EPR kinetic studies of the S−1 state in spinach thylakoids

The Y_Z decay kinetics in a formal S₋₁ state, regarded as a reduced state of the oxygen evolving complex, was determined using time-resolved EPR spectroscopy. This S₋₁ state was generated by biochemical treatment of thylakoid membranes with hydrazine. The steady-state oxygen evolution of the sample was used to optimize the biochemical procedure for performing EPR experiments. A high yield of the S₋₁ state was generated as judged by the two-flash delay in the first maximum of oxygen evolution in Joliot flash-type experiments. We have shown that the Y_Z re-reduction rate by the S₋₁ state is much slower than that of any other S-state transition in hydrazine-treated samples. This slow reduction rate in the S₋₁ to S₀ transition, which is in the order of the S₃ to S₀ transition rate, suggests that this transition is accompanied by some structural rearrangements. Possible explanations of this unique, slow reduction rate in the S₋₁ to S₀ transition are considered, in light of earlier observations by others on hydrazine/hydroxylamine reduced PS II samples.     

Effect of hydroxylamine on photosystem II: reinvestigation of electron paramagnetic resonance characteristics reveals possible S state intermediates

Previous work in many laboratories has established that hydroxylamine reduces the S(1) state of the water oxidizing complex (WOC) in one-electron steps. Significant levels of what can now be defined as the S(-1)* state are achieved by specific (concentration and incubation length) hydroxylamine treatments. This state has already been studied by electron paramagnetic resonance spectrometry (EPR), and unusual EPR signals were noted (for example, see Sivaraja, M., and Dismukes, G. C. (1988) Biochemistry 27, 3467-3475). We have now reinvestigated these initial experiments and confirmed many of the original observations. We then utilized more recent EPR markers for the S(0) and S(1) states to further explore the S(-1)* state. The broad radical "split" type EPR signal, produced by 200 K illumination of samples prepared to give a high yield of the S(-1)* state, is shown to most likely reflect a trapped intermediate state between S(-1)* and S(0)*, since samples where this signal is present can be warmed in the dark to produce S(0)*. The threshold for advancement from S(-1)* to S(0)* is near 200 K, as the yield of broad radical decreases and S(0)* multiline EPR signal increases with length of 200 K illumination. Advancement of S(0)* to S(1) is limited at 200 K, but S(1) can be restored by 273 K illumination. Illumination of these hydroxylamine-treated samples at temperatures below 77 K gives a second broad radical EPR signal. The line shape, decay, and other properties of this new radical signal suggest that it may arise from an interaction in the S(-2)* or lower S states, which are probably present in low yield in these samples. Illumination below 20 K of S(0)* state samples containing methanol, and therefore exhibiting the S(0) multiline signal, gives rise to a third broad radical with distinctive line shape. The characteristics of the three broad radicals are similar to those found from interactions between Y(Z)(*) and other S states. The evidence is presented that they do represent intermediate states in S state turnover. Further work is now needed to identify these radicals.     

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

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

pH dependence of the four individual transitions in the catalytic S-cycle during photosynthetic oxygen evolution

