Iron bound to the high-affinity Mn-binding site of the oxygen-evolving complex shifts the pK of a component controlling electron transport via Y(Z)

Flash-probe fluorescence spectroscopy was used to compare the pH dependence of charge recombination between Y(Z)(*) and Q(a)(-) in Mn-depleted, photosystem II membranes [PSII(-Mn)] and in membranes with the high-affinity (HA(Z)) Mn-binding site blocked by iron [PSII(-Mn,+Fe); Semin, B. K., Ghirardi, M. L., and Seibert, M. (2002) Biochemistry 41, 5854-5864]. The apparent half-time for fluorescence decay (t(1/2)) in PSII(-Mn) increased from 9 ms at pH 4.4 to 75 ms at pH 9.0 [with an apparent pK (pK(app)) of 7.1]. The actual fluorescence decay kinetics can be fit to one exponential component at pH <6.0 (t(1/2) = 9.5 ms), but it requires an additional component at pH >6.0 (t(1/2) = 385 ms). Similar measurements with PSII(-Mn,+Fe) membranes show that iron binding has little effect on the maximum and minimum t(1/2) values measured at alkaline and acidic pHs but that it does shift the pK(app) from 7.1 to 6.1 toward the more acidic pK(app) value typical of intact membranes. Light-induced Fe(II) blocking of the PSII(-Mn) membrane is accompanied by a decrease in buffer Fe(II) concentration. This decrease was not the result of Fe(II) binding, but rather of its oxidation at two sites, the HA(Z) site and the low-affinity site. M?ssbauer spectroscopy at 80 K on PSII(-Mn,+Fe) samples, prepared under conditions providing the maximal blocking effect but minimizing the amount of nonspecifically bound iron cations, supports this conclusion since this method detected only Fe(III) cations bound to the membranes. Correlation of the kinetics of Fe(II) oxidation with the blocking parameters showed that blocking occurs after four to five Fe(II) cations were oxidized at the HA(Z) site. In summary, the blocking of the HA(Z) Mn-binding site by iron in PSII(-Mn) membranes not only prevents the access of exogenous donors to Y(Z) but also partially restores the properties of the hydrogen bond net found in intact PS(II), which in turn controls the rate of electron transport to Y(Z).     

Blocking of electron donation by Mn(II) to Y(Z*) following incubation of Mn-depleted photosystem II membranes with Fe(II) in the light

The donation of electrons by Mn(II) and Fe(II) to Y(Z*) through the high-affinity (HA(Z)) site in Mn-depleted photosystem II (PSII) membranes has been studied by flash-probe fluorescence yield measurements. Mn(II) and Fe(II) donate electrons to Y(Z*) with about the same efficiency, saturating this reaction at the same concentration (ca. 5 microM). However, following a short incubation of the membranes with 5 microM Fe(II), but not with Mn(II) in room light, added Mn(II) or Fe(II) can no longer be photooxidized by Y(Z)(*). This blocking effect is caused by specifically bound, photooxidized Fe [> or =Fe(III)] and is accompanied by a delay in the fluorescence yield decay kinetics attributed to the slowing down of the charge recombination rate between Q(a-) and Y(Z*). Exogenously added Fe(III), on the other hand, does not donate electrons to Y(Z*), does not block the donation of electrons by added Mn(II) and Fe(II), and does not change the kinetics of the decay of the fluorescence yield. These results demonstrate that the light-dependent oxidation of Fe(II) by Y(Z*) creates an Fe species that binds at the HA(Z) site and causes the blocking effect. The pH dependence of Mn(II) electron donation to Y(Z*) via the HA(Z) site and of the Fe-blocking effect is different. These results, together with sequence homologies between the C-terminal ends of the D1 and D2 polypeptides of the PSII reaction center and several diiron-oxo enzymes, suggest the involvement of two or perhaps more (to an upper limit of four to five) bound iron cations per reaction center of PSII in the blocking effect. Similarities in the interaction of Fe(II) and Mn(II) with the HA(Z) Mn site of PSII during the initial steps of the photoactivation process are discussed. The Fe-blocking effect was also used to investigate the relationship between the HA(Z) Mn site and the HA sites on PSII for diphenylcarbazide (DPC) and NH2OH oxidation. Blocking of the HA(Z) site with specifically bound Fe leads to the total inhibition of electron donation to Y(Z*) by DPC. Since DPC and Mn(II) donation to PSII is noncompetitive [Preston, C., and Seibert, M. (1991) Biochemistry 30, 9615-9624], the Fe bound to the HA(Z) site can also block the DPC donation site. On the other hand, electron donation by NH2OH to PSII still occurs in Fe-blocked membranes. Since hydroxylamine does not reduce the Fe [> or =Fe(III)] specifically bound to the HA(Z) site, NH2OH must donate to Y(Z*) through its own site or directly to P680+.     

