Reconstitution of photosynthetic charge accumulation and oxygen evolution in CaCl₂‐treated PS II particles

Abstract

The reconstitution of high 0, evolution in CaCl,-treated PS II particles was achieved by the simultaneous addition of the 17-, 23-and 34kDa 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 nondepleted 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 0, evolution in the CaCl,-treated PS II particles was found by changing the concentration of NaCl in the assay medium, and the relationship between the amount of manganese retained in the water-splitting complex and the 0, evolving activity was examined by using the partially solubilized PS II particles with n-octyl-/I-D-glucoside.

N‐terminal sequence determination and secondary structure analysis of extrinsic membrane proteins in the water‐splitting complex of spinach

“…The extrinsic membrane proteins of the watersplitting complex were purified either as described in [18] or using the following procedure: Photosystem II peptides were prepared from spinach thylakoid membranes by treatment with Triton X-100 [19,20] exchange chromatography on Pharmacia Mono Q HR 5/S [21]. The 23 kDa protein was eluted with a linear gradient ranging from 0 to 0.3 M NaCl either in 20 mM Tris or 20 mM NI&HCOJ buffer at pH 8.0.…”

“…The 16 and 23 kDa proteins appear only partially separated under these conditions starting from the 0.5 or 1 M NaCl extract ( fig.lb). Therefore, they were first separated by an FPLC technique using anion- exchange chromatography on Pharmacia Mono Q HR 5/S [21]. The 23 kDa protein was eluted with a linear gradient ranging from 0 to 0.3 M NaCl either in 20 mM Tris or 20 mM NI&HCOJ buffer at pH 8.0.…”

Effect of alkaline pH on photosynthetic water oxidation and the association of extrinsic proteins with Photosystem Two

“…It has been reported that the inhibition of oxygen evolution by the selective removal of the 23 kDa protein can be partially overcome by increasing the concentration of Ca2l (Ghanotakis et al, 1984;Miyao & Murata, 1984;Nakatani, 1984) and Cl- (Critchley et al, 1984;Imaoka et al, 1984) in the reaction medium. In our assays the Cl-concentration was 25 mm, and further additions of this anion or CaCl2 did not eliminate the inhibition and recovery of oxygen evolution observed when the 23 kDa protein was removed and rebound.…”

The Water Oxidation Complex of Chlamydomonas

“…However, we show that the mature form of the 29 kilodalton polypeptide accumulates to wild-type levels in whole cell extracts of photosystem II deficient mutants and a water oxidation mutant of Chlamydomonas. Impaired membrane assembly has no effect on the maturation or stability of this component of the multi-subunit water oxidation complex.Three extrinsic polypeptides, with approximate mol wt of 33, 24, and 18 kD, participate in photosynthetic water oxidation in vascular plants (2,14,16). The precise function of each polypeptide is unclear, but the polypeptides are known to be organized as a complex on the lumenal side of thylakoids in close association with PSII reaction centers in higher plants (2,16,21 (19,20,24,33 (8,9,17,19).…”

“…Three extrinsic polypeptides, with approximate mol wt of 33, 24, and 18 kD, participate in photosynthetic water oxidation in vascular plants (2,14,16). The precise function of each polypeptide is unclear, but the polypeptides are known to be organized as a complex on the lumenal side of thylakoids in close association with PSII reaction centers in higher plants (2,16,21).…”