We have investigated the pH dependence for each individual redox transition in the S-cycle of the oxygen evolving complex (OEC) of photosystem II by electron paramagnetic resonance (EPR) spectroscopy. In the experiments, OEC is advanced to the appropriate S-state at normal pH. Then, the pH is rapidly changed, and a new flash is given. The ability to advance to the next S-state in the cycle at different pHs is determined by measurements of the decrease or increase of characteristic EPR signals from the OEC in different S-states. In some cases the measured EPR signals are very small (this holds especially for the S0 ML signal at pH >7.5 and pH <4.8). Therefore, we refrain from providing error limits for the determined pK's. Our results indicate that the S1 --> S2 transition is independent of pH between 4.1 and 8.4. All other S-transitions are blocked at low pH. In the acidic region, the pK's for the inhibition of the S2 --> S3, the S3 --> [S4] --> S0, and the S0 --> S1 transitions are about 4.0, 4.5, and 4.7, respectively. The similarity of these pK values indicates that the inhibition of the steady-state oxygen evolution in the acidic range, which occurs with pK approximately 4.8, is a consequence of similar pH blocks in three of the redox steps involved in the oxygen evolution. In the alkaline region, we report a clear pH block in the S3 --> [S4] --> S0 transition with a pK of about 8.0. Our study also indicates the existence of a pH block at very high pH (pK approximately 9.4) in the S2 --> S3 transition. The S0 --> S1 transition is not affected, at least up to pH 9.0. This suggests that the inhibition of the steady-state oxygen evolution, which occurs with a pK of 8.0, is dominated by the inhibition of the S3 --> [S4] --> S0 transition. Our results are obtained in the presence of 5% methanol (v/v). However, it is unlikely that the determined pK's are affected by the presence of methanol since our results also show that the pH dependence of the steady-state oxygen evolution is not affected by methanol. The results in the alkaline region are in good agreement with a model, which suggests that the redox potential of Y(Z*)/Y(Z) is directly affected by high pH. At high pH the Y(Z*)/Y(Z) potential becomes lower than that of S2/S1 and S3/S2. The acidic block, with a pK of 4-5 in three S-transitions, implies that the inhibition mechanism is similar, and we suggest that it reflects protonation of a carboxylic side chain in the proton relay that expels protons from the OEC.     

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.     

Stability of the S₃and S₂state intermediates in photosystem II directly probed by EPR spectroscopy

The stability of the S(3) and S(2) states of the oxygen evolving complex in photosystem II (PSII) was directly probed by EPR spectroscopy in PSII membrane preparations from spinach in the presence of the exogenous electron acceptor PpBQ at 1, 10, and 20 °C. The decay of the S(3) state was followed in samples exposed to two flashes by measuring the split S(3) EPR signal induced by near-infrared illumination at 5 K. The decay of the S(2) state was followed in samples exposed to one flash by measuring the S(2) state multiline EPR signal. During the decay of the S(3) state, the S(2) state multiline EPR signal first increased and then decreased in amplitude. This shows that the decay of the S(3) state to the S(1) state occurs via the S(2) state. The decay of the S(3) state was biexponential with a fast kinetic phase with a few seconds decay half-time. This occurred in 10-20% of the PSII centers. The slow kinetic phase ranged from a decay half-time of 700 s (at 1 °C) to ~100 s (at 20 °C) in the remaining 80-90% of the centers. The decay of the S(2) state was also biphasic and showed quite similar kinetics to the decay of the S(3) state. Our experiments show that the auxiliary electron donor Y(D) was oxidized during the entire experiment. Thus, the reduced form of Y(D) does not participate to the fast decay of the S(2) and S(3) states we describe here. Instead, we suggest that the decay of the S(3) and S(2) states reflects electron transfer from the acceptor side of PSII to the donor side of PSII starting in the corresponding S state. It is proposed that this exists in equilibrium with Y(Z) according to S(3)Y(Z) ? S(2)Y(Z)(?) in the case of the S(3) state decay and S(2)Y(Z) ? S(1)Y(Z)(?) in the case of the S(2) state decay. Two kinetic models are discussed, both developed with the assumption that the slow decay of the S(3) and S(2) states occurs in PSII centers where Y(Z) is also a fast donor to P(680)(+) working in the nanosecond time regime and that the fast decay of the S(3) and S(2) states occurs in centers where Y(Z) reduces P(680)(+) with slower microsecond kinetics. Our measurements also demonstrate that the split S(3) EPR signal can be used as a direct probe to the S(3) state and that it can provide important information about the redox properties of the S(3) state.     

