X-ray Detection of the Period-Four Cycling of the Manganese Cluster in Photosynthetic Water Oxidizing Enzyme.

X-ray absorption near-edge structure spectra of the manganese (Mn) cluster in physiologically native intermediate states of photosynthetic water oxidation induced by short laser flash were measured with a compact heat-insulated chamber equipped with an x-ray detector near the sample surface. The half-height energy of the Mn Kedge showed a period-four oscillation dependent on cycling of the Joliot-Kok's oxygen clock. The flash number-dependent shift in the Mn K-edge suggests that the Mn cluster is oxidized by one electron upon the S(0)-to-S(1), S(1)-to-S(2), and S(2)-to-S(3) transitions and then reduced upon the S(3)-to-S(0) transition that releases molecular oxygen.

Temperature dependence on the delayed fluorescence of chlorophyll a in blue-green algae.

1. The delayed fluorescence of chlorophyll a was measured with a phosphoroscope by changing the temperature in a range of room temperatures in intact cells of blue-green algae, Anacystis nidulans, two strains of Anabaena variabilis and Plectonema boryanum, and other kinds of algae, Cyanidium caldarium and Chlorella pyrenoidosa. The induction of delayed fluorescence remarkably depended on the temperature of measurment. Nevertheless, the induction pattern was characterized by three levels of intensity; the initial rise level at the onset of excitation light, the maximum level after a period of excitation and the steady-state level after 10 min of excitation. 2. In A. nidulans and a strain of A. variabilis grown at various temperatures, close relationship was found between the phase transition of membrane lipids and the initial rise and the steady-state levels of delayed fluorescence. The initial rise level showed the maximum at the temperature of phase transition between the liquid crystalline and the mixed solid-liquid crystalline states, The steady-state levels showed a remarkable change from a high in the liquid crystalline state to a low level in the mixed solid-liquid crystalline state. 3. The millisecond decay kinetics of the delayed fluorescence measured at the steady-state level in A. nidulans grown at 38 degrees C consisted of two components with different decay rates. The half-decay time of the fast component was about 0.17 ms and was constant throughout the temperature range of measurement. The half decay time of slow component ranged from 0.6 to 1.5 ms, depending on the temperature of measurment.

Relationship between growth temperature of Anacystis nidulans and phase transition temperature of its thylakoid membranes.

The temperatures of the lipid phase transition at which the solid phase disappears were determined by using the X-ray diffraction method in thylakoid membranes of the blue-green alga, Anacystis nidulans. The temperatures were determined as 26 and 16 degrees C for cells grown at 38 and 28 degrees C, respectively.

The functional sites of chlorophylls in D1 and D2 subunits of photosystem II identified by pulsed EPR.

The functional site of ChlZ, an auxiliary electron donor to P680+, was determined by pulsed ELDOR applied to a radical pair of YD * and Chlz+ in oriented PS II membranes from spinach. The radical-radical distance was determined to be 29.5 A and its direction was 50 degrees from the membrane normal, indicating that a chlorophyll on the D2 protein is responsible for the EPR Chlz+ signal. Spin polarized ESEEM (Electronin Spin Echo Envelop Modulation) of a 3Chl and QA - radical pair induced by a laser flash was observed in reaction center D1D2Cytb559 complex, in which QA was functionally reconstituted with DBMIB and reduced chemically. QA -ESEEM showed a characteristic oscillating time profile due to dipolar coupling with 3Chl. By fitting with the dipolar interaction parameters, the distance between 3Chl and QA - was determined to be 25.9 A, indicating that the accessory chlorophyll on the D1 protein is responsible for the 3Chl signal.

Abnormal S-state turnovers in NH3-binding Mn centers of photosynthetic O2 evolving system.

