Untreated Photosystem II Research Articles

Temperature dependence of the formation of the g ~ 5 EPR signal in the oxygen evolving complex of photosystem II.

The temperature dependence of the formation of the g ~ 5 S2 state electron paramagnetic resonance (EPR) signal in photosystem II (PSII) was investigated. The g ~ 5 signal was produced at an illumination above 200K. The half inhibition temperature of the formation of the g ~ 5 EPR signal was approximately 215K. The half inhibition temperature is close to that of the transition from the S2 state-to-S3 state in the untreated PSII, and not to that of the transition from S1 state -to-S2 state in the untreated PSII. The upshift of the half inhibition temperature of the transition from the S1 state -to-S2 state (g ~ 5) reflects the structural change upon transition from the S1 state to the S2 state. The activation energy of the g ~ 5 state formation was estimated as 40.7 ± 4.4kJ/mol, which is comparable to the reported activation energy for the S2 formation in the untreated PSII. The activation enthalpy and entropy were estimated to be 39.0 ± 4.4kJ/mol and -103 ± 19J/mol K at 210K, respectively. Based on these parameters, the formation process of the g ~ 5 state is discussed in this study.

Proton Matrix ENDOR Studies on Ca2+-depleted and Sr2+-substituted Manganese Cluster in Photosystem II

Proton matrix ENDOR spectra were measured for Ca(2+)-depleted and Sr(2+)-substituted photosystem II (PSII) membrane samples from spinach and core complexes from Thermosynechococcus vulcanus in the S2 state. The ENDOR spectra obtained were similar for untreated PSII from T. vulcanus and spinach, as well as for Ca(2+)-containing and Sr(2+)-substituted PSII, indicating that the proton arrangements around the manganese cluster in cyanobacterial and higher plant PSII and Ca(2+)-containing and Sr(2+)-substituted PSII are similar in the S2 state, in agreement with the similarity of the crystal structure of both Ca(2+)-containing and Sr(2+)-substituted PSII in the S1 state. Nevertheless, slightly different hyperfine separations were found between Ca(2+)-containing and Sr(2+)-substituted PSII because of modifications of the water protons ligating to the Sr(2+) ion. Importantly, Ca(2+) depletion caused the loss of ENDOR signals with a 1.36-MHz separation because of the loss of the water proton W4 connecting Ca(2+) and YZ directly. With respect to the crystal structure and the functions of Ca(2+) in oxygen evolution, it was concluded that the roles of Ca(2+) and Sr(2+) involve the maintenance of the hydrogen bond network near the Ca(2+) site and electron transfer pathway to the manganese cluster.

The Conserved His-144 in the PsbP Protein Is Important for the Interaction between the PsbP N-terminus and the Cyt b559 Subunit of Photosystem II

The PsbP protein regulates the binding properties of Ca(2+) and Cl(-), and stabilizes the Mn cluster of photosystem II (PSII); however, the binding site and topology in PSII have yet to be clarified. Here we report that the structure around His-144 and Asp-165 in PsbP, which is suggested to be a metal binding site, has a crucial role for the functional interaction between PsbP and PSII. The mutated PsbP-H144A protein exhibits reduced ability to retain Cl(-) anions in PSII, whereas the D165V mutation does not affect PsbP function. Interestingly, H144A/D165V double mutation suppresses the effect of H144A mutation, suggesting that these residues have a role other than metal binding. FTIR difference spectroscopy suggests that H144A/D165V restores proper interaction with PSII and induces the conformational change around the Mn cluster during the S(1)/S(2) transition. Cross-linking experiments show that the H144A mutation affects the direct interaction between PsbP and the Cyt b(559) α subunit of PSII (the PsbE protein). However, this interaction is restored in the H144A/D165V mutant. In the PsbP structure, His-144 and Asp-165 form a salt bridge. H144A mutation is likely to disrupt this bridge and liberate Asp-165, inhibiting the proper PsbP-PSII interaction. Finally, mass spectrometric analysis has identified the cross-linked sites of PsbP and PsbE as Ala-1 and Glu-57, respectively. Therefore His-144, in the C-terminal domain of PsbP, plays a crucial role in maintaining proper N terminus interaction. These data provide important information about the binding characteristics of PsbP in green plant PSII.

