Asako Kawamori

Modification of the properties of S2 state in photosynthetic O2-evolving center by replacement of chloride with other anions

Properties of the S2 state formed in photosystem II membranes in which Cl- had been replaced by various anions were investigated by means of thermoluminescence measurements and low temperature EPR spectroscopy. The Br--substituted membranes showed the normal thermoluminescence B-band arising from S2Q-B charge recombination, whereas the SO2-4-, F--, CH3COO--, and NO-3-substituted membranes showed modified B-bands with variously upshifted peak temperatures. The extent of the peak temperature upshift varied in parallel with the extent of inhibition of O2 evolution depending on the anion species. A normal EPR S2 multiline signal was induced in Br--substituted membranes, but its amplitude was reduced to less than 10% in F--, NO-3-, CH3COO--, and SO2-4-substituted membranes, In contrast, the g = 4.1 signal from S2 was markedly enhanced in F-- and NO-3-substituted membranes, not much affected in CH3COO-- and SO2-4-substituted membranes, and decreased to 70% in Br--substituted membranes. Based on these data, the effect of various types of S2 modification on the O2-evolving activity was discussed. It was suggested that anions have an important role in regulating the interaction between the Mn atoms, and thereby adjust the redox properties of the S2 state to enable further transitions beyond S2.

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.

Electron spin echo of spin-polarised radical pairs in intact and quinone-reconstituted plant photosystem I reaction centers

Abstract Light-induced spin-polarised P700+A1− pairs in intact and quinone-reconstituted photosystem I reaction centres were studied by electron spin echo (ESE) spectroscopy. The observed strong ESE envelope modulation was attributed to magnetic dipolar and exchange interactions in the pairs. The values of these interactions were derived from Fourier-transformed time traces and appeared to be D = −1.71 ± 0.05 G and J = 0.010 ± 0.015 G, respectively. A magnetic field effect on the radical pair lifetime induced by microwave pumping was observed. The reconstituted 2,3-dibromo-1,4-naphthoquinone was shown to be located in the same (A1) site as the native phylloquinone.

The distances from tyrosine D to redox-active components on the donor side of Photosystem II determined by pulsed electron-electron double resonance

Abstract A pulsed electron-electron double resonance (ELDOR) method was applied to measyre the dipole interactions between paramagnetic species on the donor side of Photosystem II. The distance between the Mn cluster and the redox-active tyrosine residue YD was determined to be 27 ± 0.2 A in the S2 state of the oxygen-evolving Photosystem II. In Ca2+-depleted preparations the same distance was obtained from the measurement of the modified multiline signal. The distance between YD and the paramagnetic species giving rise to the split S3 signal was found to be 30 ± 0.2 A .

The pulsed electron–electron double resonance and “2+1” electron spin echo study of the oriented oxygen-evolving and Mn-depleted preparations of photosystem II

A “2+1” electron spin echo method has been applied to measure the angular orientation of the radius-vector RDZ joining the redox-active tyrosine residues YD and YZ in membrane-oriented Mn-depleted preparation of photosystem II. The angle between RDZ and the normal to the photosystem II membrane was determined to be 100°±2°, where the angle of 0° corresponds to the direction from YD towards the stromal side of the membrane, along the membrane normal. A pulsed electron–electron double resonance method has been used to obtain similar information for YD and Mn cluster in oriented sample of oxygen-evolving photosystem II. The angle between the radius-vector from YD to the Mn cluster, RDMn, and the membrane normal was found to be 110°±2°.

Distance between tyrosines Z+ and D+ in plant Photosystem II as determined by pulsed EPR

Abstract A ‘2 + 1’ electron spin echo method was applied to estimate the dipole interaction between tyrosines D + and Z + in Mn-depleted preparations of plant Photosystem II. The value of dipole interaction obtained corresponds to the distance of approx. 30 A between the two types of tyrosine radical.