The inhibitory effects of NH3 on S-state turnovers were studied by curve fitting and deconvolution of thermoluminescence glow curves and low-temperature EPR spectroscopy. The following results were found: (i) High concentrations of NH3 upshifted the recombination temperatures of both S2QB- and S2QA- charge pairs, indicating formation of an abnormal S2 state having a lowered oxidation potential. (ii) The abnormal S2 was correlated to alterations in EPR multiline signal: high concentrations of NH3 induced the modified multiline signal having reduced hyperfine line spacing, accompanied by disappearance of the g = 4.1 signal, while low concentrations of NH3 reduced the line width of the g = 4.1 signal with a slight shift in its g value to 4.2 concomitant with suppression in amplitude of the normal multiline signal, both suggesting coordination of NH3 to the Mn center. (iii) More than half of the NH3-binding abnormal S2 centers underwent S-state turnover to yield S3QB- and S3QA- pairs having normal thermoluminever, the NH3-binding S3 was unable to undergo further S-state turnovers. (iv) The interruption of S-state turnover at S3 was assumed to be due to the inability of electron abstraction from the S3 state. Based on these, the mechanism of NH3 inhibition was discussed.

Chilling-Susceptibility of the Blue-Green Alga Anacystis nidulans: III. LIPID PHASE OF CYTOPLASMIC MEMBRANE.

The lipid phase of cytoplasmic membrane was studied by freeze-fracture electron microscopy in the chilling-susceptible blue-green alga, Anacystis nidulans. At growth temperatures, intramembrane particles were distributed at random in the fracture faces of cytoplasmic membrane, whereas, at chilling temperatures, the fracture faces were composed of particle-free and particle-containing regions. These findings indicate that lipids of the cytoplasmic membrane were in the liquid-crystalline state at the growth temperatures and in the phase-separation state at the chilling temperatures. Temperatures for the onset of phase separation were 5 and 16 degrees C in cells grown at 28 and 38 degrees C, respectively.In comparison, another blue-green alga, Anabaena variabilis, which is not susceptible to chilling, was also examined by the freeze-fracture electron microscopy. The intramembrane particles were distributed at random in the fracture faces of cytoplasmic membrane at the growth, as well as at the chilling, temperatures.The results in this and previous studies suggest that the chilling susceptibility of A. nidulans is a result of irreversible leakage of ions from the cytoplasm when the lipids of cytoplasmic membrane are in the phase-separation state at low temperatures.

Photoactivation of the Water-Oxidation System in Isolated Intact Chloroplasts Prepared from Wheat Leaves Grown under Intermittent Flash Illumination.

Photoactivation of the latent oxygen-evolving system in intact chloroplasts isolated from wheat (Triticum aestivum L.) leaves grown under intermittent flash illumination was investigated, and the following results were obtained: (a) The water-oxidation activity generated on illuminating the isolated intact chloroplasts was as high as that generated in intact leaves, indicating that all the machinery necessary for the activity generation is assembled within intact chloroplasts. (b) The generation of water-oxidation activity was accompanied by enhancement of the activity of diphenylcarbazide-oxidation, and both processes share the same photochemical reaction but with respective rate-limiting dark reactions of different efficiencies. (c) A23187, an ionophore for divalent cations, strongly inhibited the generation of water-oxidation activity but did not affect the activity once generated, which suggested that Mn atoms in the chloroplasts are susceptible to the ionophore before photoactivation but turn immune after photoactivation. (d) The generation of water-oxidation activity was not affected by the inhibitors of ATP formation and CO(2) fixation, but was inhibited by nitrite, methylviologen and phenylmercuric acetate which suppress or inhibit the reduction of ferredoxin in intact chloroplasts. It was inferred that some factor(s) probably present in stroma to be reduced by PSI photoreaction is involved in the process of photoactivation.

Chilling Susceptibility of the Blue-green Alga Anacystis nidulans: I. EFFECT OF GROWTH TEMPERATURE.

Effects of chilling treatment on the photosynthetic activities and the light-absorption and fluorescence spectra were investigated in intact cells of the blue-green alga Anacystis nidulans that were grown at different temperatures. When the algal cells grown at 38 C were treated at 0 C for 10 minutes, the photosynthesis and the Hill reaction with 1,4-benzoquinone were significantly inactivated and the light-absorption spectrum of carotenoids was modified. These parameters showed very similar temperature dependencies in the chilling susceptibility and the temperature regions critical for the susceptibility depended on the growth temperature. The midpoint values for the critical temperature regions were 4, 6, and 12 C in cells grown at 28, 33, and 38 C, respectively. It is proposed that a common mechanism would underlie the chilling susceptibility of the photosynthesis, the Hill reaction, and the carotenoid absorption spectrum. The decoupling of excitation transfer from allophycocyanin to chlorophyll a at the chilling temperatures occurred very slowly and is attributed to a somewhat different mechanism of the chilling susceptibility.