pH dependence of the donor side reactions in Ca2+-depleted photosystem II

We have studied how low pH affects the water-oxidizing complex in Photosystem II when depleted of the essential Ca(2+) ion cofactor. For these samples, it was found that the EPR signal from the Y(Z)(*) radical decays faster at low pH than at high pH. At 20 degrees C, Y(Z)(*) decays with biphasic kinetics. At pH 6.5, the fast phase encompasses about 65% of the amplitude and has a lifetime of approximately 0.8 s, while the slow phase has a lifetime of approximately 22 s. At pH 3.9, the kinetics become totally dominated by the fast phase, with more than 90% of the signal intensity operating with a lifetime of approximately 0.3 s. The kinetic changes occurred with an approximate pK(a) of 4.5. Low pH also affected the induction of the so-called split radical EPR signal from the S(2)Y(Z)(*) state that is induced in Ca(2+)-depleted PSII membranes because of an inability of Y(Z)(*) to oxidize the S(2) state. At pH 4.5, about 50% of the split signal was induced, as compared to the amplitude of the signal that was induced at pH 6.5-7, using similar illumination conditions. Thus, the split-signal induction decreased with an apparent pK(a) of 4.5. In the same samples, the stable multiline signal from the S(2) state, which is modified by the removal of Ca(2+), was decreased by the illumination to the same extent at all pHs. It is proposed that decreased induction of the S(2)Y(Z)(*) state at lower pH was not due to inability to oxidize the modified S(2) state induced by the Ca(2+) depletion. Instead, we propose that the low pH makes Y(Z)(*) able to oxidize the S(2) state, making the S(2) --> S(3) transition available in Ca(2+)-depleted PSII. Implications of these results for the catalytic role of Ca(2+) and the role of proton transfer between the Mn cluster and Y(Z) during oxygen evolution is discussed.     

An EPR study of the pH dependence of formate effects on Photosystem II.

Effects of formate on rates of O(2) evolution and electron paramagnetic resonance (EPR) signals were observed in the oxygen evolving PS II membranes as a function of pH. In formate treated PS II membranes, decrease in pH value resulted in the inhibition of the O(2) evolving activity, a decrease in the intensity of S(2) state multiline signal but an increase in the intensity of the Q(A)(-)Fe(2+) EPR signal. Time-resolved EPR study of the Y(Z)(*) decay kinetics showed that the light-induced intensity of Y(Z)(*) EPR signal was proportional to the formate concentration. The change in the pH affected both the light-induced intensities and the decay rates of Y(Z)(*), which was found to be faster at lower pH. At 253 K, t(1/e) value of Y(Z)(*) decay kinetics was found to be 8-10 s at pH 6.0 and 18-21 s at pH 5.0. The results presented here indicate that the extent of inhibition at the donor and the acceptor side of PS II due to formate is pH dependent, being more effective at lower pH.     

Formation of split electron paramagnetic resonance signals in photosystem II suggests that tyrosine(Z) can be photooxidized at 5 K in the S0 and S1 states of the oxygen-evolving complex

The effect of illumination at 5 K of photosystem II in different S-states was investigated with EPR spectroscopy. Two split radical EPR signals around g approximately 2.0 were observed from samples given 0 and 3 flashes, respectively. The signal from the 0-flash sample was narrow, with a width of approximately 80 G, in which the low-field peak can be distinguished. This signal oscillated with the S(1) state in the sample. The signal from the 3-flash sample was broad, with a symmetric shape of approximately 160 G width from peak to trough. This signal varied with the concentration of the S(0) state in the sample. Both signals are assigned to arise from the donor side of PSII. Both signals relaxed fast, were formed within 10 ms after a flash, and decayed with half-times at 5 K of 3-4 min. The signal in the S(0) state closely resembles split radical signals, originating from magnetic interaction between Y(Z)(*) and the S(2) state, that were first observed in Ca(2+)-depleted photosystem II samples. Therefore, we assign this signal to Y(Z)(*) in magnetic interaction with the S(0) state, Y(Z)(*)S(0). The other signal is assigned to the magnetic interaction between Y(Z)(*) and the S(1) state, Y(Z)(*)S(1). An important implication is that Y(Z) can be oxidized at 5 K in the S(0) and S(1) states. Oxidation of Y(Z) involves deprotonation of the tyrosine. This is restricted at 5 K, and we therefore suggest that the phenolic proton of Y(Z) is involved in a low-barrier hydrogen bond. This is an unusually short hydrogen bond in which proton movement at very low temperatures can occur.     

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.     