Acetate binding at the photosystem II oxygen evolving complex: an S(2)-state multiline signal ESEEM study.

Previously, using acetate deuterated in the methyl hydrogen positions, we showed that acetate binds in close proximity to the Mn cluster/Y(.)(z) tyrosine dual spin complex in acetate-inhibited photosystem II (PSII) preparations exhibiting the "split" EPR signal arising from the S(2)-Y(.)(z) interaction [Force, D. A.; Randall, D. W.; Britt, R. D. Biochemistry 1997, 36, 12062-12070]. By using paramagnetic NO to quench the paramagnetism of Y(.)(z), we are able to observe the ESEEM spectrum of deuterated acetate interacting with only the Mn cluster. A good fit of the ESEEM data indicates two (2)H dipolar hyperfine couplings of 0.097 MHz and one of 0.190 MHz. Modeling of these dipolar interactions, using our "dangler" 3 + 1 model for the S(2)-state of the Mn cluster, reveals distances consistent with direct ligation of acetate to the Mn cluster. As acetate inhibition is competitive with the essential cofactor Cl(-), this suggests that Cl(-) ligates directly to the Mn cluster. The effect of acetate binding on the structure of the Mn cluster is investigated by comparing the Mn-histidine coupling in NO/acetate-treated PSII and untreated PSII using ESEEM. We find that the addition of acetate and NO does not affect the histidine ligation to the Mn cluster. We also investigate the ability of acetate to access Y(.)(z) in Mn-depleted PSII, a PSII preparation expected to be more solvent accessible than intact PSII. We detect no coupling between Y(.)(z) and acetate. We have previously shown that small alcohols such as methanol can ligate to the Mn cluster with ease, while larger alcohols such as 2-propanol, as well as DMSO, are excluded [Force, D. A.; Randall, D. W.; Lorigan, G. A.; Clemens, K. L.; Britt, R. D. J. Am. Chem. Soc. 1998, 120, 13321-13333]. We probe the effect of acetate binding on the ability of methanol and DMSO to bind to the Mn cluster. We find that methanol is able to bind to the Mn cluster in the presence of acetate. We detect no DMSO binding in the presence of acetate. Thus, acetate binding does not increase the affinity or accessibility for DMSO binding at the Mn cluster. We also explore the possibility that the acetate binding site is also a binding site for substrate water. By comparing the ratioed three-pulse ESEEM spectra of a control, untreated PSII sample in 50% D(2)O to an NO/acetate-treated PSII sample in 50% D(2)O, we find that the binding of acetate to the oxygen evolving complex of photosystem II displaces deuterons bound very closely to the Mn cluster.

Calcium binding studies of photosystem II using a calcium-selective electrode.

