Photosynthetic water oxidation in Synechocystis sp. PCC6803: mutations D1-E189K, R and Q are without influence on electron transfer at the donor side of photosystem II.

The oxygen-evolving manganese cluster (OEC) of photosynthesis is oxidised by the photochemically generated primary oxidant (P(+*)(680)) of photosystem II via a tyrosine residue (Y(Z), Tyr161 on the D1 subunit of Synechocystis sp. PCC6803). The redox span between these components is rather small and probably tuned by protonic equilibria. The very efficient electron transfer from Y(Z) to P(+*)(680) in nanoseconds requires the intactness of a hydrogen bonded network involving Y(Z), D1-His190, and presumably D1-Glu189. We studied photosystem II core particles from photoautotrophic mutants where the residue D1-E189 was replaced by glutamine, arginine and lysine which were expected to electrostatically differ from the glutamate in the wild-type (WT). Surprisingly, the rates of electron transfer from Y(Z) to P(+*)(680) as well as from the OEC to Y(ox)(Z) were the same as in the WT. With the generally assumed proximity between D1-His190 (and thus D1-Glu189) and Y(Z), the lack of any influence on the electron transfer around Y(Z) straightforwardly implies a strongly hydrophobic environment forcing Glu (acid) and Lys, Arg (basic) at position D1-189 into electro-neutrality. As one alternative, D1-Glu189 could be located at such a large distance from the OEC, Y(Z) and P(+*)(680) that a charge on D1-189X does not influence the electron transfer. This seems less likely in the light of the drastic influence of its direct neighbour, D1-His190, on Y(Z) function. Another alternative is that D1-Glu189 is negatively charged, but is located in a cluster of acid/base groups that compensates for an alteration of charge at position 189, leaving the overall net charge unchanged in the Gln, Lys, and Arg mutants.     

No evidence from FTIR difference spectroscopy that glutamate-189 of the D1 polypeptide ligates a Mn ion that undergoes oxidation during the S0 to S1, S1 to S2, or S2 to S3 transitions in photosystem II

In the recent X-ray crystallographic structural models of photosystem II, Glu189 of the D1 polypeptide is assigned as a ligand of the oxygen-evolving Mn(4) cluster. To determine if D1-Glu189 ligates a Mn ion that undergoes oxidation during one or more of the S(0) --> S(1), S(1) --> S(2), and S(2) --> S(3) transitions, the FTIR difference spectra of the individual S-state transitions in D1-E189Q and D1-E189R mutant PSII particles from the cyanobacterium Synechocystis sp. PCC 6803 were compared with those in wild-type PSII particles. Remarkably, the data show that neither mutation significantly alters the mid-frequency regions (1800-1200 cm(-)(1)) of any of the FTIR difference spectra. Importantly, neither mutation eliminates any specific symmetric or asymmetric carboxylate stretching mode that might have been assigned to D1-Glu189. The small spectral alterations that are observed are similar in amplitude to those that are observed in wild-type PSII particles that have been exchanged into FTIR analysis buffer by different methods or those that are observed in D2-H189Q mutant PSII particles (the residue D2-His189 is located >25 A from the Mn(4) cluster and accepts a hydrogen bond from Tyr Y(D)). The absence of significant mutation-induced spectral alterations in the D1-Glu189 mutants shows that the oxidation of the Mn(4) cluster does not alter the frequencies of the carboxylate stretching modes of D1-Glu189 during the S(0) --> S(1), S(1) --> S(2), or S(2) --> S(3) transitions. One explanation of these data is that D1-Glu189 ligates a Mn ion that does not increase its charge or oxidation state during any of these S-state transitions. However, because the same conclusion was reached previously for D1-Asp170, and because the recent X-ray crystallographic structural models assign D1-Asp170 and D1-Glu189 as ligating different Mn ions, this explanation requires that (1) the extra positive charge that develops on the Mn(4) cluster during the S(1) --> S(2) transition be localized on the Mn ion that is ligated by the alpha-COO(-) group of D1-Ala344 and (2) any increase in positive charge that develops on the Mn(4) cluster during the S(0) --> S(1) and S(2) --> S(3) transitions be localized on the one Mn ion that is not ligated by D1-Asp170, D1-Glu189, or D1-Ala344. An alternative explanation of the FTIR data is that D1-Glu189 does not ligate the Mn(4) cluster. This conclusion would be consistent with earlier spectroscopic analyses of D1-Glu189 mutants, but would require that the proximity of D1-Glu189 to manganese in the X-ray crystallographic structural models be an artifact of the radiation-induced reduction of the Mn(4) cluster that occurred during the collection of the X-ray diffraction data.     