A carboxylic residue at the high-affinity, Mn-binding site participates in the binding of iron cations that block the site

The role of carboxylic residues at the high-affinity, Mn-binding site in the ligation of iron cations blocking the site [Biochemistry 41 (2000) 5854] was studied, using a method developed to extract the iron cations blocking the site. We found that specifically bound Fe(III) cations can be extracted with citrate buffer at pH 3.0. Furthermore, citrate can also prevent the photooxidation of Fe(II) cations by YZ. Participation of a COOH group(s) in the ligation of Fe(III) at the high-affinity site was investigated using 1-ethyl-3-[(3-dimethylamino)propyl] carbodiimide (EDC), a chemical modifier of carboxylic amino acid residues. Modification of the COOH groups inhibits the light-induced oxidation of exogenous Mn(II) cations by Mn-depleted photosystem II (PSII[-Mn]) membranes. The rate of Mn(II) oxidation saturates at > or = 10 microM in PSII(-Mn) membranes and > or = 500 microM in EDC-treated PSII (-Mn) samples. Intact PSII(-Mn) membranes have only one site for Mn(II) oxidation via YZ (dissociation constant, Kd = 0.64 microM), while EDC-treated PSII(-Mn) samples have two sites (Kd = 1.52 and 22 microM; the latter is the low-affinity site). When PSII(-Mn) membranes were incubated with Fe(II) before modifier treatment (to block the high-affinity site) and the blocking iron cations were extracted with citrate (pH 3.0) after modification, the membranes contained only one site (Kd = 2.3 microM) for exogenous Mn(II) oxidation by Y(Z)() radical. In this case, the rate of electron donation via YZ saturated at a Mn(II) concentration > or = 15 microM. These results indicate that the carboxylic residue participating in Mn(II) coordination and the binding of oxidized manganese cations at the HAZ site is protected from the action of the modifier by the iron cations blocking the HAZ site. We concluded that the carboxylic residue (D1 Asp-170) participating in the coordination of the manganese cation at the HAZ site (Mn4 in the tetranuclear manganese cluster [Science 303 (2004) 1831]) is also involved in the ligation of the Fe cation(s) blocking the high-affinity Mn-binding site.     

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.     

The differences in microenvironments and functions of tyrosine radicals YZ and YD in photosystem II studied by EPR

Electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) were performed to investigate the difference in microenvironments and functions between tyrosine Z (Y(Z)) and tyrosine D (Y(D)). Mn-depletion or Ca(2+)-depletion causes extension of the lifetime of tyrosine radical Y(Z)(*), which can be trapped by rapid freezing after illumination at about 250 K. Above pH 6.5, Y(Z)(*) radical in Mn-depleted PS II shows similar EPR and ENDOR spectra similar to that of Y(D)(*) radical, which are ascribed to a typical neutral tyrosine radical. Below pH 6.5, Y(Z)(*) radical shows quite different EPR and ENDOR spectra. ENDOR spectra show the spin density distribution of the low-pH form of Y(Z)(*) that has been quite different from the high-pH form of Y(Z)(*). The spin density distribution of the low-pH Y(Z)(*) can be explained by a cation radical or the neutral radical induced by strong electrostatic interaction. The pH dependence of the activation energy of the recombination rate between Y(Z)(*) and Q(A)(-) shows a gap of 4.4 kJ/mol at pH 6.0-6.5. In the Ca(2+)-depleted PS II, Y(Z)(*) signal was the mixture of the cation-like and normal neutral radicals, and the pH dependence of Y(Z)(*) spectrum in Ca(2+)-depleted PS II is considerably different from the neutral radical found in Mn-depleted PS II. Based on the recent structure data of cyanobacterial PS II, the pH dependence of Y(Z)(*) could be ascribed to the modification of the local structure and hydrogen-bonding network induced by the dissociation of ASP170 near Y(Z).     

