Yoshinori Toyoshima

The role of lipids and 17-kDa protein in enhancing the recovery of O2 evolution in cholate-treated thylakoid membranes

The recovery of O2 evolution of cholate‐treated thylakoids induced by the simultaneous addition of the 17‐ and 23‐kDa proteins was enhanced by the further addition of thylakoid total lipids up to about 75% of the non‐depleted original broken thylakoids at the optimal concentration of the lipids, through reactivation of electron transfer between the sites of DPC donation and DCIP acceptance. Novel findings clarifing the cause of the discrepancies, with regard to the requirement for the 17‐kDa protein on the O2 evolution of thylakoid membranes, were also provided, i.e., when the assay of O2 evolution for the reconstituted systems was carried out at a low level of NaCl (e.g., 0.1 mM), besides the 23‐kDa protein, the 17‐kDa protein was also required for O2 evolution, however, at a higher level of NaCI (>5 mM) the 17‐kDa protein was not. The results suggest that the 17‐kDa protein takes the place of Cl− or acts as a constituent protecting C− in the Cl− activating sites against fluctuation of the Cl− level in the inner compartment of thylakoid membranes.

Disintegration and reconstitution of Photosystem II reaction center core complex. I. Preparation and characterization of three different types of subcomplex

Abstract Disintegration of the Photosystem II (PS II) reaction center core complex (RC complex) from the spinach PS II by using mild detergents resulted in success of isolating three types of subcomplex (complexes 1–3), retaining their prosthetic groups and two pigments-binding subunit polypeptides with molecular masses of 47 kDa and 43 kDa. The polypeptide compositions of the three complexes isolated were 47 kDa/43 kDa/D1 cytochrome b-559 in complex 1, 47 kDa/D1/D2-cytochrome b-559 in complex 2 and D1/D2/cytochrome b-559 in complex 3. The complex 3 was confirmed to have four Chl a, two pheophytins a, one β-carotene and one plastoquinone A-9 (PQA) in molar ratio. These numbers were essentially the same as in the results reported by Nanba and Satoh on their D1/D2/cytochrome b-559 particle, except for PQA. All of the three subcomplexes isolated in the present work preserved one PQA at least. However, instead of the photoinduced charge separation between P-680 and QA the formation of the triplet state P-680 as a result of recombination of photoinduced P-680+ and Pheo a was observed in the three preparations. The decay time of the triplet state was determined as 25 μs. Evidence suggesting that the reconstitution of QA function was performed in complex 1 and complex 2 with the thylakoid lipids and thylakoid lipids plus PQA, respectively, was obtained.

Stoichiometry of cytochrome b-559 in Photosystem II

A careful determination of the stoichiometry of cytochrome b -559 per Photosystem II reaction centre has been made for different Photosystem II preparations. Using an extinction coefficient determined from the α-band of reduced pyridine haemochrome and chemical analyses of pigment levels it is concluded that there is one cytochrome b -559 haem per Photosystem II reaction centre. This conclusion assumes that by analogy with the purple bacterial reaction centre there are two pheophytin molecules per Photosystem II reaction centre.

A protein essential for recovering oxygen evolution in cholate-treated chloroplasts

A novel method for the reconstitution of oxygen evolution in cholate‐extracted spinach thylakoid membranes was established and a protein essential for the reconstitution was purified from cholate extracts. Purification of the protein was accomplished by chromatography on a DEAE‐Sephacel column. This protein (M r 17 000) was reinserted into vesicular membranes reconstituted from cholate‐extracted thylakoids in the presence of 25% glycerol to reactivate oxygen evolution.

Disintegration and reconstitution of Photosystem II reaction center core complex. II. Possible involvement of low-molecular-mass proteins in the functioning of QA in the PS II reaction center*

Spinach Photosystem II (PS II) reaction center complex (RC) was disintegrated in three different ways using 0.5% n-dodecyl β- d -maltoside (DM) (DM particle), 20 mM n-octyl β- d -thioglucopyranoside (OTG) (OTG particle) or 4% Triton X-100 (Triton particle and NS particle), respectively, followed by chromatography on ion-exchange columns. The average numbers of plastoquinone A-9 (PQA) molecule retained in the DM, OTG and Triton particles were 0.88, 0.63 and 0.68 per reaction center, respectively, on the basis of two pheophytin a (Pheo a) molecules and the corresponding QA activities were about 80, 15 and 45% of the original RC level. A difference in the protein pattern on SDS-PAGE between the Triton and OTG particles was observed in the low-molecular-mass region, i.e., bands of the psbL gene product (L protein) and 4.1 kDa protein detected in the former were almost lost in the latter, while the bands for the 6.1 kDa and three small proteins appeared in the latter but not in the former. By the treatment of the RC with 20 mM OTG at 10°C for 1 h, several protein components, including the proteins in the low-molecular-mass region, were extracted. Reconstitution of the OTG particle with the crude mixture of the low-molecular-mass proteins contained in the OTG extract resulted in successful recovery of the QA activity in the resulting particle at about 50% of the original RC level. From the results of the reconstitution experiments and comparison of the protein compositions among the particles, we propose that, besides the D1 and D2 proteins, two subunits of cytochrome b-559, 47 and 43 kDa chlorophyll binding proteins and the recently discovered 4.8 kDa protein, some other small proteins including the 5.0 kDa L protein are possibly involved in normal functioning of QA in the PS II reaction center.