Electrostatics and proton transfer in photosynthetic water oxidation

Photosystem II (PSII) oxidizes two water molecules to yield dioxygen plus four protons. Dioxygen is released during the last out of four sequential oxidation steps of the catalytic centre (S(0) --> S(1), S(1) --> S(2), S(2) --> S(3), S(3) --> S(4) --> S(0)). The release of the chemically produced protons is blurred by transient, highly variable and electrostatically triggered proton transfer at the periphery (Bohr effect). The extent of the latter transiently amounts to more than one H(+)/e(-) under certain conditions and this is understood in terms of electrostatics. By kinetic analyses of electron-proton transfer and electrochromism, we discriminated between Bohr-effect and chemically produced protons and arrived at a distribution of the latter over the oxidation steps of 1 : 0 : 1 : 2. During the oxidation of tyr-161 on subunit D1 (Y(Z)), its phenolic proton is not normally released into the bulk. Instead, it is shared with and confined in a hydrogen-bonded cluster. This notion is difficult to reconcile with proposed mechanisms where Y(Z) acts as a hydrogen acceptor for bound water. Only in manganese (Mn) depleted PSII is the proton released into the bulk and this changes the rate of electron transfer between Y(Z) and the primary donor of PSII P(+)(680) from electron to proton controlled. D1-His190, the proposed centre of the hydrogen-bonded cluster around Y(Z), is probably further remote from Y(Z) than previously thought, because substitution of D1-Glu189, its direct neighbour, by Gln, Arg or Lys is without effect on the electron transfer from Y(Z) to P(+)(680) (in nanoseconds) and from the Mn cluster to Y(ox)(Z).     

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.     

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.     

Short hydrogen bond between redox-active tyrosine Y(Z) and D1-His190 in the photosystem II crystal structure

The crystal structure of photosystem II (PSII) analyzed at a resolution of 1.9 ? revealed a remarkably short H-bond between redox-active tyrosine Y(Z) and D1-His190 (2.46 ? donor-acceptor distance). Using large-scale quantum mechanical/molecular mechanical (QM/MM) calculations with the explicit PSII protein environment, we were able to reproduce this remarkably short H-bond in the original geometry of the crystal structure in the neutral [Y(Z)O···H···N(ε)-His-N(δ)H···O═Asn] state, but not in the oxidized states, indicating that the neutral state was the one observed in the crystal structure. In addition to the appropriate redox/protonation state of Y(Z) and D1-His190, we found that the presence of a cluster of water molecules played a key role in shortening the distance between Y(Z) and D1-His190. The orientations of the water molecules in the cluster were energetically stabilized by the highly polarized PSII protein environment, where the Ca ion of the oxygen-evolving complex (OEC) and the OEC ligand D1-Glu189 were also involved.     

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.     

Function of redox-active tyrosine in photosystem II

Water oxidation at photosystem II Mn-cluster is mediated by the redox-active tyrosine Y(Z). We calculated the redox potential (E(m)) of Y(Z) and its symmetrical counterpart Y(D), by solving the linearized Poisson-Boltzmann equation. The calculated E(m)(Y( )/Y(-)) were +926 mV/+694 mV for Y(Z)/Y(D) with the Mn-cluster in S2 state. Together with the asymmetric position of the Mn-cluster relative to Y(Z/D), differences in H-bond network between Y(Z) (Y(Z)/D1-His(190)/D1-Asn(298)) and Y(D) (Y(D)/D2-His(189)/D2-Arg(294)/CP47-Glu(364)) are crucial for E(m)(Y(Z/D)). When D1-His(190) is protonated, corresponding to a thermally activated state, the calculated E(m)(Y(Z)) was +1216 mV, which is as high as the E(m) for P(D1/D2). We observed deprotonation at CP43-Arg(357) upon S-state transition, which may suggest its involvement in the proton exit pathway. E(m)(Y(D)) was affected by formation of P(D2)(+) (but not P(D1)(+)) and sensitive to the protonation state of D2-Arg(180). This points to an electrostatic link between Y(D) and P(D2).     