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.     

Histidine 332 of the D1 polypeptide modulates the magnetic and redox properties of the manganese cluster and tyrosine Y(Z) in photosystem II

An electron spin-echo envelope modulation study [Tang, X.-S., Diner, B. A., Larsen, B. S., Gilchrist, M. L., Jr., Lorigan, G. A., and Britt, R. D. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 704-708] and a recent Fourier transform infrared study [Noguchi, T., Inoue, Y., and Tang, X.-S. (1999) Biochemistry 38, 10187-10195], both conducted with [(15)N]histidine-labeled photosystem II particles, show that at least one histidine residue coordinates the O(2)-evolving Mn cluster in photosystem II. Evidence obtained from site-directed mutagenesis studies suggests that one of these residues may be His332 of the D1 polypeptide. The mutation D1-H332E is of particular interest because cells of the cyanobacterium Synechocystis sp. PCC 6803 that contain this mutation evolve no O(2) but appear to assemble Mn clusters in nearly all photosystem II reaction centers [Chu, H.-A., Nguyen, A. P. , and Debus, R. J. (1995) Biochemistry 34, 5859-5882]. Photosystem II particles isolated from the Synechocystis D1-H332E mutant are characterized in this study. Intact D1-H332E photosystem II particles exhibit an altered S(2) state multiline EPR signal that has more hyperfine lines and narrower splittings than the S(2) state multiline EPR signal observed in wild-type PSII particles. However, the quantum yield for oxidizing the S(1) state Mn cluster is very low, corresponding to an 8000-fold slowing of the rate of Mn oxidation by Y(Z)(*), and the temperature threshold for forming the S(2) state is approximately 100 K higher than in wild-type PSII preparations. Furthermore, the D1-H332E PSII particles are unable to advance beyond the Y(Z)(*)S(2) state, as shown by the accumulation of a narrow "split" EPR signal under multiple turnover conditions. In Mn-depleted photosystem II particles, charge recombination between Q(A)(*)(-) and Y(Z)(*) in D1-H332E is accelerated in comparison to wild-type, showing that the mutation alters the redox properties of Y(Z) in addition to those of the Mn cluster. These results are consistent with D1-His332 being located near the Mn-Y(Z) complex and perhaps ligating Mn.     

Orientation of the tetranuclear manganese cluster and tyrosine Z in the O(2)-evolving complex of photosystem II: An EPR study of the S(2)Y(Z)(*) state in oriented acetate-inhibited photosystem II membranes.

Inhibitory treatment by acetate, followed by illumination and rapid freezing, is known to trap the S(2)Y(Z)(*) state of the O(2)-evolving complex (OEC) in photosystem II (PS II). An EPR spectrum of this state exhibits broad split signals due to the interaction of the tyrosyl radical, Y(Z)(*), with the S = 1/2 S(2) state of the Mn(4) cluster. We present a novel approach to analyze S(2)Y(Z)(*) spectra of one-dimensionally (1-D) oriented acetate-inhibited PS II membranes to determine the magnitude and relative orientation of the S(2)Y(Z)(*) dipolar vector within the membrane. Although there exists a vast body of EPR data on isolated spins in oriented membrane sheets, the present study is the first of its kind on dipolar-coupled electron spin pairs in such systems. We demonstrate the feasibility of the technique and establish a rigorous treatment to account for the disorder present in partially oriented 1-D membrane preparations. We find that (i) the point-dipole distance between Y(Z)(*) and the Mn(4) cluster is 7.9 +/- 0.2 A, (ii) the angle between the interspin vector and the thylakoid membrane normal is 75 degrees, (iii) the g(z)()-axis of the Mn(4) cluster is 70 degrees away from the membrane normal and 35 degrees away from the interspin vector, and (iv) the exchange interaction between the two spins is -275 x 10(-)(4) cm(-)(1), which is antiferromagnetic. Due to the sensitivity of EPR line shapes of oriented spin-coupled pairs to the interspin distance, the present study imposes a tighter constraint on the Y(Z)-Mn(4) point-dipole distance than obtained from randomly oriented samples. The geometric constraints obtained from the 1-D oriented sample are combined with published models of the structure of Mn-depleted PS II to propose a location of the Mn(4) cluster. A structure in which Y(Z) is hydrogen bonded to a manganese-bound hydroxide ligand is consistent with available data and favors maximal orbital overlap between the two redox center that would facilitate direct electron- and proton-transfer steps.     