Nitric oxide-induced formation of the S-2 state in the oxygen-evolving complex of photosystem II from Synechococcus elongatus

In spinach photosystem II (PSII) membranes, the tetranuclear manganese cluster of the oxygen-evolving complex (OEC) can be reduced by incubation with nitric oxide at -30 degrees C to a state which is characterized by an Mn(2)(II, III) EPR multiline signal [Sarrou, J., Ioannidis, N., Deligiannakis, Y., and Petrouleas, V. (1998) Biochemistry 37, 3581-3587]. This state was recently assigned to the S(-)(2) state of the OEC [Schansker, G., Goussias, C., Petrouleas, V., and Rutherford, A. W. (2002) Biochemistry 41, 3057-3064]. On the basis of EPR spectroscopy and flash-induced oxygen evolution patterns, we show that a similar reduction process takes place in PSII samples of the thermophilic cyanobacterium Synechococcus elongatus at both -30 and 0 degrees C. An EPR multiline signal, very similar but not identical to that of the S(-)(2) state in spinach, was obtained with monomeric and dimeric PSII core complexes from S. elongatus only after incubation at -30 degrees C. The assignment of this EPR multiline signal to the S(-)(2) state is corroborated by measurements of flash-induced oxygen evolution patterns and detailed fits using extended Kok models. The small reproducible shifts of several low-field peak positions of the S(-)(2) EPR multiline signal in S. elongatus compared to spinach suggest that slight differences in the coordination geometry and/or the ligands of the manganese cluster exist between thermophilic cyanobacteria and higher plants.     

Secondary stabilization reactions and proton-coupled electron transport in photosystem II investigated by electroluminescence and fluorescence spectroscopy

The oxidized primary electron donor in photosystem II, P(680)(+), is reduced in several phases, extending over 4 orders of magnitude in time. Especially the slower phases may reflect the back-pressure exerted by water oxidation and provide information on the reactions involved. The kinetics of secondary electron-transfer reactions in the microseconds time range after charge separation were investigated in oxygen-evolving thylakoids suspended in H2O or D2O. Flash-induced changes of chlorophyll fluorescence yield and electric field-induced recombination luminescence were decomposed into contributions from oxidation states S(0), S(1), S(2), and S(3) of the oxygen-evolving complex and interpreted in terms of stabilization kinetics of the initial charge-separated state S(j)Y(Z)P(680)(+)Q(A)(-)Q(B). In approximately 10% of the centers, only charge recombination took place. Otherwise, no static heterogeneity was involved in the microsecond reduction of P(680)(+) by Y(Z) (stabilization) or Q(A)(-) (recombination). The recombination component in active centers occurs mainly upon charge separation in S(3), and, in the presence of D2O, in S(2) as well and is tentatively attributed to the presence of Y(Z)(ox)S(j-1) in equilibrium with Y(Z)S(j). A 20-30 micros stabilization occurs in all S-states, but to different extents. Possible mechanisms for this component are discussed. D2O was found to decrease: (i) the rate of the reaction Y(Z)(ox)S(1) to Y(Z)S(2), (ii) the equilibrium constant between P680(+)Y(Z)S(2) and P(680)Y(Z)(ox)S(2), (iii) the rate of the slow phase of P(680)(+) reduction for the S(3) --> S(0) transition, and (iv) the rate of electron transfer from Q(A)(-) to Q(B) /Q(B)(-). The increased 'miss probability' in D2O is due to (iii).     

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.     

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)(?).     

Reduction of the Mn cluster of the water-oxidizing enzyme by nitric oxide: formation of an S(-2) state

The manganese cluster of the oxygen-evolving enzyme of photosystem II is chemically reduced upon interaction with nitric oxide at -30 degrees C. The state formed gives rise to an S = 1/2 multiline EPR signal [Goussias, Ch., Ioannidis, N., and Petrouleas, V. (1997) Biochemistry 36, 9261] that is attributed to a Mn(II)- Mn(III) dimer [Sarrou, J., Ioannidis, N., Deligiannakis, Y., and Petrouleas, V. (1998) Biochemistry 37, 3581]. In this work, we sought to establish whether the state could be assigned to a specific, reduced S state by using flash oxymetry, chlorophyll a fluorescence, and electron paramagnetic resonance spectroscopy. With the Joliot-type O(2) electrode, the first maximum of oxygen evolution was observed on the sixth or seventh flash. Three saturating pre-flashes were required to convert the flash pattern characteristic of NO-reduced samples to that of the untreated control (i.e., O(2) evolution maximum on the third flash). Measurements of the S state-dependent level of chlorophyll fluorescence in NO-treated PSII showed a three-flash downshift compared to untreated controls. In the EPR study, the maximum S(2) multi-line EPR signal was observed after the fourth flash. The results from all three methods are consistent with the Mn cluster being in a redox state corresponding to an S(-2) state in a majority of centers after treatment with NO. We were unable to generate the Mn(II)-Mn(III) multi-line signal using hydrazine as a reductant; it appears that the valence distribution and possibly the structure of the Mn cluster in the S(-2) state are dependent on the nature of the reductant that is used.     