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.     

Iron-blocking the high-affinity Mn-binding site in photosystem II facilitates identification of the type of hydrogen bond participating in proton-coupled electron transport via YZ

Incubation of Mn-depleted PSII membranes [PSII(-Mn)] with Fe(II) is accompanied by the blocking of Y(Z)(*) at the high-affinity Mn-binding site to exogenous electron donors [Semin et al. (2002) Biochemistry 41, 5854-5864] and a shift of the pK(app) of the hydrogen bond partner for Y(Z) (base B) from 7.1 to 6.1 [Semin, B. K., and Seibert, M. (2004) Biochemistry 43, 6772-6782]. Here we calculate activation energies (E(a)) for Y(Z)(*) reduction in PSII(-Mn) and Fe-blocked PSII(-Mn) samples [PSII(-Mn, +Fe)] from temperature dependencies of the rate constants of the fast and slow components of the flash-probe fluorescence decay kinetics. At pH < pK(app) (e.g., 5.5), the decays are fit with one (fast) component in both types of samples, and E(a) is equal to 42.2 +/- 2.9 kJ/mol in PSII(-Mn) and 46.4 +/- 3.3 kJ/mol in PSII(-Mn, +Fe) membranes. At pH > pK(app), the decay kinetics exhibit an additional slow component in PSII(-Mn, +Fe) membranes (E(a) = 36.1 +/- 7.5 kJ/mol), which is much lower than the E(a) of the corresponding component observed for Y(Z)(*) reduction in PSII(-Mn) samples (48.1 +/- 1.7 kJ/mol). We suggest that the above difference results from the formation of a strong low barrier hydrogen bond (LBHB) between Y(Z) and base B in PSII(-Mn, +Fe) samples. To confirm this, Fe-blocking was performed in D(2)O to insert D(+), which has an energetic barrier distinct from H(+), into the LBHB. Measurement of the pH effects on the rates of Y(Z)(*) reduction in PSII(-Mn, +Fe) samples blocked in D(2)O shows a shift of the pK(app) from 6.1 to 7.6, and an increase in the E(a) of the slow component. This approach was also used to measure the stability of the Y(Z)(*) EPR signal at various temperatures in both kinds of membranes. In PSII(-Mn) membranes, the freeze-trapped Y(Z)(*) radical is stable below 190 K, but half of the Y(Z)(*) EPR signal disappears after a 1-min incubation when the sample is warmed to 253 K. In PSII(-Mn, +Fe) samples, the trapped Y(Z)(*) radical is unstable at a much lower temperature (77 K). However, the insertion of D(+) into the hydrogen bond between Y(Z) and base B during the blocking process increases the temperature stability of the Y(Z)(*) EPR signal at 77 K. Again, these results indicate that Fe-blocking involves Y(Z) in the formation of a LBHB, which in turn is consistent with the suggested existence of a LBHB between Y(Z) and base B in intact PSII membranes [Zhang, C., and Styring, S. (2003) Biochemistry 42, 8066-8076].     

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)(?).     

Histidine 190-D1 and glutamate 189-D1 provide structural stabilization in photosystem II

In photosynthesis, photosystem II (PSII) conducts the light-driven oxidation of water to oxygen. Tyrosine Z is Tyr 161 of the D1 polypeptide; Z acts as an intermediary electron carrier in water oxidation. In this report, EPR spectroscopy was used to study the effect of His 190 and Glu 189 on Z* yield and reduction kinetics. Neither mutation has a significant impact on the EPR line shape of Z*. At room temperature and pH 7.5, the E189Q-D1 mutation has a single turnover Z* yield that is 84% compared to wild-type. The H190Q-D1 mutation decreases the Z* yield at room temperature by a factor of 2.6 but has a more modest effect (factor of 1.6) at -10 degrees C. The temperature dependence is shown to be primarily reversible. Neither mutation has a dramatic effect on Z* decay kinetics. The Z* minus Z FT-IR spectrum, recorded at pH 7.5 on H190Q, reveals perturbations, including an increased spectral contribution from a PSII chlorophyll. The Z* minus Z FT-IR spectrum, recorded at pH 7.5 on E189Q, shows perturbations, including a decreased contribution from the carboxylate side chain of a glutamate or aspartate. Temperature-dependent changes in H190Q-D1 and E189Q-D1 Z. yield are attributed to a reversible conformational change, which alters the electron-transfer rate from Z to P(680)(+). On the basis of these results, we conclude that H190 and E189 play a role in the structural stabilization of PSII. We postulate that some or all of the phenotypic changes observed in H190Q and E189Q mutants may be caused by structural alterations in PSII.     