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.     

Light-adaptation of photosystem II is mediated by the plastoquinone pool

During the first few enzymatic turnovers after dark-adaptation of photosystem II (PSII), the relaxation rate of the EPR signals from the Mn cluster and Y(D)(*) are significantly enhanced. This light-adaptation process has been suggested to involve the appearance of a new paramagnet on the PSII donor side [Peterson, S., Ahrling, K., H?gblom, J., and Styring, S. (2003) Biochemistry 42, 2748-2758]. In the present study, a correlation is established between the observed relaxation enhancement and the redox state of the quinone pool. It is shown that the addition of quinol to dark-adapted PSII membrane fragments induces relaxation enhancement already after a single oxidation of the Mn, comparable to that observed after five oxidations in samples with quinones (PPBQ or DQ) added. The saturation behavior of Y(D)(*) revealed that with quinol added in the dark, a single flash was necessary for the relaxation enhancement to occur. The quinol-induced relaxation enhancement of PSII was also activated by illumination at 200 K. Whole thylakoids, with no artificial electron acceptor present but with an intact plastoquinone pool, displayed the same relaxation enhancement on the fifth flash as membrane fragments with exogenous quinones present. We conclude that (i) reduction of the quinone pool induces the relaxation enhancement of the PSII donor-side paramagnets, (ii) light is required for the quinol to effect the relaxation enhancement, and (iii) light-adaptation occurs in the intact thylakoid system, when the endogenous plastoquinone pool is gradually reduced by PSII turnover. It seems clear that a species on the PSII donor side is reduced by the quinol, to become a potent paramagnetic relaxer. On the basis of XANES reports, we suggest that this species may be the Mn ions not involved in the cyclic redox changes of the oxygen-evolving complex.     

Role of D1-His190 in the proton-coupled oxidation of tyrosine YZ in manganese-depleted photosystem II.

To further characterize the role of D1-His190 in the oxidation of tyrosine Y(Z) in photosystem II, the pH dependence of P(680)(*)()(+) reduction was measured in H190A and Mn-depleted wild-type PSII particles isolated from the cyanobacterium, Synechocystis sp. PCC 6803. Measurements were conducted in the presence and absence of imidazole and other small organic bases. In H190A PSII particles, rapid reduction of P(680)(*)()(+) attributed to electron transfer from Y(Z) increased dramatically above pH 9, with an apparent pK(A) of approximately 10.3. In the presence of ethanolamine and imidazole, this dramatic increase occurred at lower pH values, with the efficiency of Y(Z) oxidation correlating with the solution pK(A) value of the added base. We conclude that the pK(A) of Y(Z) is approximately 10.3 in D1-H190A PSII particles. In Mn-depleted wild-type PSII particles, P(680)(*)()(+) reduction was accelerated by all exogenous bases examined (substituted imidazoles, histidine, Tris, and 1,4-diazabicyclo[2.2.2]octane). We conclude that Y(Z) is solvent accessible in Mn-depleted wild-type PSII particles and that its pK(A) is near that of tyrosine in solution. In Mn-depleted wild-type PSII particles, over 80% of the kinetics of P(680)(*)()(+) reduction after a flash could be described by three kinetic components. The individual rate constants of these components varied slightly with pH, but their relative proportions varied dramatically with pH, showing apparent pK(A) values of 7.5 and 6.25 (6.9 and 5.8 in the presence of Ca(2+) and Mg(2+) ions). An additional pK(A) value (pK(A) < 4.5) may also be present. To describe these data, we propose (1) the pK(A) of His190 is 6.9-7.5, depending on buffer ions, (2) the deprotonation of Y(Z) is facilitated by the transient formation of a either a hydrogen bond or a hydrogen-bonded water bridge between Y(Z) and D1-His190, and (3) when protonated, D1-His190 interacts with nearby residues having pK(A) values near 6 and 4. Because Y(Z) and D1-His190 are located near the Mn cluster, these residues may interact with the Mn cluster in the intact system.     