The identification of Ca2+ as a cofactor in photosynthetic O2 evolution has encouraged research into the role of Ca2+ in photosystem II (PSII). Previous methods used to identify the number of binding sites and their affinities were not able to measure Ca2+ binding at thermodynamic equilibrium. We introduce the use of a Ca2(+)-selective electrode to study equilibrium binding of Ca2+ to PSII. The number and affinities of binding sites were determined via Scatchard analysis on a series of PSII membrane preparations progressively depleted of the extrinsic polypeptides and Mn. Untreated PSII membranes bound approximately 4 Ca2+ per PSII with high affinity (K = 1.8 microM) and a larger number of Ca2+ with lower affinity. The high-affinity sites are assigned to divalent cation-binding sites on the light-harvesting complex II that are involved in membrane stacking, and the lower-affinity sites are attributed to nonspecific surface-binding sites. These sites were also observed in all of the extrinsic polypeptide- and Mn-depleted preparations. Depletion of the extrinsic polypeptides and/or Mn exposed additional very high-affinity Ca2(+)-binding sites which were not in equilibrium with free Ca2+ in untreated PSII, owing to the diffusion barrier created by the extrinsic polypeptides. Ca2(+)-depleted PSII membranes lacking the 23 and 17 kDa extrinsic proteins bound an additional 2.5 Ca2+ per PSII with K = 0.15 microM. This number of very high-affinity Ca2(+)-binding sites agrees with the previous work of Cheniae and co-workers [Kalosaka, K., et al. (1990) in Current Research in Photosynthesis (Baltscheffsky, M., Ed.) pp 721-724, Kluwer, Dordrecht, The Netherlands] whose procedure for Ca2+ depletion was used. Further depletion of the 33 kDa extrinsic protein yielded a sample that bound only 0.7 very high-affinity Ca2+ per PSII with K = 0.19 microM. The loss of 2 very high-affinity Ca2(+)-binding sites upon depletion of the 33 kDa extrinsic protein could be due to a structural change of the O2-evolving complex which lost 2-3 of the 4 Mn ions in this sample. Finally, PSII membranes depleted of Mn and the 33, 23, and 17 kDa extrinsic proteins bound approximately 4 very high-affinity Ca2+ per PSII with K = 0.08 microM. These sites are assigned to Ca2+ binding to the vacant Mn sites.

The tetranuclear manganese cluster in photosystem II: location and magnetic properties of the S2 state as determined by saturation-recovery EPR spectroscopy.

The spin-lattice relaxation enhancement of the dark-stable tyrosine radical, YD., by the S2 state of the O2-evolving complex (OEC) of photosystem II (PSII) has been measured by using saturation-recovery EPR spectroscopy. Two forms of the S2 state have been compared: the multiline EPR signal species in untreated PSII and the altered multiline EPR signal species in NH3-treated PSII. Previous work has shown that the non-single-exponential spin-lattice relaxation kinetics of YD. in S2-state PSII result from a dipole-dipole interaction with the Mn4 cluster of the OEC. By taking into account the temperature variation of the effective magnetic moment of the S2-state multiline EPR signal form of the OEC, we provide a quantitative analysis of its temperature-dependent enhancement of the spin-lattice relaxation of YD.. Different spin states of the Mn4 cluster in the S2 state are responsible for the effect at different temperature regimes: for T </= 10 K, it is the ground spin state (S = 1/2); for T >/= 30 K, it is the first excited spin state; and at intermediate temperatures, the contributions of the two spin states are comparable. The relaxation enhancement of YD. is equivalent for both forms of the S2-state multiline EPR signal examined, indicating that the magnetic properties of the Mn4 cluster are very similar in the S2 state for both untreated and NH3-treated PSII. EPR progressive microwave-power saturation has also been used to assess the spin-lattice relaxation properties of the Mn4 cluster giving the altered S2-state multiline EPR signal in the NH3 derivative of PSII. The Orbach mechanism is shown to provide the dominant relaxation pathway; the energy difference between the ground and first excited spin states is estimated to be 30 +/- 2 cm-1, which is very similar to the value found for the S2-state multiline EPR signal species in untreated PSII. Below 4 K, the effectiveness of the S2-state multiline EPR signal species as a spin relaxation enhancer of YD. drops dramatically. This is interpreted to occur because of temperature-dependent 55Mn nuclear spin-lattice relaxation which causes averaging of the effective Larmor frequency of the S2-state multiline EPR signal species during the time scale for spin-lattice relaxation of YD.; because the line shape of the S2-state multiline EPR signal is dominated by isotropic 55Mn nuclear hyperfine splittings, such nuclear relaxation processes allow frequencies in near resonance with that of YD. to be accessed, thereby producing a greater relaxation enhancement. By using a dipolar model that includes the line shapes of both the YD. and S2-state multiline EPR signals, the spin-lattice relaxation enhancement of YD. is analyzed to obtain a lower limit of 22 A for the distance between YD. and the OEC. Together with recent studies showing a close proximity of the Mn4 cluster to YZ., these results provide further support for an asymmetric location of the Mn4 cluster with respect to the two redox-active tyrosines in PSII.