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.     

A functional role for tyrosine-D in assembly of the inorganic core of the water oxidase complex of photosystem II and the kinetics of water oxidation.

The role of D2-Tyr160 (Y(D)), a photooxidizable residue in the D2 reaction center polypeptide of photosystem II (PSII), was investigated in both wild type and a mutant strain (D2-Tyr160Phe) in which phenylalanine replaces Y(D) in the cyanobacterium Synechocystis sp. (strain PCC 6803). Y(D) is the symmetry-related tyrosine that is homologous to the essential photoactive Tyr161(Y(Z)) of the D1 polypeptide of PSII. We compared the flash-induced yield of O(2) in intact, functional PSII centers from both wild-type and mutant PSII core complexes. The yield of O(2) in the intact holo-enzyme was found to be identical in the mutant and wild-type PSII cores using long (saturating) pulses or continuous illumination, but was observed to be appreciably reduced in the mutant using short (nonsaturating) light pulses (<50 ms). We also compared the rates of the first two kinetically resolved steps of photoactivation. Photoactivation is the assembly process for binding of the inorganic cofactors to the apo-water oxidation/PSII complex (apo-WOC-PSII) and their light-induced photooxidation to form the functional Mn(4)Ca(1)Cl(x)() core required for O(2) evolution. We show that the D2-Tyr160Phe mutant cores can assemble a functional WOC from the free inorganic cofactors, but at a much slower rate and with reduced quantum efficiency vs wild-type PSII cores. Both of these observations imply that the presence of Y(D)(*) leads to a more efficient photooxidation of the Mn cluster relative to deactivation (reductive processes). One possible explanation for this behavior is that the phenolic proton on Y(D) is retained within the reaction center following Y(D) oxidation. The positive charge, likely shared by D2-His189 and other residues, raises the reduction potential of P(680)(+)/P(680), thereby increasing the driving force for the oxidation of Mn(4)Y(Z). There is, therefore, a competitive advantage to organisms that retain the Y(D) residue, possibly explaining its retention in all sequences of psbD (encoding the D2 polypeptide) known to date. We also find that the sequence of metal binding steps during assembly of apo-WOC-PSII centers in cyanobacteria cores differs from that in higher plants. This is seen by a reduced calcium affinity at its effector site and reduced competition for binding to the Mn(II) site, resulting in acceleration of the initial lagtime by Ca(2+), in contrast to retardation in spinach. Ca(2+) binding to its effector site promotes the stability of the photointermediates (IM1 and above) by suppressing unproductive decay.     

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.     

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.     

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.     

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

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

Participation of glutamate-354 of the CP43 polypeptide in the ligation of manganese and the binding of substrate water in photosystem II