Does histidine 332 of the D1 polypeptide ligate the manganese cluster in photosystem II? An electron spin echo envelope modulation study

The tetranuclear manganese cluster in photosystem II is ligated by one or more histidine residues, as shown by an electron spin echo envelope modulation (ESEEM) study conducted with [(15)N]histidine-labeled photosystem II particles isolated from the cyanobacterium Synechocystis sp. strain PCC 6803 [Tang, X.-S., Diner, B. A., Larsen, B. S., Gilchrist, M. L., Jr., Lorigan, G. A., and Britt, R. D. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 704-708]. One of these residues may be His332 of the D1 polypeptide. Photosystem II particles isolated from the Synechocystis mutant D1-H332E exhibit an altered S(2) state multiline EPR signal that has more hyperfine lines and narrower splittings than the corresponding signal in wild-type PSII particles [Debus, R. J., Campbell, K. A., Peloquin, J. M., Pham, D. P., and Britt, R. D. (2000) Biochemistry 39, 470-478]. These D1-H332E PSII particles are also unable to advance beyond an altered S(2)Y(Z)(*) state, and the quantum yield for forming the S(2) state is very low, corresponding to an 8000-fold slowing of the rate of Mn oxidation by Y(Z)(*). These observations are consistent with His332 being close to the Mn cluster and modulating the redox properties of both the Mn cluster and tyrosine Y(Z). To determine if D1-His332 ligates the Mn cluster, we have conducted an ESEEM study of D1-H332E PSII particles. The histidyl nitrogen modulation observed near 5 MHz in ESEEM spectra of the S(2) state multiline EPR signal of wild-type PSII particles is substantially diminished in D1-H332E PSII particles. This result is consistent with ligation of the Mn cluster by D1-His332. However, alternate explanations are possible. These are presented and discussed.     

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.     

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.     

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)(?).     

Ca2+ effects on Fe(II) interactions with Mn-binding sites in Mn-depleted oxygen-evolving complexes of photosystem II and on Fe replacement of Mn in Mn-containing,Ca-depleted complexes

