongly indicates that the wings and the middle part of the split spectrum are parts of the same signal, since their decay kinetics in the dark at 5 K and microwave relaxation behavior are indistinguishable. In addition, our decay-associated spectra indicate that the g approximately 2.0 part of the "Split S1" EPR spectrum contains a contribution from magnetically uncoupled Y(Z)* as judged from the g value and 22 G line width of the EPR signal. The g value, 2.0033-2.0040, suggests that the oxidation of Y(Z) at 5 K results in a partially protonated radical. Irrespective of the S state, a small amount of a carotenoid or chlorophyll radical was formed by the illumination. However, this had relaxation and decay characteristics that clearly distinguish this radical from the split signal spectra. In this paper, we present the "clean" spectra from the low-temperature illumination-induced split EPR signals from higher plants, which will provide the basis for further simulation studies.     

Effects of pH on the S(3) state of the oxygen evolving complex in photosystem II probed by EPR split signal induction

The electrons extracted from the CaMn(4) cluster during water oxidation in photosystem II are transferred to P(680)(+) via the redox-active tyrosine D1-Tyr161 (Y(Z)). Upon Y(Z) oxidation a proton moves in a hydrogen bond toward D1-His190 (His(Z)). The deprotonation and reprotonation mechanism of Y(Z)-OH/Y(Z)-O is of key importance for the catalytic turnover of photosystem II. By light illumination at liquid helium temperatures (～5 K) Y(Z) can be oxidized to its neutral radical, Y(Z)(?). This can be followed by the induction of a split EPR signal from Y(Z)(?) in a magnetic interaction with the CaMn(4) cluster, offering a way to probe for Y(Z) oxidation in active photosystem II. In the S(3) state, light in the near-infrared region induces the split S(3) EPR signal, S(2)'Y(Z)(?). Here we report on the pH dependence for the induction of S(2)'Y(Z)(?) between pH 4.0 and pH 8.7. At acidic pH the split S(3) EPR signal decreases with the apparent pK(a) (pK(app)) ～ 4.1. This can be correlated to a titration event that disrupts the essential H-bond in the Y(Z)-His(Z) motif. At alkaline pH, the split S(3) EPR signal decreases with the pK(app) ～ 7.5. The analysis of this pH dependence is complicated by the presence of an alkaline-induced split EPR signal (pK(app) ～ 8.3) promoted by a change in the redox potential of Y(Z). Our results allow dissection of the proton-coupled electron transfer reactions in the S(3) state and provide further evidence that the radical involved in the split EPR signals is indeed Y(Z)(?).     

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.     

Trapping of metalloradical intermediates of the S-states at liquid helium temperatures. Overview of the phenomenology and mechanistic implications

The oxygen-evolving complex (OEC) of photosystem II (PSII) consists of a Mn cluster (believed to be tetranuclear) and a tyrosine (Tyr Z or Y(Z)). During the sequential absorption of four photons by PSII, the OEC undergoes four oxidative transitions, S(0) to S(1), ..., S(3) to (S(4))S(0). Oxygen evolves during the S(3) to S(0) transition (S(4) being a transient state). Trapping of intermediates of the S-state transitions, particularly those involving the tyrosyl radical, has been a goal of ultimate importance, as that can test critically models employing a role of Tyr Z in proton (in addition to electron) transfer, and also provide important clues about the mechanism of water oxidation. Until very recently, however, critical experimental information was lacking. We review and evaluate recent observations on the trapping of metalloradical intermediates of the S-state transitions, at liquid helium temperatures. These transients are assigned to Tyr Z(*) magnetically interacting with the Mn cluster. Besides the importance of trapping intermediates of this unique catalytic mechanism, liquid helium temperatures offer the additional advantage that proton motions (unlike electron transfer) are blocked except perhaps across strong hydrogen bonds. This paper summarizes the recent observations and discusses the constraints that the phenomenology imposes.     

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.     

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

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

Assignment of the g = 4.1 EPR 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.     

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)     

Visible light induction of an electron paramagnetic resonance split signal in Photosystem II in the S(2) state reveals the importance of charges in the oxygen-evolving center during catalysis: a unifying model

Cryogenic illumination of Photosystem II (PSII) can lead to the trapping of the metastable radical Y(Z)(?), the radical form of the redox-active tyrosine residue D1-Tyr161 (known as Y(Z)). Magnetic interaction between this radical and the CaMn(4) cluster of PSII gives rise to so-called split electron paramagnetic resonance (EPR) signals with characteristics that are dependent on the S state. We report here the observation and characterization of a split EPR signal that can be directly induced from PSII centers in the S(2) state through visible light illumination at 10 K. We further show that the induction of this split signal takes place via a Mn-centered mechanism, in the same way as when using near-infrared light illumination [Koulougliotis, D., et al. (2003) Biochemistry 42, 3045-3053]. On the basis of interpretations of these results, and in combination with literature data for other split signals induced under a variety of conditions (temperature and light quality), we propose a unified model for the mechanisms of split signal induction across the four S states (S(0), S(1), S(2), and S(3)). At the heart of this model is the stability or instability of the Y(Z)(?)(D1-His190)(+) pair that would be formed during cryogenic oxidation of Y(Z). Furthermore, the model is closely related to the sequence of transfers of protons and electrons from the CaMn(4) cluster during the S cycle and further demonstrates the utility of the split signals in probing the immediate environment of the oxygen-evolving center in PSII.     

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.     

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)     

The S(3) state of the oxygen-evolving complex in photosystem II is converted to the S(2)Y(Z)* state at alkaline pH

Here we report an EPR signal that is induced by a pH jump to alkaline pH in the S(3) state of the oxygen-evolving complex in photosystem II. The S(3) state is first formed with two flashes at pH 6. Thereafter, the pH is changed in the dark prior to freezing of the sample. The EPR signal is 90-100 G wide and centered around g = 2. The signal is reversibly induced with a pK = 8.5 +/- 0.3 and is very stable with a decay half-time of 5-6 min. If the pH is changed in the dark from pH 8.6 to 6.0, the signal disappears although the S(3) state remains. We propose that the signal arises from the interaction between the Mn cluster and Y(Z), resulting in the spin-coupled S(2)Y(Z)(*) signal. Our data suggest that the potential of the Y(Z)(*)/Y(Z) redox couple is sensitive to the ambient pH in the S(3) state. The alkaline pH decreases the potential of the Y(Z)(*)/Y(Z) couple so that Y(Z) can give back an electron to the S(3) state, thereby obtaining the S(2)Y(Z)(*) EPR signal. The tyrosine oxidation also involves proton release from Y(Z), and the results support a mechanism where this proton is released to the bulk medium presumably via a close-lying base. Thus, the equilibrium is changed from S(3)Y(Z) to S(2)Y(Z)(*) by the alkaline pH. At normal pH (pH 5.5-7), this equilibrium is set strongly to the S(3)Y(Z) state. The results are discussed in relation to the present models of water oxidation. Consequences for the relative redox potentials of Y(Z)(*)/Y(Z) and S(3)/S(2) at different pH values are discussed. We also compare the pH-induced S(2)Y(Z)(*) signal with the S(2)Y(Z)(*) signal from Ca(2+)-depleted photosystem II.