In the current X-ray crystallographic structural models of photosystem II, Glu354 of the CP43 polypeptide is the only amino acid ligand of the oxygen-evolving Mn(4)Ca cluster that is not provided by the D1 polypeptide. To further explore the influence of this structurally unique residue on the properties of the Mn(4)Ca cluster, the CP43-E354Q mutant of the cyanobacterium Synechocystis sp. PCC 6803 was characterized with a variety of biophysical and spectroscopic methods, including polarography, EPR, X-ray absorption, FTIR, and mass spectrometry. The kinetics of oxygen release in the mutant were essentially unchanged from those in wild type. In addition, the oxygen flash yields exhibited normal period four oscillations having normal S state parameters, although the yields were lower, correlating with the mutant's lower steady-state rate (approximately 20% compared to wild type). Experiments conducted with H(2)(18)O showed that the fast and slow phases of substrate water exchange in CP43-E354Q thylakoid membranes were accelerated 8.5- and 1.8-fold, respectively, in the S(3) state compared to wild type. Purified oxygen-evolving CP43-E354Q PSII core complexes exhibited a slightly altered S(1) state Mn-EXAFS spectrum, a slightly altered S(2) state multiline EPR signal, a substantially altered S(2)-minus-S(1) FTIR difference spectrum, and an unusually long lifetime for the S(2) state (>10 h) in a substantial fraction of reaction centers. In contrast, the S(2) state Mn-EXAFS spectrum was nearly indistinguishable from that of wild type. The S(2)-minus-S(1) FTIR difference spectrum showed alterations throughout the amide and carboxylate stretching regions. Global labeling with (15)N and specific labeling with l-[1-(13)C]alanine revealed that the mutation perturbs both amide II and carboxylate stretching modes and shifts the symmetric carboxylate stretching modes of the α-COO(-) group of D1-Ala344 (the C-terminus of the D1 polypeptide) to higher frequencies by 3-4 cm(-1) in both the S(1) and S(2) states. The EPR and FTIR data implied that 76-82% of CP43-E354Q PSII centers can achieve the S(2) state and that most of these can achieve the S(3) state, but no evidence for advancement beyond the S(3) state was observed in the FTIR data, at least not in a majority of PSII centers. Although the X-ray absorption and EPR data showed that the CP43-E354Q mutation only subtly perturbs the structure and spin state of the Mn(4)Ca cluster in the S(2) state, the FTIR and H(2)(18)O exchange data show that the mutation strongly influences other properties of the Mn(4)Ca cluster, altering the response of numerous carboxylate and amide groups to the increased positive charge that develops on the cluster during the S(1) to S(2) transition and weakening the binding of both substrate water molecules (or water-derived ligands), especially the one that exchanges rapidly in the S(3) state. The FTIR data provide evidence that CP43-Glu354 coordinates to the Mn(4)Ca cluster in the S(1) state as a bridging ligand between two metal ions but provide no compelling evidence that this residue changes its coordination mode during the S(1) to S(2) transition. The H(2)(18)O exchange data provide evidence that CP43-Glu354 interacts with the Mn ion that ligates the substrate water molecule (or water-derived ligand) that is in rapid exchange in the S(3) state.     

Low-barrier hydrogen bond plays key role in active photosystem II--a new model for photosynthetic water oxidation

The function and mechanism of Tyr(Z) in active photosystem II (PSII) is one of the long-standing issues in the study of photosynthetic water oxidation. Based on recent investigations on active PSII and theoretical studies, a new model is proposed, in which D1-His190 acts as a bridge, to form a low-barrier hydrogen bond (LBHB) with Tyr(Z), and a coordination bond to Mn or Ca ion of the Mn-cluster. Accordingly, this new model differs from previous proposals concerning the mechanism of Tyr(Z) function in two aspects. First, the LBHB plays a key role to decrease the activation energy for Tyr(Z) oxidation and Tyr(Z)(.) reduction during photosynthetic water oxidation. Upon the oxidation of Tyr(Z), the hydrogen bond between Tyr(Z) and His190 changes from a LBHB to a weak hydrogen bond, and vice versa upon Tyr(Z)(.) reduction. In both stages, the electron transfer and proton transfer are coupled. Second, the positive charge formed after Tyr(Z) oxidation may play an important role for water oxidation. It can be delocalized on the Mn-cluster, thus helps to accelerate the proton release from substrate water on Mn-cluster. This model is well reconciled with observations of the S-state dependence of Tyr(Z) oxidation and Tyr(Z)(.) reduction, proton release, isotopic effect and recent EPR experiments. Moreover, the difference between Tyr(Z) and Tyr(D) in active PSII can also be readily rationalized. The His190 binding to the Mn-cluster predicted in this model is contradictious to the recent structure data, however, it has been aware that the crystal structure of the Mn-cluster and its environment are significantly modified by X-ray due to radiation damage and are different from that in active PSII. It is suggested that the His190 may be protonated during the radiation damage, which leads to the loss of its binding to Mn-cluster and the strong hydrogen bond with Tyr(Z). This type of change arising from radiation damage has been confirmed in other enzyme systems.