An electron spin echo envelope modulation study of the primary acceptor quinone in Zn‐substituted plant photosystem II

A Fe2+ ion on the acceptor side of plant photosystem II has been substituted by Zn2+ and an anion radical of the primary acceptor quinone, Q−A, has been studied by electron spin echo method. The electron spin echo modulation shows the interaction of the unpaired electron of Q−A with nitrogen nuclei of the histidine and, probably, alanine residues situated nearby. The comparison of the modulation spectra of Q−A with those of the anion radical of plastoquinone‐9 stabilized in protonated and deuterated isopropanol matrices allows one to distinguish between the spectrum lines due to the quinone protons and due to the protons of other molecules that form hydrogen bonds with the oxygen atoms of the quinones.

EPR STUDY OF TRAPPED TYROSINE Z+ IN CA-DEPLETED PHOTOSYSTEM II

Abstract The dependence of the light-induced transient EPR signal intensity of Y Z + on the illumination temperature was measured in Ca-depleted PS II in the presence of DCMU. The maximum yield of Y Z + , about 70% of Y D + EPR intensity, was obtained by illumination at 245 K, and trapped by immediate freezing in liquid nitrogen. Analysis of the relative intensities of Y Z + and modified multiline EPR signals under various illumination periods and temperatures shows that Y Z + has been trapped in the S 1 state of the oxygen evolving center, and the trapped Y Z + EPR converted to the multiline signal through the advancement from the S 1 to S 2 state in the dark at 273 K. Spin-lattice relaxation times of Y D + and Y Z + in the Ca-depleted and Mn-depleted PS II were measured by pulsed EPR. From the relaxation enhancements of these radicals due to the dipole interaction with the Mn cluster, the distance from Y Z + to the Mn cluster was estimated to be 15–20 A. Using the 2 + 1 ESE method, the distance between Y Z + and Y D + was determined to be 29–30 A.

Microenvironments of tyrosine D+ and tyrosine Z+ in Photosystem II studied by proton matrix ENDOR

Abstract Proton matrix ENDOR spectra of tyrosines Z + (Y + Z ) and D + (Y + D ) were studied in the frozen Tris-treated PS II. ENDOR spectra of the tyrosines in oriented membranes observed at 10 K have shown similar angular dependence with similar line pair separations, suggesting that the local arrangement of protons surrounding both radical species is similar. The angular dependence of EPR spectra showed the C-O bonds in Y + Z and Y + D lie parallel to the membrane normal. With increasing temperature, the intensity of the line pair with 1.2 MHz separation decreased above 25 K for Y + Z , and above 110 K for Y + D , respectively. Assuming a motion of the proton responsible to the line pair, the activation energies for the motional broadening were estimated to be 86 ± 20 J/mol for Y + Z , and 810 ± 200 J/mol for Y + D , respectively. The broadening may be ascribed to small amplitude vibrations of protons surrounding tyrosine radicals. The intensities for the peaks with about 7 MHz separation assigned to the OH proton of the tyrosine decreased, which was ascribed to a similar motional broadening. The difference in temperature dependence of line broadening of the two radicals suggests that the functional difference between them may be ascribable to differences in flexibility in their microenvironment.

ENDOR study on the position of hydrogens close to the manganese cluster in S2 state of photosystem II

Proton matrix ENDOR of the manganese multiline in the S2, state of photosystem II membranes from spinach has been investigated. The spectral structure over a range of frequencies of ± 2 MHz centered on the position for a free proton was analyzed through employing a simulation method based on the dipolar interaction between electron and proton magnetic moments. The 6 pairs of lines resolved were attributed to the surrounding proton populations at distances varying within the range 2.7–6.0 Å from the putative manganese center. Two of the 6 pairs, namely those corresponding to protons at distances of 2.7 and 3.2 Å from the manganese center were eliminated on washing of the membranes with deuterated buffer. This suggests that these protons belong to the water molecules coordinating to the manganese cluster. The presence of ethylene glycol instead of glycerol and sucrose in the buffer broadened both EPR and ENDOR spectra. This suggests that ethylene glycol molecules are also accessible to the two‐manganese cluster. A possible model of water association to the manganese ions in the photosynthetic water‐oxidation system is proposed.