Electronic structure of S(2) state of the oxygen-evolving complex of photosystem II studied by PELDOR.

Photosynthetic water splitting is catalyzed by a Mn(4)CaO(5) cluster in photosystem II, whose structure was recently determined at a resolution of 1.9Å [Umena, Y. et al. 2011, Nature, 473:55-60]. To determine the electronic structure of the Mn(4)CaO(5) cluster, pulsed electron-electron double resonance (PELDOR) measurements were performed for the tyrosine residue Y(D)() and S(2) state signals with non-oriented and oriented photosystem II (PS II) samples. Based on these measurements, the spin density distributions were calculated by comparing with the experimental results. The best fitting parameters were obtained with a model in which Mn1 has a large positive projection, Mn3 has a small positive projection, and Mn2 and Mn4 have negative projections (the numbering of Mni (i=1-4) is based on the crystal structure at a 1.9Å resolution), which yielded spin projections of 1.97, -1.20, 1.19 and -0.96 for Mn1-4 ions. The results show that the Mn1 ion, which is coordinated by H332, D342 and E189, has a valence of Mn(III) in the S(2) state. The sign of the exchange interactions J(13) is positive, and the other signs are negative.

Proton-coupled electron-transfer processes in photosystem II probed by highly resolved g-anisotropy of redox-active tyrosine YZ.

The oxidation of a redox-active tyrosine residue Y(Z) in photosystem II (PSII) is coupled with proton transfer to a hydrogen-bonded D1-His190 residue. Because of the apparent proximity of Y(Z) to the water-oxidizing complex and its redox activity, it is believed that Y(Z) plays a significant role in water oxidation in PSII. We investigated the g-anisotropy of the tyrosine radical Y(Z)(•) to provide insight into the mechanism of Y(Z)(•) proton-coupled electron transfer in Mn-depleted PSII. The anisotropy was highly resolved by electron paramagnetic resonance spectroscopy at the W-band (94.9 GHz) using PSII single crystals. The g(X)-component along the phenolic C-O bond of Y(Z)(•) was calculated by density functional theory (DFT). It was concluded from the highly resolved g-anisotropy that Y(Z) loses a phenol proton to D1-His190 upon tyrosine oxidation, and D1-His190 redonates the same proton back to Y(Z)(•) upon reduction.

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).

Comparative study of the water oxidizing reactions and the millisecond delayed chlorophyll fluorescence in photosystem II at different pH.

Water splitting activity, the multiline EPR signal associated with S(2)-state of the CaMn(4)-cluster and the fast and slow phases of the induction curve of the millisecond delayed chlorophyll fluorescence from photosystem II (PSII) in the pH range of 4.5-8.5 were studied in the thylakoid membranes and purified PSII particles. It has been found that O(2) evolution and the multiline EPR signal were inhibited at acidic (pK approximately 5.3) and alkaline (pK approximately 8.1) pH values, and were maximal at pH 6.0-7.0. Our results indicate that the loss of O(2) evolution and the S(2)-state multiline EPR signal associated with the decrease of the millisecond delayed chlorophyll fluorescence only in alkaline region (pH 7.0-8.5). Possible correlations of the millisecond delayed chlorophyll fluorescence components with the donor side reactions in PSII are discussed.

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

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

g-Anisotropy of the S2-state manganese cluster in single crystals of cyanobacterial photosystem II studied by W-band electron paramagnetic resonance spectroscopy.

The multiline signal from the S2-state manganese cluster in the oxygen evolving complex of photosystem II (PSII) was observed in single crystals of a thermophilic cyanobacterium Thermosynechococcus vulcanus for the first time by W-band (94 GHz) electron paramagnetic resonance (EPR). At W-band, spectra were characterized by the g-anisotropy, which enabled the precise determination of the tensor. Distinct hyperfine splittings (hfs's) as seen in frozen solutions of PSII at X-band (9.5 GHz) were detected in most of the crystal orientations relative to the magnetic field. In some orientations, however, the hfs's disappeared due to overlapping of a large number of EPR lines from eight crystallographic symmetry-related sites of the manganese cluster within the unit cell of the crystal. Analysis of the orientation-dependent spectral features yielded the following g-tensor components: g(x) = 1.988, g(y) = 1.981, g(z) = 1.965. The principal values suggested an approximate axial symmetry around the Mn(III) ion in the cluster.

EPR characteristics of chloride-depleted photosystem II membranes in the presence of other anions.

Chloride is an essential cofactor for the oxidation of water to oxygen. Anion substitution (Br(-), I(-), NO(2)(-), F(-)) in Cl(-)-depleted PS II membranes brings out significant changes in the EPR signals arising from the S(2) state and from the iron-quinone complex of PS II. On the basis of the changes observed in the S(2) state multiline signal and the Q(A)Fe(3+) EPR signal in Cl(-)-depleted PS II membranes after substituting with various anions, we report a possible binding site of anions such as chloride and bromide at the PS II donor side as well as at the acceptor side.

Interactions of chloride and formate at the donor and the acceptor side of photosystem II.

Chloride is required for the maximum activity of the oxygen evolving complex (OEC) while formate inhibits the function of OEC. On the basis of the measurements of oxygen evolution rates and the S(2) state multiline EPR signal, an interaction between the action of chloride and formate at the donor side of PS II has been suggested. Moreover, the Fe(2)+Q-A EPR signals were measured to investigate a common binding site of both these anions at the PS II acceptor side. Other monovalent anions like bromide, nitrate etc. could influence the effects of formate to a small extent at the donor side of PS II, but not significantly at the acceptor side of PS II. The results presented in this paper clearly suggest a competitive binding of formate and chloride at the PS II acceptor side.

X-ray snapshots of quinone cofactor biogenesis in bacterial copper amine oxidase.

The quinone cofactor TPQ in copper amine oxidase is generated by posttranslational modification of an active site tyrosine residue. Using X-ray crystallography, we have probed the copper-dependent autooxidation process of TPQ in the enzyme from Arthrobacter globiformis. Apo enzyme crystals were anaerobically soaked with copper; the structure determined from this crystal provides a view of the initial state: the unmodified tyrosine coordinated to the bound copper. Exposure of the copper-bound crystals to oxygen led to the formation of freeze-trapped intermediates; structural analyses indicate that these intermediates contain dihydroxyphenylalanine quinone and trihydroxyphenylalanine. These are the first visualized intermediates during TPQ biogenesis in copper amine oxidase.