Fe(II) cations bind with high efficiency and specificity at the high-affinity (HA), Mn-binding site (termed the “blocking effect” since Fe blocks further electron donation to the site) of the oxygen-evolving complex (OEC) in Mn-depleted, photosystem II (PSII) membrane fragments (Semin et al. in Biochemistry 41:5854, 2002). Furthermore, Fe(II) cations can substitute for 1 or 2Mn cations (pH dependent) in Ca-depleted PSII membranes (Semin et al. in Journal of Bioenergetics and Biomembranes 48:227, 2016; Semin et al. in Journal of Photochemistry and Photobiology B 178:192, 2018). In the current study, we examined the effect of Ca²⁺ cations on the interaction of Fe(II) ions with Mn-depleted [PSII(-Mn)] and Ca-depleted [PSII(-Ca)] photosystem II membranes. We found that Ca²⁺ cations (about 50 mM) inhibit the light-dependent oxidation of Fe(II) (5 µM) by about 25% in PSII(-Mn) membranes, whereas inhibition of the blocking process is greater at about 40%. Blocking of the HA site by Fe cations also decreases the rate of charge recombination between Q_A^? and Y_Z^?+ from t_1/2?=?30 ms to 46 ms. However, Ca²⁺ does not affect the rate during the blocking process. An Fe(II) cation (20 µM) replaces 1Mn cation in the Mn₄CaO₅ catalytic cluster of PSII(-Ca) membranes at pH 5.7 but 2 Mn cations at pH 6.5. In the presence of Ca²⁺ (10 mM) during the substitution process, Fe(II) is not able to extract Mn at pH 5.7 and extracts only 1Mn at pH 6.5 (instead of two without Ca²⁺). Measurements of fluorescence induction kinetics support these observations. Inhibition of Mn substitution with Fe(II) cations in the OEC only occurs with Ca²⁺ and Sr²⁺ cations, which are also able to restore oxygen evolution in PSII(-Ca) samples. Nonactive cations like La³⁺, Ni²⁺, Cd²⁺, and Mg²⁺ have no influence on the replacement of Mn with Fe. These results show that the location and/or ligand composition of one Mn cation in the Mn₄CaO₅ cluster is strongly affected by calcium depletion or rebinding and that bound calcium affects the redox potential of the extractable Mn4 cation in the OEC, making it resistant to reduction.

     

Electron paramagnetic resonance study of the electron transfer reactions in photosystem II membrane preparations from Arabidopsis thaliana

Arabidopsis thaliana is widely used as a model organism in plant biology as its genome has been sequenced and transformation is known to be efficient. A large number of mutant lines and genomic resources are available for Arabidopsis. All this makes Arabidopsis a useful tool for studies of photosynthetic reactions in higher plants. In this study, photosystem II (PSII) enriched membranes were successfully isolated from thylakoids of Arabidopsis plants and for the first time the electron transfer cofactors in PSII were systematically studied using electron paramagnetic resonance (EPR) spectroscopy. EPR signals from both of the donor and acceptor sides of PSII, as well as from auxiliary electron donors were recorded. From the acceptor side of PSII, EPR signals from Q(A)- Fe2(+) and Phe- Q(A)- Fe2(+) as well as from the free Phe- radical were observed. The multiline EPR signals from the S?- and S?-states of CaMn?O(x)-cluster in the water oxidation complex were characterized. Moreover, split EPR signals, the interaction signals from Y(Z) and CaMn?O(x)-cluster in the S?-, S?-, S?-, and the S?-state were induced by illumination of the PSII membranes at 5K and characterized. In addition, EPR signals from auxiliary donors Y(D), Chl(+) and cytochrome b??? were observed. In total, we were able to detect about 20 different EPR signals covering all electron transfer components in PSII. Use of this spectroscopic platform opens a possibility to study PSII reactions in the library of mutants available in Arabidopsis.     

Kinetics of dissociation of reduced nicotinamide adenine dinucleotide phosphate from its complexes with malic enzyme in relation to substrate inhibition and half-of-the-sites reactivity

Malic enzyme of pigeon liver binds NADPH at four equivalent enzyme sites and binds Mn2+ and malate each at two sets of "tight" and "weak" sites with negative cooperativity [Pry, T. A., & Hsu, R. Y. (1980) Biochemistry 19, 951-962]. Stopped-flow studies on the displacement of NADPH from the malate-enzyme complexes E4-NADPH4, E4-Mn2(2+)-NADPH4, E4-Mn2(2+)-NADPH4-dimalate, and E4-Mn2(2+)-NADPH4-tetramalate by large excess NADP+ or its analogue phosphoadenosine(2')diphospho(5')ribose show that NADPH dissociates from the binary complex rapidly with a first-order rate constant of 427 s-1. Dissociation from the ternary E4-Mn2(2+)-NADPH4 complex containing two tightly bound Mn2+ ions can be described by a single first-order process with a rate constant of 135 s-1, or more satisfactorily by two simultaneous first-order processes attributable to the reactions of Mn2+-deficient (k congruent to 427 s-1) and Mn2+-liganded (k = 96 s-1) subunits. The latter equals twice the maximum steady-state turnover rate of 53.2 + 3.0 s-1 assigned to dissociation of the reduced nucleotide from transient E-Mn2+-NADPH, and this 2:1 ratio strongly supports our proposed "half-of-the-sites" model [Hsu, R. Y., & Pry, T. A. (1980) Biochemistry 19, 962-968]. Dissociation from the E4-Mn2(2+)-NADPH4-dimalate complex (k = 100 s-1) follows only the slower process, suggesting that occupancy of malate at two sites tightens enzyme-bound NADPH on the adjacent sites. Binding of malate at two additional weak sites yields E4-Mn2(2+)-NADPH4-tetramalate and a NADPH dissociation rate constant of 2.69 s-1. The 97% decrease in NADPH dissociation parallels the observed 93% maximal inhibition by malate and is the cause of substrate inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)     