Photosystem II 3-D structure and the role of the extrinsic subunits in photosynthetic oxygen evolution

Two-dimensional (2-D) crystals of photosystem II (PSII) from spinach ( Spinacia oleracea) have been subjected to Tris-washing in order to selectively remove the three oxygen-evolution enhancing (OEE) subunits. Electron microscopy of the 2-D crystals of Tris-treated PSII (tPSII) confirmed the previously reported unit cell parameters a = 17.7 nm, b = 20.1 and γ = 91° (cf. Holzenburg et al., 1994) and, in conjuction with Fourier reconstruction techniques, was used to calculate the three-dimensional (3-D) structure of tPSII at 1.8 nm resolution which is in good agreement with our earlier tPSII data in projection. A comparison between the 3-D structures of tPSII and untreated PSII (Holzenburg et al., 1993) by Fourier difference analysis revealed directly and unambiguously the existence of a micro-cavity as part of the complex. We demonstrate that this intramolecular microenvironment is formed by the OEE subunits and discuss its importance for oxygen evolution.

Photooxidation of cytochrome b559 in oxygen-evolving photosystem II

Cytochrome b559 (cyt b559) is an intrinsic and essential component of the photosystem II (PSII) protein complex, but its function, stoichiometry, and electron-transfer kinetics in the physiological system are not well-defined. In this study, we have used flash-detection optical spectroscopy to measure the kinetics and yields of photooxidation and dark reduction of cyt b559 in untreated, O2-evolving PSII-enriched membranes at room temperature. The dark redox states of cyt b559 and the primary electron acceptor, QA, were determined over the pH range 5.0-8.5. Both the fraction of dark-oxidized cyt b559 and dark-reduced QA increased with increasing acidity. Consistent with these results, an acid-induced drop in pH from 8.5 to 4.9 in a dark-adapted sample caused the oxidation of cyt b559, indicating a shift in the redox state during the dark reequilibration. As expected from the dark redox state of cyt b559, the rate and extent of photooxidation of cyt b559 during continuous illumination decreased toward more acidic pH values. After a single, saturating flash, the rate of photooxidation of cyt b559 was of the same order of magnitude as the rate of S2QA- charge recombination. In untreated PSII samples at pH 8.0 with 42% of cyt b559 oxidized and 15% of QA reduced in the dark, 4.7% of one copy of cyt b559 was photooxidized after one flash with a t1/2 of 540 +/- 90 ms. On the basis of our previous work [Buser, C. A., Thompson, L. K., Diner, B. A., & Brudvig, G. W (1990) Biochemistry 29, 8977] and the data presented here, we conclude that Sn+1, YZ., and P680+ are in redox equilibrium and cyt b559 (and YD) are oxidized via P680+. After a period of illumination sufficient to fully reduce the plastoquinone pool, we also observed the pH-dependent dark reduction of photooxidized cyt b559, where the rate of reduction decreased with decreasing pH and was not observed at pH < 6.4. To determine the direct source of reductant to oxidized cyt b559, we studied the dark reduction of cyt b559 and the reduction of the PQ pool as a function of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) concentration. We find that DCMU inhibits the reduction of cyt b559 under conditions where the plastoquinone pool and QA are reduced. We conclude that QB-. (H+) or QBH2 is the most likely source of the electron required for the reduction of oxidized cyt b559.(ABSTRACT TRUNCATED AT 400 WORDS)

Detection of structural changes upon S1-to-S2 transition in the oxygen-evolving manganese cluster in photosystem II by light-induced Fourier transform infrared difference spectroscopy.