EPR studies of the water oxidizing complex in the S1 and the higher S states: the manganese cluster and Y(Z) radical

The parallel polarization electron paramagnetic resonance (EPR) method has been applied to investigate manganese EPR signals of native S1 and S3 states of the water oxidizing complex (WOC) in photosystem (PS) II. The EPR signals in both states were assigned to thermally excited states with S=1, from which zero-field interaction parameters D and E were derived. Three kinds of signals, the doublet signal, the singlet-like signal and g=11-15 signal, were detected in Ca2+-depleted PS II. The g=11-15 signal was observed by parallel and perpendicular modes and assigned to a higher oxidation state beyond S2 in Ca2+-depleted PS II. The singlet-like signal was associated with the g=11-15 signal but not with the Y(Z) (the tyrosine residue 161 of the D1 polypeptide in PS II) radical. The doublet signal was associated with the Y(Z) radical as proved by pulsed electron nuclear double resonance (ENDOR) and ENDOR-induced EPR. The electron transfer mechanism relevant to the role of Y(Z) radical was discussed.     

Glutamate 189 of the D1 polypeptide modulates the magnetic and redox properties of the manganese cluster and tyrosine Y(Z) in photosystem II

Recent models for water oxidation in photosystem II postulate that the tyrosine Y(Z) radical, Y(Z)(*), abstracts both an electron and a proton from the Mn cluster during one or more steps in the catalytic cycle. This coupling of proton- and electron-transfer events is postulated to provide the necessary driving force for oxidizing the Mn cluster in its higher oxidation states. The formation of Y(Z)(*) requires the deprotonation of Y(Z) by His190 of the D1 polypeptide. For Y(Z)(*) to abstract both an electron and a proton from the Mn cluster, the proton abstracted from Y(Z) must be transferred rapidly from D1-His190 to the lumenal surface via one or more proton-transfer pathways. The proton acceptor for D1-His190 has been proposed to be either Glu189 of the D1 polypeptide or a group positioned by this residue. To further define the role of D1-Glu189, 17 D1-Glu189 mutations were constructed in the cyanobacterium Synechocystis sp. PCC 6803. Several of these mutants are of particular interest because they appear to assemble Mn clusters in 70-80% of reaction centers in vivo, but evolve no O(2). The EPR and electron-transfer properties of PSII particles isolated from the D1-E189Q, D1-E189L, D1-E189D, D1-E189N, D1-E189H, D1-E189G, and D1-E189S mutants were examined. Intact PSII particles isolated from mutants that evolved no O(2) also exhibited no S(1) or S(2) state multiline EPR signals and were unable to advance beyond an altered Y(Z)(*)S(2) state, as shown by the accumulation of narrow "split" EPR signals under multiple turnover conditions. In the D1-E189G and D1-E189S mutants, the quantum yield for oxidizing the S(1) state Mn cluster was very low, corresponding to a > or =1400-fold slowing of the rate of Mn oxidation by Y(Z)(*). In Mn-depleted D1-Glu189 mutant PSII particles, charge recombination between Q(A)(*)(-) and Y(Z)(*) in the mutants was accelerated, showing that the mutations alter the redox properties of Y(Z) in addition to those of the Mn cluster. These results are consistent with D1-Glu189 participating in a network of hydrogen bonds that modulates the properties of both Y(Z) and the Mn cluster and are consistent with proposals that D1-Glu189 positions a group that accepts a proton from D1-His190.     

