Taka-aki Ono

Direct detection of a carboxylate bridge between Mn and Ca2+ in the photosynthetic oxygen-evolving center by means of Fourier transform infrared spectroscopy

Abstract Calcium is an indispensable cofactor for photosynthetic oxygen evolution. We have studied structural relevance of Ca2+ to the oxygen-evolving center (OEC) of Photosystem II (PS II) by means of Fourier transform infrared (FTIR) spectroscopy. The single-pulse induced FTIR difference spectra of PS II membranes reflecting solely the structural changes of OEC between in the S1 and S2 states were measured by controlling the redox potential and pH of the buffer. Comparison between the two S2/S1 difference spectra using untreated and Ca2+-depleted PS II membranes showed that the negative bands at 1560 and 1403 cm−1 (belonging to S1) and the positive bands at 1587 and 1364 cm−1 (belonging to S2) were lost upon Ca2+ depletion. These bands were assigned to the asymmetric (higher frequency bands) and symmetric (lower frequency bands) COO− stretching modes of a certain carboxylate group in Asp, Glu or the C-termini, based on the infrared data of 20 amino acids and the S2/S1 spectra of 15N-labeled PS II membranes. The frequency differences of the asymmetric and symmetric COO− bands, i.e., 157 cm−1 for S1 and 223 cm−1 for S2, indicated that this carboxylate group possesses the structure of bridging bidentate coordination in the S1 state and that of unidentate coordination in the S2 state. Taking together the observation of disappearance of these bands upon Ca2+ depletion, it was concluded that (i) this carboxylate serves as a bridging ligand between the redox-active Mn and the Ca2+ ions, (ii) upon S2 formation, the coordination bond of this carboxylate to Ca2+ is selectively broken, and (iii) upon depletion of Ca2+, this carboxylate ligand is liberated even from the Mn ion. Along with the changes of COO− bands, several intense bands in 1680–1630 cm−1, which were assigned to the amide I modes of backbone amide groups, were lost upon Ca2+ depletion. This indicates that some perturbations on the protein conformations around the Mn-cluster induced by the S2 formation require the presence of Ca2+ in OEC. Possible roles of Ca2+ in the oxygen-evolving reactions are discussed based on these findings.

Requirement of divalent cations for photoactivation of the laten water-oxidation system in intact chloroplasts from flashed leaves

Abstract The effects of divalent cations on photoactivation of the latent water-oxidation system in intact chloroplasts isolated from wheat (Triticum aestivum L.) leaves grown under intermittent flash illumination were investigated by using A23187, an ionophore for divalent cations, and the following results were obtained. (a) Photoactivation in the intact chloroplasts was inhibited by A23187, but was restored on addition of a low concentration of Mn2+ (10 μM). (b) A high concentration of Mn2+ (70 μM) was inhibitory, in contrast, for photoactivation, but the inhibition was restored by the coexistence of a suitable concentration of Ca2+ (5 mM). (c) The Ca2+-dependent restoration was inhibited by a high concentration of Mg2+ or Sr2+, but the inhibition was restored by the coexistence of Ca2+. (d) Kinetic analyses of these competitive effects between divalent cations revealed that: (i) High concentration of Ca2+ inhibits photoactivation in competition with Mn2+. (ii) High concentration of Mn2+ inhibits photoactivation in competition with Ca2+. (iii) High concentration of Mg2+ affects photoactivation by inhibiting Ca2+-dependent restoration in competition with Ca2+. Based on these results, we propose that the latent water-oxidation center has two binding sites, each specific for Mn2+ and Ca2+, and that photoactivation takes place in the center having both Mn2+ and Ca2+ on their respective binding sites.

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.

A CARBOXYLATE LIGAND INTERACTING WITH WATER IN THE OXYGEN-EVOLVING CENTER OF PHOTOSYSTEM II AS REVEALED BY FOURIER TRANSFORM INFRARED SPECTROSCOPY

Abstract The effect of H/D exchange on a Fourier transform infrared (FTIR) difference spectrum between the S1 and S2 states of the oxygen-evolving center (OEC) in Photosystem II (PS II) has been investigated. Upon deuteration, a large upshift of a differential band by about 18 cm−1 was observed in the 1600-1500 cm−1 region, where asymmetric COO− stretching and amide II modes mainly have intensities. This upshift was basically unaffected by 15N substitution of the PS II membranes. FTIR measurements of three model carboxylate compounds showed that asymmetric COO− stretching bands upshift upon deuteration when the carboxylate groups form hydrogen bonds with water; for free acetate and Mn-EDTA in which the carboxylate groups interact with bulk water, the upshift was 7 cm−1, whereas (CH3COO)2Ni · 4H2O whose carboxylate ligands form hydrogen bonds with water molecules coordinated to the metal ion, exhibits a much larger upshift of 20 cm−. The above observations indicate that the most plausible assignment of the upshifting band in the S 2 S 1 spectrum is the asymmetric COO− mode of a carboxylate group interacting with water in OEC. The large upshift of 18 cm−1 found in PS II may suggest that this carboxylate group, which is possibly a ligand of a redox-active Mn ion, forms a hydrogen bond with a water molecule bound to the Mn-cluster, although the possibility that it forms a strong hydrogen bond with an exchangeable hydrogen atom of the neighboring amino acid residue cannot be excluded. In the amide I region of the S 2 S 1 spectrum, the bands around 1659 cm−1 exhibited large downshifts to around 1645 cm−1 upon deuteration. This frequency shift upon deuteration is typical of a random coil structure, and hence it is suggested that hydrophilic domains with random coil conformations are present in the close vicinity of the Mn-cluster.