The light-induced Fourier transform infrared (FT-IR) difference spectrum between the S1 and S2 states of the O2-evolving photosystem II (PSII) was obtained for the first time. Detection of an S2/S1 difference spectrum virtually free from contributions by the acceptor-side signals was achieved by employing an exogenous electron acceptor, potassium ferricyanide, to trypsin-treated PSII membranes and using one-flash excitation at 250 K. A synthetic difference spectrum obtained by adding this S2/S1 spectrum to the QA-/QA spectrum measured with Mn-depleted PSII was almost identical with the difference spectrum of the S2QA-/S1QA charge separation measured with untreated PSII. This successful simulation verifies the correctness of the S2/S1 spectrum thus obtained. The observed S2/S1 spectrum reflects the structural changes within the water-oxidizing Mn cluster upon the S1-to-S2 transition, most probably changes in vibrational modes of ligands coordinating to the Mn ion(s) that is (are) oxidized upon the S2 formation and/or changes in protein conformation. The present results demonstrate that FT-IR difference spectroscopy is a promising method to investigate the structure of the intermediates of the Mn cluster involved in photosynthetic water oxidation.

The S0 state of photosystem II induced by hydroxylamine: differences between the structure of the manganese complex in the S0 and S1 states determined by x-ray absorption spectroscopy

Hydroxylamine at low concentrations causes a two-flash delay in the first maximum flash yield of oxygen evolved from spinach photosystem II (PSII) subchloroplast membranes that have been excited by a series of saturating flashes of light. Untreated PSII membrane preparations exhibit a multiline EPR signal assigned to a manganese cluster and associated with the S2 state when illuminated at 195 K, or at 273 K in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). We used the extent of suppression of the multiline EPR signal observed in samples illuminated at 195 K to determine the fraction of PSII reaction centers set back to a hydroxylamine-induced S0-like state, which we designate S0*. The manganese K-edge X-ray absorption edges for dark-adapted PSII preparations with or without hydroxylamine are virtually identical. This indicates that, despite its high binding affinity to the oxygen-evolving complex (OEC) in the dark, hydroxylamine does not reduce chemically the manganese cluster within the OEC in the dark. After a single turnover of PSII, a shift to lower energy is observed in the inflection of the Mn K-edge of the manganese cluster. We conclude that, in the presence of hydroxylamine, illumination causes a reduction of the OEC, resulting in a state resembling S0. This lower Mn K-edge energy of S0*, relative to the edge of S1, implies the storage and stabilization of an oxidative equivalent within the manganese cluster during the S0----S1 state transition. An analysis of the extended X-ray absorption fine structure (EXAFS) of the S0* state indicates that a significant structural rearrangement occurs between the S0* and S1 states. The X-ray absorption edge position and the structure of the manganese cluster in the S0* state are indicative of a heterogeneous mixture of formal valences of manganese including one Mn(II) which is not present in the S1 state.

Characterization of the multiple forms of cytochrome b559 in photosystem II

Cytochrome b559 is an essential component of the photosystem II (PSII) protein complex. Its function, which has long been an unsolved puzzle, is likely to be related to the unique ability of PSII to oxidize water. We have used EPR spectroscopy and spectrophotometric redox titrations to probe the structure of cytochrome b559 in PSII samples that have been treated to remove specific components of the complex. The results of these experiments indicate that the low-temperature photooxidation of cytochrome b559 does not require the presence of the 17-, 23-, or 33-kDa extrinsic polypeptides or the Mn complex (the active site in water oxidation). We observe a shift in the g value of the EPR signal of cytochrome b559 upon warming a low-temperature photooxidized sample, which presumably reflects a change in conformation to accommodate the oxidized state. At least three redox forms of cytochrome b559 are observed. Untreated PSII membranes contain one high-potential (375 mV) and one intermediate-potential (230 mV) cytochrome b559 per PSII. Thylakoid membranes also appear to contain one high-potential and one intermediate-potential cytochrome b559 per PSII, although this measurement is more difficult due to interference from other cytochromes. Removal of the 17- and 23-kDa extrinsic polypeptides from PSII membranes shifts the composition to one intermediate-potential (170 mV) and one low-potential (5 mV) cytochrome b559. This large decrease in potential is accompanied by a very small g-value change (0.04 at gz), indicating that it is the environment and not the ligand field of the heme which changes significantly upon the removal of the 17- and 23-kDa polypeptides.