Ca(2+) function in photosynthetic oxygen evolution studied by alkali metal cations substitution

Effects of adding monovalent alkali metal cations to Ca(2+)-depleted photosystem (PS)II membranes on the biochemical and spectroscopic properties of the oxygen-evolving complex were studied. The Ca(2+)-dependent oxygen evolution was competitively inhibited by K(+), Rb(+), and Cs(+), the ionic radii of which are larger than the radius of Ca(2+) but not inhibited significantly by Li(+) and Na(+), the ionic radii of which are smaller than that of Ca(2+). Ca(2+)-depleted membranes without metal cation supplementation showed normal S(2) multiline electron paramagnetic resonance (EPR) signal and an S(2)Q(A)(-) thermoluminescence (TL) band with a normal peak temperature after illumination under conditions for single turnover of PSII. Membranes supplemented with Li(+) or Na(+) showed properties similar to those of the Ca(2+)-depleted membranes, except for a small difference in the TL peak temperatures. The peak temperature of the TL band of membranes supplemented with K(+), Rb(+), or Cs(+) was elevated to approximately 38 degrees C which coincided with that of Y(D)(+)Q(A)(-) TL band, and no S(2) EPR signals were detected. The K(+)-induced high-temperature TL band and the S(2)Q(A)(-) TL band were interconvertible by the addition of K(+) or Ca(2+) in the dark. Both the Ca(2+)-depleted and the K(+)-substituted membranes showed the narrow EPR signal corresponding to the S(2)Y(Z)(+) state at g = 2 by illuminating the membranes under multiple turnover conditions. These results indicate that the ionic radii of the cations occupying Ca(2+)-binding site crucially affect the properties of the manganese cluster.     

EPR/ENDOR characterization of the physical and electronic structure of the OEC Mn cluster

Electron paramagnetic resonance (EPR) spectroscopy has often played a crucial role in characterizing the various cofactors and processes of photosynthesis, and photosystem II and its oxygen evolving chemistry is no exception. Until recently, the application of EPR spectroscopy to the characterization of the oxygen evolving complex (OEC) has been limited to the S2-state of the Kok cycle. However, in the past few years, continuous wave-EPR signals have been obtained for both the S0- and S1-state as well as for the S2 (radical)(Z)-state of a number of inhibited systems. Furthermore, the pulsed EPR technique of electron spin echo electron nuclear double resonance spectroscopy has been used to directly probe the 55Mn nuclei of the manganese cluster. In this review, we discuss how the EPR data obtained from each of these states of the OEC Kok cycle are being used to provide insight into the physical and electronic structure of the manganese cluster and its interaction with the key tyrosine, Y(Z).     

Molecular interference of Cd(2+) with Photosystem II

Many heavy metals inhibit electron transfer reactions in Photosystem II (PSII). Cd(2+) is known to exchange, with high affinity in a slow reaction, for the Ca(2+) cofactor in the Ca/Mn cluster that constitutes the oxygen-evolving center. This results in inhibition of photosynthetic oxygen evolution. There are also indications that Cd(2+) binds to other sites in PSII, potentially to proton channels in analogy to heavy metal binding in photosynthetic reaction centers from purple bacteria. In search for the effects of Cd(2+)-binding to those sites, we have studied how Cd(2+) affects electron transfer reactions in PSII after short incubation times and in sites, which interact with Cd(2+) with low affinity. Overall electron transfer and partial electron transfer were studied by a combination of EPR spectroscopy of individual redox components, flash-induced variable fluorescence and steady state oxygen evolution measurements. Several effects of Cd(2+) were observed: (i) the amplitude of the flash-induced variable fluorescence was lost indicating that electron transfer from Y(Z) to P(680)(+) was inhibited; (ii) Q(A)(-) to Q(B) electron transfer was slowed down; (iii) the S(2) state multiline EPR signal was not observable; (iv) steady state oxygen evolution was inhibited in both a high-affinity and a low-affinity site; (v) the spectral shape of the EPR signal from Q(A)(-)Fe(2+) was modified but its amplitude was not sensitive to the presence of Cd(2+). In addition, the presence of both Ca(2+) and DCMU abolished Cd(2+)-induced effects partially and in different sites. The number of sites for Cd(2+) binding and the possible nature of these sites are discussed.