Chilling Susceptibility of the Blue-green Alga Anacystis nidulans: II. STIMULATION OF THE PASSIVE PERMEABILITY OF CYTOPLASMIC MEMBRANE AT CHILLING TEMPERATURES.

Potassium ions and amino acids were found to leak from the cytoplasm to the outer medium when the blue-green alga, Anacystis nidulans, was exposed to the chilling temperatures. The leakage was marked below the critical temperature regions, the midpoint values for which were around 5 and 14 C in cells grown at 28 and 38 C, respectively. These temperature regions coincided with those critical for the susceptibility of the photosynthetic activities and the carotenoid absorption spectrum previously studied (Ono TA, N Murata 1981 Plant Physiol 67: 176-181).Potassium and magnesium ions in the cell suspension medium protected the algal cells from the chilling-induced damage of the Hill reaction with 1,4-benzoquinone. The activity of the Hill reaction which had been diminished by the first chilling treatment in a low salt medium was restored by the second chilling treatment of a high salt medium. The chilling susceptibility of the Hill reaction could be attributed to the leakage of cations from the cytoplasm due to increased permeability of the cytoplasmic membrane at the chilling temperatures.A mechanism is proposed to interpret the chilling susceptibility of A. nidulans: (a) at chilling temperatures, the bilayer lipids of the cytoplasmic membrane are in the phase separation state; (b) ions and solutes having low molecular weights leak from the cytoplasm to the outer medium when the lipids of the cytoplasmic membrane are in the phase separation state; (c) decreases in the intracellular concentrations of ions and solutes degrade the physiological activities of the cells.

Unique binding site for Mn2+ ion responsible for reducing an oxidized YZ tyrosine in manganese-depleted photosystem II membranes.

Binding of Mn2+ to manganese-depleted photosystem II and electron donation from the bound Mn2+ to an oxidized YZ tyrosine were studied under the same equilibrium conditions. Mn2+ associated with the depleted membranes in a nonsaturating manner when added alone, but only one Mn2+ ion per photosystem II (PS II) was bound to the membranes in the presence of other divalent cations including Ca2+ and Mg2+. Mn2+-dependent electron donation to photosystem II studied by monitoring the decay kinetics of chlorophyll fluorescence and the electron paramagnetic resonance (EPR) signal of an oxidized YZ tyrosine (YZ+) after a single-turnover flash indicated that the binding of only one Mn2+ ion to the manganese-depleted PS II is sufficient for the complete reduction of YZ+ induced by flash excitation. The results indicate that the manganese-depleted membranes have only one unique binding site, which has higher affinity and higher specificity for Mn2+ compared with Mg2+ and Ca2+, and that Mn2+ bound to this unique site can deliver an electron to YZ+ with high efficiency. The dissociation constant for Mn2+ of this site largely depended on pH, suggesting that a single amino acid residue with a pKa value around neutral pH is implicated in the binding of Mn2+. The results are discussed in relation to the photoactivation mechanism that forms the active manganese cluster.

Changes in Protein Composition and Mn Abundance in Photosystem II Particles on Photoactivation of the Latent O(2)-Evolving System in Flash-Grown Wheat Leaves.

Protein composition and Mn abundance were compared between the two photosystem II (PSII) particle preparations obtained before and after photoactivation of the latent O(2)-evolving system in intermittently flashed wheat leaves. The following results have been obtained: (a) nonphotoactivated PSII particles were devoid of two extrinsic proteins which corresponded to the 24 and 16 kilodalton proteins in spinach particles, although the particles contained all the intrinsic proteins and the 33 kilodalton extrinsic protein. (b) The two extrinsic proteins absent in nonphotoactivated PSII particles were present in nonphotoactivated thylakoids, but were easily removed by a hypotonic shock followed by brief sonication. Such removal of the proteins did not occur in photoactivated thylakoids. (c) Nonphotoactivated PSII particles contained 1.5 Mn/400 chlorophyll, while photoactivated particles contained 8 Mn/400 chlorophyll. (d) Nonphotoactivated thylakoids contained 6 Mn/400 chlorophyll, but most of them were removed from thylakoids by a hypotonic shock in the presence of ethylenediaminetetraacetate. Such removal of Mn did not occur in photoactivated thylakoids.