Formation of the S2 state and structure of the Mn complex in photosystem II lacking the extrinsic 33 kilodalton polypeptide

Electron paramagnetic resonance (EPR) spectroscopy and O2 evolution assays were performed on photosystem II (PSII) membranes which had been treated with 1 M CaCl2 to release the 17, 23 and 33 kilodalton (kDa) extrinsic polypeptides. Manganese was not released from PSII membranes by this treatment as long as a high concentration of chloride was maintained. We have quantitated the EPR signals of the several electron donors and acceptors of PSII that are photooxidized or reduced in a single stable charge separation over the temperature range of 77 to 240 K. The behavior of the samples was qualitatively similar to that observed in samples depleted of only the 17 and 23 kDa polypeptides (de Paula et al. (1986) Biochemistry25, 6487-6494). In both cases, the S2 state multiline EPR signal was observed in high yield and its formation required bound Ca(2+). The lineshape of the S2 state multiline EPR signal and the magnetic properties of the manganese site were virtually identical to those of untreated PSII membranes. These results suggest that the structure of the manganese site is unaffected by removal of the 33 kDa polypeptide. Nevertheless, in samples lacking the 33 kDa polypeptide a stable charge separation could only be produced in about one half of the reaction centers below 160 K, in contrast to the result obtained in untreated or 17 and 23 kDa polypeptide-depleted PSII membranes. This suggests that one function of the 33 kDa polypeptide is to stabilize conformations of PSII that are active in secondary electron transfer events.

Effect of the 17- and 23-kilodalton polypeptides, calcium, and chloride on electron transfer in photosystem II.

Electron paramagnetic resonance (EPR) measurements were performed on photosystem II (PSII) membranes that were treated with 2 M NaCl to release the 17- and 23-kilodalton (kDa) polypeptides. By using 75 microM 3-(3,4-dichlorophenyl)-1,1-dimethylurea to limit the photosystem II samples to one stable charge separation in the temperature range of 77-273 K, we have quantitated the EPR signals of the several electron donors and acceptors of photosystem II. It was found that removal of the 17- and 23-kDa polypeptides caused low potential cytochrome b559 to become fully oxidized during the course of dark adaptation. Following illumination at 77-130 K, one chlorophyll molecule per reaction center was oxidized. Between 130 and 200 K, both a chlorophyll molecule and the S1 state were photooxidized and, together, accounted for one oxidation per reaction center. Above 200 K, the chlorophyll radical was unstable. Oxidation of the S1 state gave rise to the S2-state multiline EPR signal, which arises from the Mn site of the O2-evolving center. The yield of the S2-state multiline EPR signal in NaCl-washed PSII membranes was as high as 93% of the control, untreated PSII membranes, provided that both Ca2+ and Cl- were bound. Furthermore, the 55Mn nuclear hyperfine structure of the S2-state multiline EPR signal was unaltered upon depletion of the 17- and 23-kDa polypeptides. In NaCl-washed PSII samples where Ca2+ and/or Cl- were removed, however, the intensity of the S2-state multiline EPR signal decreased in parallel with the fraction of PSII lacking bound Ca2+ and Cl-.(ABSTRACT TRUNCATED AT 250 WORDS)