Replacement of tyrosine D with phenylalanine affects the normal proton transfer pathways for the reduction of P680+ in oxygen-evolving photosystem II particles from Chlamydomonas

We have probed the electrostatics of P680(+) reduction in oxygenic photosynthesis using histidine-tagged and histidine-tagged Y(D)-less Photosystem II cores. We make two main observations: (i) that His-tagged Chlamydomonas cores show kinetics which are essentially identical to those of Photosystem II enriched thylakoid membranes from spinach; (ii) that the microsecond kinetics, previously shown to be proton/hydrogen transfer limited [Schilstra et al. (1998) Biochemistry 37, 3974-3981], are significantly different in Y(D)-less Chlamydomonas particles when compared with both the His-tagged Chlamydomonas particles and the spinach membranes. The oscillatory nature of the kinetics in both Chlamydomonas samples is normal, indicating that S-state cycling is unaffected by either the histidine-tagging or the replacement of tyrosine D with phenylalanine. We propose that the effects on the proton-coupled electron transfers of P680(+) reduction in the absence of Y(D) are likely to be due to pK shifts of residues in a hydrogen-bonded network of amino acids in the vicinity of Y(Z). Tyrosine D is 35 A from Y(Z) and yet has a significant influence on proton-coupled electron transfer events in the vicinity of Y(Z). This finding emphasizes the delicacy of the proton balance that Photosystem II has to achieve during the water splitting process.     

Oxidation of the non-heme iron complex in photosystem II

In photosystem II (PSII), the redox properties of the non-heme iron complex (Fe complex) are sensitive to the redox state of quinones (Q(A/)(B)), which may relate to the electron/proton transfer. We calculated the redox potentials for one-electron oxidation of the Fe complex in PSII [E(m)(Fe)] based on the reference value E(m)(Fe) = +400 mV at pH 7 in the Q(A)(0)Q(B)(0) state, considering the protein environment in atomic detail and the associated changes in protonation pattern. Our model yields the pH dependence of E(m)(Fe) with -60 mV/pH as observed in experimental redox titration. We observed significant deprotonation at D1-Glu244 in the hydrophilic loop region upon Fe complex oxidation. The calculated pK(a) value for D1-Glu244 depends on the Fe complex redox state, yielding a pK(a) of 7.5 and 5.5 for Fe(2+) and Fe(3+), respectively. To account for the pH dependence of E(m)(Fe), a model involving not only D1-Glu244 but also the other titratable residues (five Glu in the D-de loops and six basic residues near the Fe complex) seems to be needed, implying the existence of a network of residues serving as an internal proton reservoir. Reduction of Q(A/B) yields +302 mV and +268 mV for E(m)(Fe) in the Q(A)(-)Q(B)(0) and Q(A)(0)Q(B)(-) states, respectively. Upon formation of the Q(A)(0)Q(B)(-) state, D1-His252 becomes protonated. Forming Fe(3+)Q(B)H(2) by a proton-coupled electron transfer process from the initial state Fe(2+)Q(B)(-) results in deprotonation of D1-His252. The two EPR signals observed at g = 1.82 and g = 1.9 in the Fe(2+)Q(A)(-) state of PSII may be attributed to D1-His252 with variable and fixed protonation, respectively.