Temperature dependence of 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.

Temperature dependence of the photosynthetic activities in the thylakoid membranes from the blue-green alga Anacystis nidulans

Abstract The photosynthetic electron transport and phosphorylation reactions were measured in the room temperature region in the thylakoid membranes prepared from the blue-green alga, Anacystis nidulans . The Arrhenius plot of the Hill reaction with 2,6-dichlorophenolindophenol showed a distinct break of straight lines at 21°C in the membranes from cells grown at 38°C, and at 12°C in those from cells grown at 28°C. The Arrhenius plot of the Hill reaction with ferricyanide showed a break at 13°C in the membranes from cells grown at 38°C, and at 7°C in those from cells grown at 28°C. On the other hand, the Arrhenius plot of the System I reaction with methylviologen as an electron acceptor and 2,6-dichlorophenolindophenol and ascorbate as an electron donor system was composed of a straight line in the membranes from cells grown at 28°C as well as at 38°C. The Arrhenius plot of the System II reaction measured by the ferricyanide reduction mediated by silicotungstate in the presence of 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea also showed a break at 11°C in the membranes from cells grown at 38°C. The Arrhenius plot of the phosphorylation mediated by N -methylphenazonium methylsulfate showed a break at 21°C in the membranes from cells grown at 38°C and at 12°C in those from cells grown at 28°C. The Arrhenius plot of the phosphorylation mediated by the System I reaction showed a break at 24°C in the membranes from cells grown at 38°C. The characteristic features in the Arrhenius plots of the photosynthetic electron transport and phosphorylation reactions are discussed in terms of the transition of physical phase of the thylakoid membrane lipids.

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.

Photosynthetic electron transport and phosphorylation reactions in thylakoid membranes from the blue-green alga Anacystis nidulans

Thylakoid membranes were prepared from the blue-green alga, Anacystis nidulans with lysozyme treatment and a short period of sonic oscillation. The thylakoid membrane preparation was highly active in the electron transport reactions such as the Hill reactions with ferricyanide and with 2,6-dichlorophenolindophenol, the Mehler reaction mediated by methyl viologen and the system 1 reaction with methyl viologen as an electron acceptor and 2,6-dichlorophenolindophenol and ascorbate as an electron donor system. The Hill reaction with ferricyanide and the system 1 reaction was stimulated by the phosphorylating conditions. The cyclic and non-cyclic phosphorylation was also active. These findings suggest that the preparation of thylakoid membranes retained the electron transport system from H2O to reaction center 1, and that the phosphorylation reaction was coupled to the Hill reaction and the system 1 reaction.

Temperature dependence of the S1→S2 transition in the oxygen-evolving complex of photosystem II studied by FT-IR spectroscopy

The temperature dependence of the single-turnover redox reaction on the electron-donor side of Photosystem II (PS II) has been investigated by measuring light-induced FTIR difference spectra of DCMU-treated PS II membranes at various temperatures of 80–240 K. The negative band at 1404 cm−1 in the difference spectra exhibited the same temperature dependence as the S2 formation in the oxygen-evolving center previously determined by EPR spectroscopy (De Paula, J.C., Innes, J.B. and Brudvig, G.W. (1985) Biochemistry 24, 8114–8120). This observation strongly supports our previous assignment of the 1404 cm−1 band to the structural change of proteins upon the S1 → S2 transition (Noguchi, T., Ono, T. and Inoue, Y. (1992) Biochemistry 31, 5953–5956). The temperature dependence of the 1660 cm−1 band due to cytochrome b559 (Cyt b559) oxidation has shown a complementary relationship to that of the 1404 cm−1 band, consistent with the view that the oxidation of Cyt b559 competes with the S2 formation on the electron-donor side of PS II. The present FTIR data also suggest that the structural difference between the two S2 states exhibiting the g = 4.1 EPR signal and the multiline signal is not very large as far as the protein moiety around the Mn cluster responsible for the 1404 cm−1 band is concerned.

Applications of pulsed ELDOR-detected NMR measurements to studies of photosystem II: Magnetic characterization of YD tyrosine radical and Mn2+ bound to the high-affinity site

The pulsed electron-electron double resonance (ELDOR)-detected nuclear magnetic resonance (NMR) technique has been applied to the investigation of the hyperfine structure of the oxidized radical of YD tyrosine (YD·) and Mn2+ ion bound to the high-affinity site in photosystem II. The resulting ELDOR spectrum of YD· was found to correspond with the pulsed electron-nuclear double resonance (ENDOR) spectrum except for a slightly larger linewidth (by a factor of 1.7). The spectrum showed marked anisotropy and revealed three peaks which can be assigned to matrix protons, C-3 and −5 protons and one of the β-protons in the tyrosine molecule. The results demonstrate that the pulsed ELDOR-detected NMR technique is applicable to the study of organic radicals in biological systems. The Mn2+ ion bound to the high-affinity site in photosystem II yielded well-resolved ELDOR signals spreading over ±1000 MHz. The magnetic properties of the Mn2+ were characterized on the basis of the calculation of the ENDOR transitions and the anisotropy of the ELDOR spectrum.