Photoinduced charge separation in liposomes containing chlorophyll a. I. Photoreduction of copper(II) by potassium ascorbate through liposome bilayer containing purified chlorophylla a

Photosensitivity of dispersion of phosphatidylcholine bilayer liposomes containing purified chlorophyll alpha was examined. The reduction of Cu(II) in the solution outside liposomes was observed upon illumination with visible light under anaerobic condition by means of ESR. The rate of photoreduction was significantly increased by a reductant, potassium ascorbate, localized in the solution of the opposite side of the membrane. The aciton spectrum of the reduction agreed with the absorption spectrum of chlorphyll a in the dispersion. The amount of bleach chlorophyll a was negligible compared with that of reduced (Cu(II). These facts lead to the conclusion that the potoinduced redox reactions at both the membrane-solution interfaces are coupled with each other through the bilayer of each liposome. Kinetic analysis of the reactions based on a possible reaction scheme was carried out and some of the kinetic parameters were determined.

Reconstitution of photosynthetic charge accumulation and oxygen evolution in CaCl2-treated PS II particles: I: Establishment of a high recovery of O2 evolution and examination of the roles of the 17-, 23- and 34-kDa proteins, focusing on the effect of Cl− on

The reconstitution of high O2 evolution in CaCl2‐treated PS II particles was achieved by the simultaneous addition of the 17‐, 23‐ and 34‐kDa proteins and total thylakoid lipids in the presence of 25% glycerol and 15 mM sodium cholate. The activity of the reconstituted membranes recovered to 85% of that of the non‐depleted original PS II particles at the optimal condition. By means of this reconstitution method, evidence for the cooperation of the three proteins in the recovery of O2 evolution in the CaCl2‐treated PS II particles was found by changing the concentration of NaCI in the assay medium, and the relationship between the amount of manganese retained in the water‐splitting complex and the O2 evolving activity was examined by using the partially solubilized PS II particles with n‐octyl‐β‐D‐glucoside.

Direct evidence for cooperation of the 17- and 23-kDa proteins in the recovery of oxygen evolution in cholate-treated thylakoids

Repeated extractions of spinach thylakoid membranes with a solution containing 50 mM sodium cholate, 1 M NaCl, 3 mM MgCl2, 0.2 M sucrose and 20 mM tricine at pH 8.4 for 15 min perfectly inhibited the O2 evolution of the thylakoids, concomitant with a complete release of the 17‐ and 23‐kDa proteins and partial release of many other proteins from the thylakoid membranes. Recovery of O2 evolution in the cholate‐treated thylakoids was achieved up to about 40% of that in the original thylakoids by the simultaneous reinsertion of the 17‐ and 23‐kDa proteins, but not by the reinsertion of one of them only. The recovery of O2 evolution induced by the reinsertion of the 17‐ and 23‐kDa proteins was enhanced by the further addition of a certain fraction of the crude thylakoid extract up to about 70% of the non‐depleted control, suggesting that in addition to the 17‐ and 23‐kDa proteins, one or more unknown component(s) released partially from the thylakoids upon cholate treatment is (are) also (a) constituent(s) of the O2 evolving apparatus. The purified 34‐kDa protein did not replace the unknown component.

Roles of three lumen-surface proteins in the formation of S2 state and O2 evolution in Photosystem II particles from spinach thylakoid membranes

Abstract Reconstitution of O 2 evolution in CaCl 2 -treated PS II at the level of the original PS II was achieved by simultaneous reinsertion of the three lumen-surface proteins, 17, 23 and 34 kDa proteins, and total thylakoid lipids in the presence of 25% glycerol and 15 mM sodium cholate. By means of this reconstitution method, the following was found, concerning the roles of the three proteins in the formation of the S 2 state and O 2 evolution of the water-splitting complex. (1) Complete removal of the three proteins from the PS II particles by 1 M CaCl 2 treatment caused a rapid release of Mn from the particles when incubated at 25°C, but not at 0°C. The rapid release of Mn at 25°C was prevented by the reinsertion of the 34 kDa protein but not by the other two proteins. (2) O 2 evolution was recovered in CaCl 2 -treated PS II by reinserting the 34 kDa protein, when Cl − was present at a level of 50 mM, but it declined when the Cl − concentration was decreased. Upon further addition of the 23 kDa protein, O 2 -evolving activity was enhanced to the original PS II level at concentrations of Cl − higher than 10 mM. Below this concentration, besides the 23 and 34 kDa proteins, the 17 kDa protein was required for maximal O 2 evolution. (3) The effects of the three proteins and Cl − on the recovery of the low-temperature multiline EPR signal in the CaCl 2 -treated PS II were essentially the same as those on the recovery of the O 2 evolution, indicating that the three proteins and Cl − contribute to the advancement of the water-splitting complex to the S 2 state.

Photoinduced charge separation in liposomes containing chlorophyll a. II the effect of ion transport across membrane on the photoreduction of Fe(CN)63

Abstract The reduction of Fe(CN)63− in the solution of outside liposome incorporating chlorophyll a was observed upon illumination with visible light under unaerobic condition. The photoreduction of Fe(CN)63− was enhanced by the presence of ion carrier, such as carbonylcyanide m-chlorophenylhydrazone, in the dispersion. The action spectrum of the photoreduction agreed with the absorption spectrum of chlorophyll a in liposome bilayer. The carrier-mediated proton transport was also observed upon illumination, occuring in the direction to compensate the excess charge produced by the photoinduced electron transport through the bilayer.