The role of light in formation of modified S2-state upon low pH-treatment of oxygen-evolving Photosystem II.

An abnormal, structurally modified, kinetically stable S2-state has been reported to be induced when Photosystem II was treated with NaCl-EGTA (or EDTA) in the light or with pH in darkness, both are assumed to release functional Ca(2+). In order to compare the mechanism of induction of modified S2-state between the two treatments, effects of illumination during or before low pH-treatment on formation of the abnormal S2-state were investigated by means of thermoluminescence measurements and low temperature EPR spectroscopy. Following results have been obtained: Flash illumination during low pH-treatment did not practically induce the abnormal S2-state, whereas preflash illumination given immediately before low pH-treatment efficiently induced the abnormal S2-state, and its amplitude showed a period-four oscillation on varying the preflash number with maxima at the second and sixth flashes. The abnormal S2-state thus induced by preflashes was identical with the modified S2-state that could be induced in dark-low pH-treated Photosystem II by excitation at 0°C after neutralization to pH 6.5. It was inferred that in low pH-treatment, modified S2-state can be formed from both S2- and S3-states, but its yield from the latter is much higher than from the former, consistent with the early results by Boussac et al. obtained for NaCl-EGTA-light or NaCl-citrate-light treatment.

Simulation of S2-state multiline EPR signal in oriented photosystem II membranes: structural implications for the manganese cluster in an oxygen-evolving complex.

High-quality angular-dependent spectra of multiline electron paramagnetic resonance (EPR) signals from the S2-state Mn cluster in a photosynthetic oxygen-evolving complex (OEC) were obtained for partially oriented photosystem (PS) II membranes, and the magnetic structure of the Mn cluster has been studied by simulation analysis. The angular-dependent multiline spectra were simulated by taking into account the anisotropic properties of both hyperfine tensors of intrinsic Mn ions and g-tensor of the cluster in a tetranuclear model. The best-fit parameters for the simulation indicate that (a) the oxidation state of the S2-state Mn cluster is Mn(III, IV, IV, IV), (b) the electronic orbital configuration of the Mn(III) ion is (dpi)3[dz2(sigma))]1, (c) the effective g-tensor of the Mn cluster and the hyperfine tensor of the Mn(III) ion are axially symmetric, and their principal z-axes are nearly collinear each other, and (d) the z-axis of the dz2 orbital of the Mn(III) ion and the normal of the thylakoid membrane are at an angle of 50.1 +/- 1.8 degrees. The results are compatible with those of the oriented XAFS study [Mukerji, I., et al. (1994) Biochemistry 33, 9712-9721], and indicate that the O-O vector of the putative di-mu-oxo bridged Mn(III)-Mn(IV) dimer unit in the Mn cluster tilts by 43-56 degrees with respect to the normal of thylakoid membrane. A model of the arrangement of the di-mu-oxo bridged Mn(III)-Mn(IV) unit with respect to the thylakoid membrane is proposed.

Positions of Q(A)and Chl(Z)Relative to Tyrosine Y(Z)and Y(D)in Photosystem II Studied by Pulsed EPR.

PELDOR (Pulsed Electron eLectron DOuble Resonance) was applied to determinethe distance of between Y(Z)and Q(A) (-)inY(D)-less mutant of Chlamydomonas reinhardtiiin Tris-treatedand Zn-substituted preparation of photosystem II. The value of distance wasfound to be 34.5 ± 1 Â. A '2+1' electron spin echo method has beenapplied to measure the orientation of the radius-vector RfomY(D)to Chl(Z)in a membrane-oriented photosystem II. The anglebetween Rand the membrane normal nwas determined to be 50 ±5(°), using the distance 29.4 ± 0.5 Â determined in non-orientedPS II.